Nanomaterial composite and its anti-tumor use

Abstract

The present application relates to a kind of anti-tumor nanomaterial complex, it includes the SP94-PEG modified graphite alkyne carrier and antitumor drug, the SP94-PEG is multi-arm polyethylene glycol connection targeted peptide SP94, the antitumor drug includes sorafenib, siRNA and doxorubicin and ferrous ion complex, the siRNA is the nucleotide sequence of targeting SLC7A11 gene.The drug-loaded graphite alkyne nanocomposite of the present application has special advantage and good dispersibility, solves the toxic side effect and drug resistance problem brought by traditional chemotherapy drug, it belongs to the nanomedicine of cancer cell by ferroptosis targeted, can make in vitro and in vivo antitumor treatment efficiency significantly improve, is a kind of promising tumor targeted therapy strategy.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceuticals, specifically relating to a nanomaterial composite and its anti-tumor applications. Background Technology

[0002] Cancer is a significant public health issue worldwide, ranking as the leading cause of death globally. Among these, liver cancer has garnered significant attention from the medical community due to its severe impact. According to the latest cancer statistics in 2022, although the incidence of new liver cancer cases is only 2%, it remains the fastest-growing cancer in decades, accounting for an estimated 5% of all cancer deaths worldwide. Most notably, hepatocellular carcinoma (HCC) is the most common type of liver cancer, accounting for 85%-90% of all primary liver cancer pathological types.

[0003] Clinical treatment for hepatocellular carcinoma mainly includes local ablation, surgical resection, and drug therapy. Although significant progress has been made in resection combined with drug therapy, long-term use of chemotherapy drugs leads to significant drug resistance, resulting in poor clinical prognosis. Furthermore, while the relative survival rate for most cancers has improved to 68%, the 5-year overall relative survival rate for hepatocellular carcinoma remains only 20%. Therefore, there is an urgent need to develop a more effective and stable targeted therapy strategy.

[0004] Graphdiyne oxide (GDYO) is a two-dimensional carbon nanomaterial composed of sp and sp2 hybridized carbon atoms. It is obtained by oxidizing the alkynyl groups of graphdiyne (GDY) with concentrated nitric acid, and its surface is composed of ordered carbon oxygen and carbon hydroxyl groups. These groups have a high affinity for proteins driven by hydrogen bonding and salt bridge formation, and the depressions formed on their surface provide great potential for loading small molecule drugs and genes, making graphdiyne oxide a promising candidate for applications in the pharmaceutical field.

[0005] Sorafenib is a multi-target tyrosine kinase inhibitor and is currently considered the first-line chemotherapy drug for treating hepatocellular carcinoma. Notably, sorafenib is not only a kinase inhibitor but also an inducer of ferrous degradation. By inhibiting the expression level of the cystine transporter SLC7A11, it reduces intracellular GSH levels and induces iron sagging.

[0006] As a broad-spectrum chemotherapy drug, doxorubicin (DOX), after being modified with ferrous ions, can induce a new form of cell death. DOX-Fe 2+ The complex is a compound drug in which ferrous ions preferentially replace the phenolic hydrogen sites in DOX. DOX-Fe 2+It can increase the unstable iron pool in tumor cells and induce excessive lipid peroxidation in mitochondria. Unlike other forms of cell death, sideroptosis is caused by the abnormal accumulation of lipid peroxides due to excess iron. The regulation of the SLC7A11 and GPX4 genes is the main molecular mechanism of sideroptosis. Inducing sideroptosis in iron-rich tumors is a challenging problem with great potential. However, drug resistance is a major cause of chemotherapy failure and a major obstacle to clinical application. Previous studies have shown that sorafenib combined with copper disulfiram-induced hepatocyte sideroptosis enhances hepatocyte sensitivity. But these findings are far from sufficient, and it is imperative to find a new treatment strategy to improve the efficacy of chemotherapy drugs. Summary of the Invention

[0007] To solve the above-mentioned technical problems, the present invention includes the following aspects:

[0008] A first aspect of the present invention provides an anti-tumor drug, said drug being an siRNA targeting the SLC7A11 gene.

[0009] Preferably, the siRNA is an SLC7A11-i-1 or SLC7A11-i-2 nucleotide sequence, wherein the sense strand nucleotide sequence of SLC7A11-i-1 has at least 90% sequence homology with SEQ ID NO.1; the antisense strand nucleotide sequence of SLC7A11-i-1 has at least 90% sequence homology with SEQ ID NO.2; the sense strand nucleotide sequence of SLC7A11-i-2 has at least 90% sequence homology with SEQ ID NO.3; and the antisense strand nucleotide sequence of SLC7A11-i-2 has at least 90% sequence homology with SEQ ID NO.4.

[0010] Further preferably, the sense strand nucleotide sequence of SLC7A11-i-1 has at least 95% sequence homology, or at least 98% sequence homology, or at least 99% sequence homology with SEQ ID NO.1; the antisense strand nucleotide sequence of SLC7A11-i-1 has at least 95% sequence homology, or at least 98% sequence homology, or at least 99% sequence homology with SEQ ID NO.2; the sense strand nucleotide sequence of SLC7A11-i-2 has at least 95% sequence homology, or at least 98% sequence homology, or at least 99% sequence homology with SEQ ID NO.3; and the antisense strand nucleotide sequence of SLC7A11-i-2 has at least 95% sequence homology, or at least 98% sequence homology, or at least 99% sequence homology with SEQ ID NO.4.

[0011] Most preferably, the sense strand nucleotide sequence of SLC7A11-i-1 is: 5'- GGAAGAGAUUCAAGUAUUA-3' (SEQ ID NO.1); the antisense strand nucleotide sequence of SLC7A11-i-1 is: 3'- CCUUCUCUAAGUUCAUAAU-5' (SEQ ID NO.2).

[0012] Most preferably, the sense strand nucleotide sequence of SLC7A11-i-2 is: 5'-CUUGCAAUAUGUAUAUCCA-3' (SEQ ID NO.3); the antisense strand nucleotide sequence of SLC7A11-i-2 is: 3'-GAACGUUAUACAUAUAGGU-5' (SEQ ID NO.4).

[0013] A second aspect of the present invention provides an antitumor drug-loaded nanomaterial composite comprising an SP94-PEG modified graphdiyne oxide carrier and an antitumor drug.

[0014] Preferably, the SP94-PEG is a multi-arm polyethylene glycol (PEG) linked target peptide SP94, and the sequence of the target peptide SP94 is NH2-CGGSFSIIHTPILPL-COOH.

[0015] Preferably, the antitumor drug includes sorafenib, siRNA, and doxorubicin-ferrous ion complex (DOX-Fe). 2+ ).

[0016] Preferably, the molar ratio of doxorubicin to ferrous ions in the doxorubicin-ferrous ion complex is 1:3.

[0017] Preferably, the sorafenib and doxorubicin complex with ferrous ions (DOX-Fe) 2+ The molar ratio of sorafenib and doxorubicin to ferrous ions (DOX-Fe) is (1:5)-(5:1). More preferably, the sorafenib and doxorubicin complex with ferrous ions (DOX-Fe) is... 2+ The molar ratio of sorafenib and doxorubicin to ferrous ions is (1:3)-(3:1). Most preferably, the sorafenib and doxorubicin complex with ferrous ions (DOX-Fe) is... 2+ The molar ratio of ) is 1:1.

[0018] Preferably, the sorafenib and doxorubicin complex with ferrous ions (DOX-Fe) 2+ The weight ratio of ) is 1:1.

[0019] Preferably, the siRNA is a nucleotide sequence that targets the SLC7A11 gene.

[0020] More preferably, the siRNA is an SLC7A11-i-1 or SLC7A11-i-2 nucleotide sequence, wherein the sense strand nucleotide sequence of SLC7A11-i-1 has at least 90% sequence homology with SEQ ID NO.1; the antisense strand nucleotide sequence of SLC7A11-i-1 has at least 90% sequence homology with SEQ ID NO.2; the sense strand nucleotide sequence of SLC7A11-i-2 has at least 90% sequence homology with SEQ ID NO.3; and the antisense strand nucleotide sequence of SLC7A11-i-2 has at least 90% sequence homology with SEQ ID NO.4.

[0021] More preferably, the sense strand nucleotide sequence of SLC7A11-i-1 has at least 95% sequence homology, or at least 98% sequence homology, or at least 99% sequence homology with SEQ ID NO.1; and the antisense strand nucleotide sequence of SLC7A11-i-1 has at least 95% sequence homology, or at least 98% sequence homology, or at least 99% sequence homology with SEQ ID NO.2.

[0022] More preferably, the sense strand nucleotide sequence of SLC7A11-i-2 has at least 95% sequence homology, or at least 98% sequence homology, or at least 99% sequence homology with SEQ ID NO.3; and the antisense strand nucleotide sequence of SLC7A11-i-2 has at least 95% sequence homology, or at least 98% sequence homology, or at least 99% sequence homology with SEQ ID NO.4.

[0023] Most preferably, the sense strand nucleotide sequence of SLC7A11-i-1 is: 5'- GGAAGAGAUUCAAGUAUUA-3' (SEQ ID NO.1); the antisense strand nucleotide sequence of SLC7A11-i-1 is: 3'- CCUUCUCUAAGUUCAUAAU-5' (SEQ ID NO.2).

[0024] Most preferably, the sense strand nucleotide sequence of SLC7A11-i-2 is: 5'-CUUGCAAUAUGUAUAUCCA-3' (SEQ ID NO.3); the antisense strand nucleotide sequence of SLC7A11-i-2 is: 3'-GAACGUUAUACAUAUAGGU-5' (SEQ ID NO.4).

[0025] Preferably, the tumor is liver cancer or pancreatic cancer.

[0026] More preferably, the liver cancer is a human hepatocellular carcinoma cell line Huh7 or SMMC-7721; and the pancreatic cancer is a human pancreatic carcinoma cell line SW1990.

[0027] A third aspect of the present invention provides a method for preparing the above-mentioned drug-loaded nanomaterial composite, comprising the following steps:

[0028] (1) Mix GDYO powder with H2N-PEG-NH2, add EDC and N-hydroxysuccinimide to the mixture, stir at low temperature, and collect the dark brown precipitate by centrifugation;

[0029] (2) Disperse the dark brown precipitate with solvent, add EDC and N-hydroxysuccinimide to the suspension, stir and add SP94 short peptide powder, continue stirring at low temperature, and centrifuge to obtain graphdiyne oxide linked with SP94-PEG.

[0030] (3) Disperse the SP94-PEG-linked graphyne oxide obtained in step (2) with a solvent, add branched PEI and EDC to the suspension, stir, and then add DOX-Fe. 2+ Sorafenib was subjected to low-temperature ultrasonic treatment, stirred, centrifuged, and the precipitate was collected, freeze-dried, and the precipitate was obtained.

[0031] (4) Disperse the freeze-dried precipitate obtained in step (3) with a solvent, add siRNA, mix well, and obtain drug-loaded graphdiyne oxide nanocomposite.

[0032] Preferably, in step (1), the weight ratio of GDYO powder to H2N-PEG-NH2 is 1:3.

[0033] Preferably, the weight ratio of GDYO powder in step (1) to SP94 short peptide powder in step (2) is 4:1.

[0034] Preferably, in step (3) DOX-Fe 2+ The preparation method is as follows: Dissolve doxorubicin in an organic solvent, add anhydrous FeCl2 to the doxorubicin solution, mix well, and the product is obtained.

[0035] Preferably, the DOX-Fe 2+ The organic solvent used in the preparation method is DMSO.

[0036] Preferably, the DOX-Fe 2+ The concentration of doxorubicin solution in the preparation method is 10 mg / ml.

[0037] Preferably, the DOX-Fe 2+ In the preparation method, the molar ratio of doxorubicin to FeCl2 is 1:3.

[0038] Preferably, the DOX-Fe2+ The mixing time in the preparation method is 30 minutes.

[0039] Preferably, the DOX-Fe 2+ The weight ratio of sorafenib to surafenib is 1:1.

[0040] Preferably, the DOX-Fe 2+ The molar ratio of sorafenib to sorafenib is 1:1.

[0041] Preferably, in step (4), the reaction is carried out at a ratio of 5g of lyophilized precipitate per 1 nmol of siRNA.

[0042] A fourth aspect of the present invention provides the application of the above-described drug-loaded nanomaterial composite in the preparation of antitumor drugs.

[0043] Preferably, the tumor is liver cancer or pancreatic cancer.

[0044] More preferably, the hepatocellular carcinoma is a human hepatocellular carcinoma cell line Huh7 or SMMC-7721; and the pancreatic cancer is a human pancreatic carcinoma cell line SW1990.

[0045] A fifth aspect of the present invention provides a pharmaceutical composition having a synergistic antitumor effect, comprising sorafenib and doxorubicin with a ferrous ion complex, wherein the sorafenib and doxorubicin with a ferrous ion complex (DOX-Fe) 2+ The molar ratio of ) is (1:5)-(5:1).

[0046] Preferably, the sorafenib and doxorubicin complex with ferrous ions (DOX-Fe) 2+ The molar ratio of sorafenib and doxorubicin to ferrous ions is (1:3)-(3:1). Most preferably, the sorafenib and doxorubicin complex with ferrous ions (DOX-Fe) is... 2+ The molar ratio of ) is 1:1.

[0047] Preferably, the molar ratio of doxorubicin to ferrous ions in the doxorubicin-ferrous ion complex is 1:3.

[0048] Preferably, the DOX-Fe 2+ The preparation method is as follows: Dissolve doxorubicin in an organic solvent, add anhydrous FeCl2 to the doxorubicin solution, mix well, and the product is obtained.

[0049] Preferably, the DOX-Fe 2+ The organic solvent used in the preparation method is DMSO.

[0050] Preferably, the DOX-Fe 2+ The concentration of doxorubicin solution in the preparation method is 10 mg / ml.

[0051] Preferably, the DOX-Fe 2+ The mixing time in the preparation method is 30 minutes.

[0052] Due to the specific nature of nucleotide sequences, any nucleic acid sequence containing the indicated SLC7A11-i-1 or SLC7A11-i-2, or a variant thereof, provided that its fragment shares more than 90% homology with the aforementioned nucleic acid sequence and has the same function, falls within the scope of protection of this invention. These polynucleotide variants include substitution variants, deletion variants, and insertion variants. For example, an allelic variant is a substitution of a polynucleotide; it may involve the substitution, deletion, or insertion of multiple nucleotides, but it does not substantially alter the function of the amino acid it encodes.

[0053] In the drug-loaded nanomaterial composite synthesized in this invention, graphyne oxide (GDYO) serves as the carrier, loading SP94-PEG and DOX-Fe. 2+ Sorafenib and siRNA enter hepatocellular carcinoma cells for targeted therapy; SP94-PEG has targeting properties and plays a role in targeting hepatocellular carcinoma in vivo; DOX-Fe 2+ It is a complex of the chemotherapy drug doxorubicin (DOX) and ferrous ions, which is used to carry ferrous ions into the cell, increase the unstable iron pool in tumor cells, and thus induce effective ferroptosis in cancer cells; sorafenib can effectively induce intracellular ferroptosis in cancer cells; siRNA can interfere with the expression of SLC7A11 in cancer cells, block the transport function of SystemXC- receptor, and initiate the ferroptosis process in cells, which can be used for precision treatment of cancer.

[0054] The beneficial effects of this invention are:

[0055] 1. This invention is the first to synthesize SP94-PEG linked and loaded with sorafenib and DOX-Fe. 2+ Graphdiyne oxide ultrathin nanosheets with siRNA can be used for the precision treatment of cancer, especially liver or pancreatic cancer.

[0056] 2. The graphyne oxide used in this invention is a nanosheet-structured carrier with high drug loading capacity and high dispersibility. After being linked with SP94-PEG, it can be used to target cancers such as liver cancer. In addition, the graphyne oxide framework has no obvious toxicity in vitro, exhibiting special biocompatibility and safety.

[0057] 3. The traditional chemotherapy drug doxorubicin binds to ferrous ions, increasing the intracellular pool of unstable iron, while sorafenib can induce ferroptosis. The combined use of these two drugs can enhance cytotoxicity against hepatocellular carcinoma cells and exhibits a synergistic effect.

[0058] 4. siRNA-SLC7A11 can interfere with the expression of SLC7A11 in cancer cells, inhibit the transport function of SystemXC- receptor, and initiate the ferroptosis process in cells. Through this form of programmed cell death, drug resistance in cancer cells can be eliminated and the efficiency of drug treatment can be improved. Attached Figure Description

[0059] Figure 1 This is a transmission electron microscope image of the drug-loaded graphdiyne oxide nanocomposite of this invention;

[0060] Figure 2 This is a fluorescence microscope image of the drug-loaded graphyne oxide nanocomposite of the present invention;

[0061] Figure 3 This invention relates to sorafenib and DOX-Fe 2+ The combined use resulted in a synergistic killing effect on Huh7 cells;

[0062] Figure 4 This is a protein expression diagram of siRNA transfection of Huh7 and SMMC-7721 cells and knockdown of SLC7A11 expression in this invention;

[0063] Figure 5 This invention describes the cytotoxic effect of the drug-loaded graphdiyne oxide nanocomposite on Huh7 cells.

[0064] Figure 6 This invention describes the cytotoxic effect of the drug-loaded graphdiyne oxide nanocomposite on SW1990 cells.

[0065] Figure 7 This is a graph showing the results of the ability of the drug-loaded graphdiyne oxide nanocomposite of the present invention to inhibit the proliferation of Huh7 cells;

[0066] Figure 8 This is a histogram of the plate cloning experiment of the drug-loaded graphynylene oxide nanocomposite of the present invention inhibiting Huh7 cells;

[0067] Figure 9 This is an experimental result showing the ability of the drug-loaded graphdiyne oxide nanocomposite of the present invention to inhibit the invasion of Huh7 cells.

[0068] Figure 10 This is a histogram showing the number of Huh7 cells invaded by the drug-loaded graphdiyne oxide nanocomposite of this invention.

[0069] Figure 11 This is a scratch assay result showing the effect of the drug-loaded graphdiyne oxide nanocomposite of the present invention on inhibiting the migration ability of Huh7 cells.

[0070] Figure 12This is a histogram of scratch damage distances showing the effect of the drug-loaded graphynylene oxide nanocomposite of the present invention on inhibiting the migration ability of Huh7 cells.

[0071] Figure 13 The present invention relates to the cell viability of Huh7 cells treated with drug-loaded graphdiyne oxide nanocomposite with or without ferroptosis inhibitors.

[0072] Figure 14 This involves using Western blotting to detect the expression levels of ferroptosis-related proteins in Huh7 cells under different drug concentrations. Detailed Implementation

[0073] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the drug-loaded nanocomposites in the experimental examples of the present invention all use the drug-loaded graphdiyne oxide nanocomposites prepared in Preparation Example 1.

[0074] Preparation Example 1: Preparation of Drug-Loaded Graphdiyne Oxide Nanocomposite

[0075] Based on the properties of graphynylene oxide, SP94-PEG and DOX-Fe were used. 2+ Sorafenib and siRNA FAM The drug and gene are attached to graphyne oxide, which acts as a carrier to load the aforementioned drugs and genes into cells to exert their effects. The specific preparation method is as follows:

[0076] Step 1: Synthesize graphyne oxide linked to SP94-PEG (GDYO@SP94-PEG)

[0077] 5 mg of GDYO powder was mixed with H2N-PEG-NH2 (15 mg / mL, 1 mL, pH=8.0) and sonicated for 5 minutes. EDC (5 mg / mL, 1 mL) and N-hydroxysuccinimide (NHS, 6 mg / mL, 1 mL, pH=8.0) were added to the mixture, and the mixture was sonicated for 30 minutes. After stirring at 4°C for 12 hours, the resulting dark brown precipitate was collected by centrifugation at 13000 rpm for 15 minutes and redispersed in 500 μL DMSO. 5 mg of EDC and 6 mg of NHS were added to the suspension, and the pH was adjusted to 6.0. After stirring the suspension for another 15 minutes, 1.25 mg of SP94 short peptide powder was added, and stirring was continued at 4°C for 24 hours. Centrifugation yielded SP94-PEG-linked graphyne oxide.

[0078] Step 2: Graphdiyne oxide loaded with drug (GDYO@SP94-PEG / DOX-Fe) 2+ +Sorafini)

[0079] Doxorubicin (DOX) was dissolved in DMSO to prepare a 100 mg / ml solution. 0.2 ml of the doxorubicin solution was then mixed with 14 mg of anhydrous FeCl2 (the molar ratio of FeCl2 to DOX was 3:1) at 37°C in an incubator to prepare DOX-FeCl2. 2+ Solution, for later use.

[0080] Branched PEI (12.5 mg / mL, 1 mL) and EDC (3.75 mg) were added to the GDYO@SP94-PEG suspension redispersed in 500 μL LDMSO obtained in step 1 and stirred for 6 hours. DOX-Fe was then taken... 2+ (100 mg / ml, 200 μL) and sorafenib (100 mg / ml, 200 μL) were added to the above GDYO@SP94-PEG suspension and mixed. The mixture was sonicated at 4 °C for 12 hours and stirred for 24 hours. After centrifugation at 13000 rpm for 15 min, the supernatant and the lower precipitate were collected. The lower precipitate was freeze-dried for 5 hours to obtain the lyophilized precipitate.

[0081] Step 3: Graphdiyne oxide loaded with siRNA (GDYO@SP94-PEG / DOX-Fe) 2+ +Sorafenib+siRNA)

[0082] The precipitate obtained in step 2 was redispersed in DMSO to prepare a drug dispersion of 100 mg / ml. After calculating the required drug concentration, it was mixed with 20 nM siRNA at a volume ratio of 1:1 and allowed to stand for 15 minutes to finally obtain the drug-loaded graphdiyne oxide nanocomposite.

[0083] The ultrathin sheet structure of the drug-loaded graphdiyne oxide nanocomposite prepared in Example 1 was captured by transmission electron microscopy as follows: Figure 1 As shown; fluorescence of the drug and gene loaded in the drug-loaded graphdiyne oxide nanocomposite was captured by fluorescence microscopy (see...). Figure 2 ), including siRNA FAM With green fluorescence ( Figure 2 A), DOX-Fe 2+ With red fluorescence ( Figure 2 B).

[0084] Experimental Example 1: Sorafenib and DOX-Fe 2+ synergistic effect verification of combined use

[0085] 1. Test Methods

[0086] 10,000 Huh7 cells / well were seeded into a 96-well plate, and sorafenib and DOX-Fe prepared in Preparation Example 1 were added. 2+Dissolve in DMSO to prepare 10 mM stock solutions, then dilute with serum-free medium to the experimental concentrations. Sorafenib and DOX-Fe at concentrations of 0, 2.5, 5, 7.5, 10, 12.5, and 15 μM respectively. 2+ 100 μL each, and sorafenib and DOX-Fe at the above concentrations. 2+ A mixture of 50 μL each (e.g., 50 μL of 15 μM sorafenib + 15 μM DOX-Fe) 2+ 50 μL, mixture containing sorafenib and DOX-Fe 2+ The reagents (in a 1:1 molar ratio) were added to serum-free medium and cultured for 24 hours. Finally, 10 μL of CCK-8 reagent was added to each well, and the mixture was incubated for 1.5 hours. The optical density at 450 nm was then measured.

[0087] 2. Test Results

[0088] Table 1 shows the survival rates of Huh7 cells in each experimental group, based on the Huh7 cell survival data. Figure 3 .

[0089] Table 1. Survival rate data of Huh7 cells in different drug test groups

[0090]

[0091] Table 1 and Figure 3 The results showed that, using the CCK-8 assay, the toxicity of the drugs to Huh7 cells was analyzed. The results indicated that the combined use of both drugs in an equimolar ratio inhibited hepatocellular carcinoma cells, and that the combined use of the two drugs exhibited a stronger killing effect on Huh7 cells than the single drug, demonstrating a significant synergistic effect. These experimental results suggest that DOX-Fe... 2+ Sorafenib, as a candidate drug loaded on GDYO nanosheets, can synergistically enhance the therapeutic effect on hepatocellular carcinoma when used in combination.

[0092] Experimental Example 2: Experiment on the interference of siRNA with SLC7A11 expression level in Huh7 and SMMC-7721 cells according to the present invention.

[0093] We constructed siRNAs that specifically recognize SLC7A11, namely SLC7A11-i-1 (sense and antisense strand sequences are SEQ ID NO.1 and SEQ ID NO.2, respectively) and SLC7A11-i-2 (sense and antisense strand sequences are SEQ ID NO.3 and SEQ ID NO.4, respectively). The control siRNA (si-NC) was used as a non-homologous negative control vector. This is a scrambled, non-targeted whole-genome nucleic acid sequence nucleotide sequence. After large-scale data analysis and experimental verification, it does not target any known human, mouse, or rat genes.

[0094] Hepatocellular carcinoma cell lines Huh7 and SMMC-7721 were cultured separately and seeded into 6-well plates (300,000 cells / well). When the cell density reached 50%, SLC7A11-i-1 and SLC7A11-i-2, as well as the control si-NC, were transfected using liposomes. Specifically, 5 μl of 20 nM siRNA plasmid and 5 μl of lipofectamin 3000 (Invitrogen) were mixed and added to the cell culture dish of the 6-well plate. The medium was replaced with fresh culture medium after 12 hours. The interference effect was assessed after 48 hours of transfection.

[0095] Huh7 and SMMC-7721 cells transfected for 48 hours were collected, lysed with RIPA buffer, and protein solutions were extracted. Protein concentration was determined using a BCA kit. Proteins were then separated by 10% SDS-PAGE and blotted onto PVDF membranes. The blotted PVDF membranes were blocked in 5% skim milk, incubated overnight with primary antibody, and then removed for incubation with HRP-labeled secondary antibody. Western blot results showed that, compared to the si-NC control group, interference with SLC7A11-i-1 and SLC7A11-i-2 reduced SLC7A11 protein expression levels in both Huh7 and SMMC-7721 cells. Figure 4 ).

[0096] Experimental Example 3: Inhibitory Effect of Drug-Loaded Graphdiyne Oxide Nanocomposite on Tumor Cell Proliferation and Migration

[0097] The effects of drug-loaded graphdiyne oxide nanocomposite on hepatocellular carcinoma cells (HCC cells 7 and SW1990) were studied using these two cell lines. HCC cells 7 and SW1990 were cultured and seeded into 96-well plates (10,000 cells / well). The drug-loaded graphdiyne oxide nanocomposite was dissolved in DMSO to prepare a 100 mg / ml solution, which was then diluted with serum-free DMEM. The nanocomposite was added at concentrations of 0, 10, 20, 30, 40, and 50 μg / ml to HCC cells and SW1990 cells at a 50% cell density, respectively. After 24 hours of culture, cell viability was assessed using a CCK-8 assay. The results showed that cell viability decreased with increasing drug concentration, with the lowest viability observed at a drug concentration of 50 μg / ml. Figure 5 and Figure 6 ).

[0098] The hepatocellular carcinoma cell line Huh7 was seeded in 6-well plates (500 cells / well) and cultured at 37°C with 5% CO2 until cell adhesion. Then, 5 μg / ml of graphyne oxide, 2 μg / ml, and 5 μg / ml of drug-loaded graphyne oxide nanocomposite were added. After 14 days of treatment, a plate colony assay was performed to observe cell proliferation. This cell proliferation model experiment demonstrated that the drug-loaded graphyne oxide nanocomposite of this invention can effectively inhibit the proliferation of hepatocellular carcinoma cells. Figure 7 and Figure 8 ).

[0099] Invasion experiments were conducted using Huh7 cells. 50,000 cells were mixed with 200 μl of serum-free culture medium and added to the upper chamber of an invasion chamber, while the lower chamber was filled with complete culture medium containing 10% serum. After 6 hours of culture until cell adhesion, drug-loaded graphyne oxide nanocomposite at concentrations of 20 μg / ml, 10 μg / ml, and 20 μg / ml were added, respectively. After 24 hours of drug treatment, cells were fixed and stained, and cell invasion was observed under a microscope. This cell invasion experiment confirmed that the drug-loaded graphyne oxide nanocomposite of this invention can effectively inhibit the invasive ability of hepatocellular carcinoma cells. Figure 9 and 10 ).

[0100] Huh7 cells were seeded at the same density in 6-well plates. After complete cell adhesion, uniform lesions were made using a pipette tip, and drug-loaded graphyne oxide nanocomposites at concentrations of 20 μg / ml, 10 μg / ml, and 20 μg / ml were added, respectively, and cultured under the same conditions. Cell migration was then observed under a microscope at 0 and 48 hours. This cell migration model experiment demonstrates that the drug-loaded graphyne oxide nanocomposites of this invention can effectively inhibit the migration of hepatocellular carcinoma cells. Figure 11 and12 ).

[0101] In the experiment to restore cell viability, 1×10 5 Huh7 cells were seeded at 100 cells / well in 96-well plates and treated with the drug-loaded graphdiyne oxide nanocomposite along with the drug inhibitors ferrostatin-1 (1 μM), DFO (100 μM), and GSH (1 mM) for 24 hours. 10 μL of CCK-8 reagent was added to each well, and the plates were incubated at 37°C for 1.5 hours. Absorbance values ​​were measured using a microplate reader, and the ratio of cell viability in each group to the control group was calculated. This cell viability model experiment demonstrates that the efficacy of the drug-loaded graphdiyne oxide nanocomposite of this invention is inhibited by ferroptosis inhibitors. Figure 13 ).

[0102] Huh7 cells treated with different concentrations of drug-loaded graphdiyne oxide nanocomposite for 24 hours were collected. Protein samples were prepared, and changes in ferroptosis-related genes were verified by Western blotting. Western blotting results showed that after ferroptosis was induced by the drug-loaded graphdiyne oxide nanocomposite, the expression of the target genes SLC7A11 and GPX4 was significantly inhibited. Figure 14 ).

[0103] The above experimental results confirm that, through gene interference, drug synergy, and short peptide targeting, this invention has obtained a highly efficient drug-loaded graphdiyne oxide nanocomposite that induces cancer cells to enter the ferroptosis mechanism. This nanocomposite effectively inhibits the proliferation, migration, and invasion of cancer cells (especially liver cancer cells and pancreatic cancer cells) and exhibits strong cytotoxicity. The experimental results also revealed that the drug-loaded graphdiyne oxide nanocomposite can be affected by ferroptosis reversal agents, thus reducing its cytotoxic effect. Furthermore, the expression levels of ferroptosis target genes SLC7A11 and GPX4 were significantly reduced, indicating that the drug-loaded graphdiyne oxide nanocomposite ultimately leads to ferroptosis.

[0104] The aforementioned research data provides strong evidence that the drug-loaded graphdiyne oxide nanocomposite of this invention can successfully load drugs and genes, act on hepatocellular carcinoma sites, successfully synthesize and induce ferroptosis, and improve the efficiency of targeted antitumor therapy. Therefore, the drug-loaded graphdiyne oxide nanocomposite of this invention has unique advantages and good dispersibility, solving the problems of toxic side effects and drug resistance caused by traditional chemotherapy drugs. It belongs to the category of nanomedicines that preferentially target liver or pancreatic cancer cells through ferroptosis, which can significantly improve the efficiency of in vitro and in vivo antitumor therapy, showing great potential in the field of targeted tumor therapy and potentially becoming a very promising targeted tumor therapy strategy.

[0105] This invention can also be applied to the treatment and drug development of tumors in other systems. Any use of the drug-loaded graphdiyne oxide nanocomposite of this invention for tumor treatment and drug development falls within the scope of protection of this invention.

Claims

1. An antitumor drug-loaded nanomaterial complex, characterized in that, The nanomaterial composite comprises an SP94-PEG modified graphdiyne carrier and an antitumor drug, wherein the antitumor drug is a complex of sorafenib, siRNA and doxorubicin-ferrous ion DOX-Fe 2+ The composition comprises sorafenib and doxorubicin-ferrous ion complex DOX-Fe 2+ The molar ratio of sorafenib and doxorubicin-ferrous ion complex DOX-Fe 2+ The molar ratio of doxorubicin and ferrous ion in the doxorubicin-ferrous ion complex DOX-Fe is 1:3, the SP94-PEG is a multi-arm polyethylene glycol linked to a targeting peptide SP94, the sequence of the targeting peptide SP94 is NH2-CGGSFSIIHTPILPL-COOH, the siRNA is a SLC7A11-i-1 or SLC7A11-i-2 nucleotide sequence, the sense strand nucleotide sequence of SLC7A11-i-1 is shown in SEQ ID NO. 1, the antisense strand nucleotide sequence of SLC7A11-i-1 is shown in SEQ ID NO. 2, the sense strand nucleotide sequence of SLC7A11-i-2 is shown in SEQ ID NO. 3, and the antisense strand nucleotide sequence of SLC7A11-i-2 is shown in SEQ ID NO.

4.

2. The preparation method of the drug-loaded nanomaterial complex of claim 1, comprising the following steps: (1) mixing the graphdiyne oxide powder with H2N-PEG-NH2, adding EDC and N-hydroxysuccinimide in the mixed solution, stirring at low temperature, and collecting the dark brown precipitate by centrifugation; (2) dispersing the dark brown precipitate with a solvent, adding EDC and N-hydroxysuccinimide in the suspension, adding the SP94 short peptide powder after stirring, continuing to stir at low temperature, and obtaining the graphdiyne oxide connected with SP94-PEG after centrifugation; (3) dispersing the SP94-PEG connected graphdiyne obtained in step (2) with a solvent to obtain a suspension, adding branched PEI and EDC to the suspension, stirring, and then adding DOX-Fe 2+ and sorafenib, low-temperature ultrasonic treatment, stirring, collecting the precipitate after centrifugation, and freeze-drying to obtain a freeze-dried precipitate; (4) dispersing the freeze-dried precipitate obtained in step (3) with a solvent, adding siRNA, mixing uniformly, and obtaining the drug-loaded graphdiyne oxide nanocomposite.

3. The use of the drug-loaded nanomaterial complex of claim 1 in the preparation of an antitumor drug.

4. Use according to claim 3, characterized in that, The tumor is liver cancer or pancreatic cancer.

Images