A nano preparation for enhancing the therapeutic effect of ferroptosis and its preparation method and application

Abstract

The present invention discloses a nano - preparation for enhancing the therapeutic effect of ferroptosis, its preparation method and application. The nano - preparation uses a ferroptosis inducer and an antiparasitic drug atovaquone as active ingredients. The ferroptosis inducer can trigger cellular ferroptosis by inhibiting the System Xc ‑ -GSH - GPX4 pathway or the FSP1 - CoQH2 pathway, and atovaquone can enhance the therapeutic effect of ferroptosis by inhibiting DHODH, improving the hypoxic microenvironment, and inhibiting the metabolic repair of target cells. The present invention proposes a novel ferroptosis treatment strategy with dual - drug combination, which can significantly improve the therapeutic effect of ferroptosis and has good practical application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and in particular relates to a nano preparation for enhancing the therapeutic effect of ferroptosis, a preparation method thereof, and an application thereof in tumor treatment. Background Art

[0002] Cancer is a major public health problem worldwide and the second leading cause of death in my country after heart disease, posing a great threat to human life, health and safety. Ferroptosis is a non-apoptotic cell death method proposed in recent years. It was first proposed by Brent R. Stockwell's research group in 2012. It is characterized by an iron-dependent reactive oxygen species (ROS) and lipid peroxidation product accumulation-induced cell death. It is a new type of cell death that is different from apoptosis, cell necrosis, and cell autophagy. Due to "ironophilia" and high H2O2 content in tissues, some highly invasive malignant tumors have been determined to be susceptible to ferroptosis. At the same time, due to the characteristics of the non-apoptotic form, cancer therapy based on ferroptosis is expected to bypass the shortcomings of traditional chemotherapy mediated by apoptotic pathways, and therefore shows great application prospects in the treatment of drug-resistant malignant tumors.

[0003] Ferroptosis depends on iron-mediated oxidative damage. Increased intracellular iron accumulation, free radical production, unsaturated lipid supply and increased lipid peroxides are key to inducing ferroptosis. The core molecular mechanism of ferroptosis is the imbalance between oxidative damage and antioxidant defense. There are multiple antioxidant defense mechanisms in cells to escape ferroptosis and promote tumor development, including System Xc - The GSH-GPX4 pathway, FSP1-CoQH2 axis, DHODH-CoQH2 pathway and GCH1-BH4 pathway, among which GSH, CoQH2 and BH4 can reduce lipid peroxides to non-toxic hydroxyphospholipids, effectively detoxifying ferroptosis. Therefore, by inhibiting the clearance of lipid peroxides by defense mechanisms, tumor cell ferroptosis can be induced.

[0004] As an antiparasitic drug, atovaquone is approved for the treatment of helminths. As an inhibitor of mitochondrial oxidative phosphorylation, it can inhibit dihydrolactate dehydrogenase (DHODH). DHODH is a key defense mechanism for ferroptosis in mitochondria, which can reduce ubiquinone (Coenzyme Q, CoQ) to ubiquinol (CoQH2) for the removal of lipid peroxides. Atovaquone's inhibition of DHODH can increase the therapeutic effect of ferroptosis. Atovaquone can inhibit the synthesis of pyrimidines by inhibiting DHODH, thereby inhibiting the proliferation of tumor cells. In addition, mitochondria, as the main site of cellular energy generation, are organelles for cellular aerobic respiration. Therefore, inhibiting the electron transfer of the mitochondrial respiratory chain can reduce the cell's utilization of oxygen, provide sufficient oxygen for the peroxidation of unsaturated lipids, promote the accumulation of lipid peroxides, and effectively promote the ferroptosis of tumor cells.

[0005] In summary, the present invention designs and develops a nanoformulation that is simultaneously loaded with atovaquone and a ferroptosis inducer, and increases the killing effect on tumor cells by combining the two drugs to enhance the ferroptosis treatment effect. Summary of the invention

[0006] The purpose of the present invention is to provide a nano preparation for enhancing the therapeutic effect of ferroptosis, a preparation method thereof and an application thereof in tumor treatment.

[0007] In order to achieve the above object, the present invention adopts the following scheme:

[0008] A nanoformulation for enhancing the therapeutic effect of ferroptosis, comprising an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is a ferroptosis inducer and atovaquone;

[0009] The ferroptosis inducer is a cystine uptake inhibitor, a GPX4 inhibitor or a FSP1 inhibitor.

[0010] Furthermore, the cystine uptake inhibitor is selected from sorafenib, erastin or sulfasalazine (SAS); the GPX4 inhibitor is selected from RSL3, ML162, ML210, FIN56 or FINO2. The FSP1 inhibitor is iFSP1.

[0011] As a preferred embodiment, sorafenib is used as a ferroptosis inducer.

[0012] Furthermore, the carrier is albumin, hemoglobin or transferrin.

[0013] Furthermore, the albumin is human serum albumin, bovine serum albumin, ovalbumin, mouse serum albumin; preferably bovine serum albumin or human serum albumin.

[0014] Sorafenib targets and inhibits System Xc - The subunit SLC7A11 in ferroptosis induces ferroptosis by reducing the intracellular cystine level, inhibiting the synthesis of GSH, destroying the intracellular redox balance, promoting the excessive accumulation of lipid peroxides.

[0015] Atovaquone inhibits electron transfer in the mitochondrial respiratory chain, reduces the use of oxygen by cells, provides sufficient oxygen for the peroxidation of unsaturated lipids, and promotes the accumulation of lipid peroxides. At the same time, atovaquone inhibits DHODH from reducing CoQ to CoQH2, reducing the clearance of lipid peroxides. At the same time, it downregulates the synthesis of pyrimidines, inhibits the metabolic repair process of tumor cells, and effectively induces ferroptosis of tumor cells.

[0016] The preparation method of the above-mentioned nano preparation comprises the following steps:

[0017] Step 1, preparing an organic solvent solution of atovaquone;

[0018] Step 2, preparing an organic solvent solution of a ferroptosis inducing agent;

[0019] Step 3, preparing a phosphate buffer solution of albumin;

[0020] Step 4, mixing the organic solvent solution of atovaquone with the organic solvent solution of the ferroptosis inducer, and adding the mixed solution dropwise to the phosphate buffer solution of albumin under stirring conditions to obtain a nanoformulation. Further, probe ultrasound can be used to enhance the loading efficiency of serum albumin for the ferroptosis inducer and atovaquone.

[0021] Furthermore, the obtained nanoformulation solution can be transferred to a dialysis bag (14K is selected in the embodiment of the present invention), dialyzed in a phosphate buffer to remove the dimethyl sulfoxide organic solvent, and the dialyzed nanoformulation is centrifuged to remove the unencapsulated hydrophobic drug.

[0022] In one embodiment of the present invention, the concentration of atovaquone in the atovaquone organic solvent solution is controlled to be 1-25 mg / mL, preferably 5 mg / mL.

[0023] In one embodiment of the present invention, the ferroptosis inducing agent is sorafenib, and the concentration of sorafenib in the sorafenib organic solvent solution is controlled to be 1 to 40 mg / mL, preferably 10 mg / mL.

[0024] In one embodiment of the present invention, the organic solvent may be any one or more of dimethyl sulfoxide, N,N-dimethylformamide, chloroform, dichloromethane, methanol, ethanol, and acetonitrile; preferably dimethyl sulfoxide.

[0025] In one embodiment of the present invention, the type of albumin can be selected from human serum albumin, bovine serum albumin, ovalbumin, and mouse serum albumin; preferably, bovine serum albumin or human serum albumin.

[0026] In one embodiment of the present invention, the concentration of bovine serum albumin in the phosphate solution of bovine serum albumin is controlled to be 0.5-30 mg / mL, preferably 3 mg / mL.

[0027] In one embodiment of the present invention, the molar ratio of atovaquone to sorafenib is 0.25:1 to 4:1, preferably 1:1.

[0028] In one embodiment of the present invention, the volume ratio of the mixed solution of atovaquone and sorafenib to the phosphate buffer solution of bovine serum albumin is 1:5 to 1:30, preferably 1:15.

[0029] Furthermore, the nanoformulation may be subjected to step 5, wherein the atovaquone and sorafenib self-assembled albumin nanoformulation is freeze-dried.

[0030] As a preferred embodiment, the freeze-drying step in the preparation method of the atovaquone and sorafenib self-assembled albumin nanoformulation is as follows: the atovaquone and sorafenib self-assembled albumin nanoformulation obtained according to the preferred embodiment is mixed with a lyoprotectant in a certain proportion, pre-frozen, and fully freeze-dried to obtain a freeze-dried drug of the atovaquone and sorafenib self-assembled albumin nanoformulation.

[0031] In step 5, the lyoprotectant includes excipients (sucrose, trehalose, mannitol, lactose, glucose, maltose), cryoprotectants (glycerol, DMSO, PVP), antioxidants (vitamin D, sodium thiosulfate), and pH regulators.

[0032] In step 5, the ratio of the lyophilization protectant to the nanoparticle preparation is in the range of 10 to 100 g / L;

[0033] In step 5, the pre-freezing temperature ranges from -20 to -80°C, preferably -80°C;

[0034] In step 5, the pre-freezing time ranges from 12 to 48 hours, preferably 48 hours;

[0035] In step 5, the pre-freezing speed includes low-temperature quick freezing and low-temperature slow freezing, and low-temperature quick freezing is preferred.

[0036] Application of the above-mentioned nanoformulations in tumor treatment.

[0037] Furthermore, the tumor is breast cancer, liver cancer, cervical cancer or colon cancer.

[0038] The present invention provides a method for preparing a nano-preparation for enhancing the therapeutic effect of ferroptosis and its application in tumor treatment. The nano-preparation uses serum albumin with good biocompatibility and strong tumor targeting to load atovaquone and ferroptosis inducer. The ferroptosis inducer inhibits System Xc - -GSH-GPX4 pathway or FSP1-CoQH2 pathway downregulates intracellular ferroptosis resistance and induces cell ferroptosis by increasing the accumulation of intracellular lipid peroxides. As an auxiliary drug, atovaquone inhibits the mitochondrial respiratory chain, can improve the hypoxic microenvironment of tumor tissues, provide sufficient oxygen for the generation of lipid peroxides, promote ROS production, improve the efficiency of lipid peroxide generation, and efficiently induce tumor cell ferroptosis. At the same time, atovaquone inhibits the DHODH-CoQH2 pathway's ability to reduce lipid peroxides. In addition, atovaquone can inhibit the synthesis of pyrimidines by inhibiting DHODH, thereby inhibiting the repair process of tumor cell metabolism. This ferroptosis-inducing nanoformulation has excellent application prospects in tumor treatment.

[0039] Compared with the prior art, the atovaquone and sorafenib self-assembled albumin nanoformulation provided by the present invention has the following advantages:

[0040] 1. The present invention prepares for the first time the atovaquone and sorafenib self-assembled albumin nanoformulation (ATO / SRF@BSA), which is a nanoformulation formed by self-assembly of atovaquone (ATO) and sorafenib (SRF) in an albumin solution. The preparation method is simple, the operation is convenient, and it has the potential for clinical transformation.

[0041] 2. The atovaquone and sorafenib self-assembled albumin nanoformulation prepared by the present invention has the advantages of high drug loading, uniform particle size, good stability, good biocompatibility, etc.

[0042] 3. In cell studies and animal levels, compared with single-drug nanoformulations loaded with only atovaquone or sorafenib, albumin nanoformulations loaded with both atovaquone and sorafenib showed more potent induction of tumor cell death and enhanced ferroptosis therapeutic effects; this nanoformulation provides more possibilities for the ferroptosis treatment of breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The particle size, potential, stability and transmission electron microscope images of the albumin nanoformulations loaded with different drugs in the embodiments of the present invention are shown.

[0044] Figure 2 This is the detection of cell survival rate after different groups of different cell lines were treated under hypoxia and normoxia conditions according to the embodiments of the present invention.

[0045] Figure 3 These are the results of measuring the levels of intracellular ROS, GSH, MDA and lipid peroxides in 4T1 cells after administration of atovaquone and sorafenib self-assembled albumin nanoformulations according to the examples of the present invention.

[0046] Figure 4 These are the results of the oxygen content in the culture medium, cellular oxygen consumption rate (OCR), mitochondrial membrane potential, the ratio of CoQH2 to CoQ, and DNA replication ability of 4T1 cells after intervention with different nanoformulations in the embodiments of the present invention.

[0047] Figure 5 This is a diagram showing the anti-tumor results of an embodiment of the present invention in mice.

[0048] Figure 6 The following are the results of acute toxicity experiments of the embodiments of the present invention in mice. DETAILED DESCRIPTION

[0049] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly and comprehensively understood. The present invention will be further described below in conjunction with the drawings and specific embodiments.

[0050] The present invention is achieved through the following technical solution, and the specific steps are as follows:

[0051] A 1-25 mg / mL atovaquone dimethyl sulfoxide solution and a 1-25 mg / mL sorafenib dimethyl sulfoxide solution were prepared; a 3-20 mg / mL bovine serum albumin phosphate buffer solution was prepared; the atovaquone and sorafenib dimethyl sulfoxide solutions were mixed at a molar ratio of 1:1 to form a mixed solution, and the mixed solution was added dropwise to the bovine serum albumin phosphate buffer solution at a volume ratio of 1:5-1:30 under stirring conditions to obtain atovaquone and sorafenib albumin nanoparticles by self-assembly.

[0052] The atovaquone and sorafenib albumin nanoparticle preparation can be freeze-dried to obtain atovaquone and sorafenib self-assembled albumin nanoparticle medicine.

[0053] The above-mentioned atovaquone and sorafenib albumin nanoformulation is used for the treatment of triple-negative breast cancer, and the administration method is intravenous injection.

[0054] The above-mentioned preparations can be used for monotherapy or combination therapy of malignant tumors and drug-resistant tumor types, including breast cancer, liver cancer, cervical cancer and colon cancer; they can be combined with various treatment strategies such as photodynamic therapy, photothermal therapy, immunotherapy, radiotherapy and chemotherapy.

[0055] Example 1

[0056] The specific preparation scheme of atovaquone and sorafenib albumin nanoformulation is as follows:

[0057] A 5 mg / mL atovaquone dimethyl sulfoxide solution, a 10 mg / mL sorafenib dimethyl sulfoxide solution, and a 3 mg / mL bovine serum albumin phosphate buffer solution were prepared; the atovaquone and sorafenib dimethyl sulfoxide solutions were fully mixed at a molar ratio of 1:1 to form a mixed solution, and under stirring, the mixed solution was dropwise added into the bovine serum albumin phosphate buffer solution at a volume ratio of 1:15 of the drug-containing organic solvent to the albumin solution to obtain sorafenib and atovaquone albumin nanoformulation 1 (ATO / SRF@BSA) by self-assembly.

[0058] The albumin was bovine serum albumin, and atovaquone was used as a drug component. With reference to the above preparation method, an albumin nanoformulation 2 (ATO@BSA) was obtained.

[0059] The albumin was bovine serum albumin, and sorafenib was used as a drug component. With reference to the above preparation method, the albumin nanoformulation 3 (SRF@BSA) was obtained.

[0060] The albumin used was bovine serum albumin, and atovaquone and sorafenib were used as drug ingredients. Referring to the above-mentioned preparation method, after obtaining the albumin nanoparticle preparation, it was placed in a -80°C refrigerator for pre-freezing for 24 hours, and then freeze-dried in a -40°C environment in the dark, and the albumin nanoparticle preparation 4 was obtained after 48 hours.

[0061] Figure 1 The characterization results of the albumin nanoformulations 1-3 prepared in Example 1 are shown in FIG. Figure 1 As shown in Figure A, the dynamic light scattering particle size of ATO / SRF@BSA is 106.3±1.60nm, and the transmission electron microscopy results show that ATO / SRF@BSA is a regular sphere. Figure 1 As shown in Figure B, the dynamic light scattering particle sizes of ATO@BSA and SRF@BSA are 266.79±5.80nm and 122.03±2.22nm, respectively. Figure 1 C is the potential results of the three nanoformulations. The potentials of the three nanoformulations are all around -10 mV. Figure 1D is the 7-day stability of ATO / SRF@BSA in different media. The results show that it can be stably present in DMEM, glucose solution, saline and PBS for 7 days, indicating that ATO / SRF@BSA still has good stability under physiological conditions.

[0062] Example 2

[0063] The cell survival rate experiment after atovaquone and sorafenib albumin nanoformulations were co-incubated with different cell lines under normoxic conditions and hypoxic conditions. The mouse breast cancer cell line 4T1 was selected as the research object. The cells were cultured in DMEM culture medium containing 1% penicillin-streptomycin solution and 10% fetal bovine serum at 37°C and 5% CO2. The cells were evenly spread in a 96-well plate. After the nanoformulation solution was diluted to different concentrations using serum-free DMEM culture medium, 100 μL of the drug solution was added to each well, and the culture was continued for 24 hours under normoxic conditions or hypoxic conditions, and the final cell survival rate was detected. This example uses cell thiazole blue (MTT) staining, determines the absorbance at 490nm and calculates the cell survival rate. In addition, human cervical cancer cell line HeLa and human liver cancer cell line HepG2 were used as research objects, and the survival rate was determined after administration according to the above operation under normoxic conditions.

[0064] Figure 2 The results are the survival rates of each cell line after drug administration. Figure 2 A and 2B are the cell survival rates of 4T1 cells after treatment with each nanoformulation under normoxic conditions and hypoxic conditions, respectively. The results showed that each nanoformulation exhibited concentration-dependent cytotoxicity, among which the cytotoxicity of ATO / SRF@BSA was significantly higher than that of the other two single-drug preparations. Figure 2 C and 2D are the cytotoxicity of each nanoformulation on HeLa cells and HepG2 cells under normoxic conditions, respectively. The results show that ATO / SRF@BSA still exhibits excellent cell killing effects in cervical cancer cell lines and liver cancer cell lines.

[0065] Example 3

[0066] Detection of the effect of atovaquone and sorafenib albumin nanoformulations in inducing ferroptosis in 4T1 cells. During ferroptosis, the level of reactive oxygen species is positively correlated with the level of lipid peroxides and the degree of ferroptosis, but negatively correlated with the level of intracellular reducing substance GSH. As an oxidation-sensitive fluorescent probe, DCFH-DA itself has no fluorescence, can freely pass through the cell membrane, and is hydrolyzed by intracellular esterases into DCFH and stored in the cell. It can be oxidized by intracellular reactive oxygen species into a strong green fluorescent substance DCF. Therefore, the level of intracellular ROS can be quantified according to its fluorescence intensity. Liperfluo is a lipid peroxidation probe that will be specifically oxidized by lipid peroxides to emit green fluorescence and is often used as a specific probe for detecting intracellular lipid peroxidation levels. Malondialdehyde (MDA) is a degradation product of lipid peroxides. By detecting the level of MDA, the level of intracellular lipid peroxidation can effectively reflect the level of intracellular lipid peroxidation. Detecting intracellular ROS, GSH and other indicators can effectively indicate the effect of nanoformulations in inducing cell ferroptosis. The mouse breast cancer cell line 4T1 was selected as the research object. The cells were evenly plated in a 6-well plate, and 1 mL of drug solution was administered to each well. After 6 hours of drug administration, DCFH-DA and Liperfluo staining were performed, and the fluorescence intensity of each group of cells after staining was observed by fluorescence microscopy or laser confocal microscopy, and photographed and recorded. The above drug administration operation was repeated, the cells were collected and fully lysed, and the MDA and GSH contents in the cells were determined.

[0067] Figure 3 The results of the detection of intracellular reactive oxygen species, lipid peroxides, MDA and GSH levels in 4T1 cells 6 hours after drug administration. Figure 3 As shown in A, after ATO / SRF@BSA intervened in 4T1 cells, the fluorescence intensity of intracellular DCF was significantly enhanced, and a large amount of ROS was generated. Figure 3 B is the result of intracellular Liperfluo staining, which shows that SRF can induce the production of intracellular LPO; after co-delivery of ATO, a large amount of intracellular lipid peroxides accumulated in the ATO / SRF@BSA group, and the fluorescence of Liperfluo was significantly enhanced. Figure 3 C is the quantitative result of intracellular MDA level. Compared with the single-drug nanoformulation group, the MDA level in the ATO / SRF@BSA group was significantly increased, which corresponds to the Liperfluo result. Figure 3 D is the quantitative result of intracellular GSH level. SRF can significantly downregulate GSH level. ATO / SRF@BSA administration will further reduce intracellular GSH level, destroy redox balance, accumulate lipid peroxides, and effectively induce ferroptosis.

[0068] Example 4

[0069] Detection of the effect of atovaquone on enhancing ferroptosis. Atovaquone inhibits DHODH from reducing CoQ to CoQH2, reducing the clearance of lipid peroxides. At the same time, by inhibiting DHODH, the synthesis of pyrimidines is downregulated, inhibiting the metabolic repair process of tumor cells. In addition, atovaquone inhibits the electron transfer of the mitochondrial respiratory chain, reduces the utilization of oxygen by cells, provides sufficient oxygen for the peroxidation of unsaturated lipids, promotes the accumulation of lipid peroxides, and effectively induces ferroptosis of tumor cells. Therefore, after resuspending the cells in culture medium containing different nanoformulations, the oxygen content in the culture medium was measured by a dissolved oxygen meter; the oxygen consumption rate (OCR) of the cells was measured by SeahorseXF96. The decrease in mitochondrial membrane potential was detected by the transition of the JC-1 probe from red fluorescence to green fluorescence to evaluate mitochondrial activity. As a substrate of DHODH, ubiquinone can be effectively reduced to ubiquinol, detoxify lipid peroxides, and inhibit ferroptosis. The contents of CoQ and CoQH2 were detected by ELISA kits, and the inhibitory effect of atovaquone on DHODH was evaluated by calculating the ratio of the two. EdU (5-ethynyl-2'-deoxyuridine) is a new thymidine analog that can replace thymine and be incorporated into newly synthesized DNA during DNA replication. It is used to detect cell proliferation ability.

[0070] Figure 4 These are the results of oxygen content in the culture medium, cellular oxygen consumption rate (OCR), mitochondrial membrane potential, the ratio of CoQH2 to CoQ, and DNA replication ability of 4T1 cells after intervention with different nanoformulations. Figure 4 A is the oxygen content in the culture medium of 4T1 cells after intervention with the nanoformulation. The results show that after the cells were treated with ATO@BSA and ATO / SRF@BSA, the oxygen consumption rate in the culture medium was significantly slowed down, indicating that cell respiration and oxygen consumption were inhibited. Figure 4 The detection results of intracellular OCR in B showed that ATO / SRF@BSA significantly inhibited the OCR of basal respiration and ATP-related respiration. Figure 4 The results of mitochondrial JC-1 staining in C showed that ATO / SRF@BSA induced a decrease in the red fluorescence intensity of mitochondria and an increase in the green fluorescence intensity, indicating that ATO / SRF@BSA significantly induced the depolarization of mitochondrial membrane potential and damaged mitochondria. Figure 4 D is the ratio of CoQH2 to CoQ. The results showed that after administration of ATO / SRF@BSA, intracellular CoQH2 / CoQ was downregulated and the content of the reducing substance CoQH2 in mitochondria was reduced. Figure 4The results of the intracellular DNA replication ability test in E show that the fluorescence density and fluorescence ratio of EdU staining in cells are significantly reduced after ATO / SRF@BSA administration, indicating that DNA replication is inhibited. The results in this figure show that ATO / SRF@BSA damages mitochondria, inhibits cell respiration, and reduces the proportion of intracellular CoQH2, breaking the intracellular oxidation balance and promoting ferroptosis. At the same time, ATO / SRF@BSA reduces pyrimidine synthesis by inhibiting DHODH, inhibits DNA replication, blocks cell repair, and further aggravates ferroptosis.

[0071] Example 5

[0072] Anti-tumor therapy was studied using atovaquone and sorafenib albumin nanoformulations. One million 4T1 cell suspensions were inoculated into the lower back of Balb / c mice and the tumor volume grew to 100 mm. 3 The mice were divided into groups for drug administration, with 5 mice in each group, including the untreated group (Untreated), ATO@BSA, SRF@BSA, and ATO / SRF@BSA. Atovaquone and sorafenib albumin nanoformulation solution were injected through the tail vein every two days for a total of 5 times. During the experiment, the size of the mouse tumor was measured with a vernier caliper every other day and the mice were weighed, recorded and counted. After the experiment, the mouse tumor tissue was removed and photographed. Figure 5 The figure shows the weight curve of mice, tumor growth curve, tumor tissue anatomy and tumor tissue weight during the period of anti-tumor treatment. Figure 5 As shown in A, during the drug administration period, the body weight of tumor-bearing mice did not fluctuate significantly, indicating that the animals were in good survival condition. Figure 5 B is the tumor growth curve of mice during the administration period, and the results showed that ATO / SRF@BSA administration significantly inhibited tumor growth. Figure 5 C and 5D are the tumor tissue anatomical diagram and tumor tissue weight results, respectively. The results showed that the tumor volume and weight of mice in the SRF@BSA group were significantly reduced, and the tumor volume and weight of the ATO / SRF@BSA group were further reduced, indicating that ATO / SRF@BSA treatment can effectively inhibit the growth of tumor tissue.

[0073] Example 6

[0074] Acute toxicity experiments were conducted in mice using atovaquone and sorafenib albumin nanoformulations. Atovaquone and sorafenib albumin nanoformulations were injected through the tail vein. In the acute toxicity experiment, the nanoformulations were administered every other day for a total of 5 times. During the experiment, the mice were weighed, recorded, and counted every other day. After the experiment, the main organ tissues of the mice were removed, fixed, paraffin-embedded, sectioned, and H&E stained. The results of the acute toxicity experiment are shown in the figure. Figure 6 shown. Figure 6A is the survival curve of mice in the acute toxicity experiment. The results show that all the mice in the SRF@BSA group died on the 9th day, and only 20% of the mice in the ATO / SRF@BSA group survived on the 9th day, indicating that SRF itself is highly toxic, but ATO / SRF@BSA can relatively alleviate the toxicity problem caused by SRF. Figure 6 B is the weight curve of mice in the acute toxicity experiment. There was no significant change in the weight of mice in different groups. Figure 6 C is the result of H&E staining of multiple organs in mice, which shows that SRF@BSA has multi-organ toxicity, while the same dose of ATO / SRF@BSA can alleviate the hepatocyte swelling and alveolar wall thickening caused by sorafenib.

[0075] In this embodiment, atovaquone and sorafenib are used as drug ingredients to self-assemble to form an albumin nanoformulation with good biocompatibility. After administration by tail vein injection, since the particle size of the nanoformulation is about 100nm, through the EPR effect, the nanoformulation passively targets and accumulates in the tumor site, and at the same time, with the help of the high expression of cysteine-rich acidic secretory protein (Secreted Protein Acidic and Rich in Cysteine, SPARC) on the surface of the tumor cell membrane, the specific binding with albumin increases the internalization of tumor cells to albumin nanoformulations. Sorafenib induces ferroptosis by inhibiting the uptake of cystine by tumor cells, reducing the synthesis of the reducing substance GSH in tumor tissues; at the same time, atovaquone acts on the mitochondrial respiratory chain, inhibits the reduction of lipid peroxides by the DHODH pathway, and improves the hypoxic microenvironment of tumor tissues, providing sufficient oxygen for the generation of lipid peroxides, and improving the generation efficiency of lipid peroxides. In addition, atovaquone inhibits the synthesis of pyrimidines by inhibiting DHODH, inhibits the metabolic repair process of tumor cells, and significantly increases the ferroptosis treatment effect of malignant tumors and drug-resistant tumor tissues.

[0076] The above specific examples are used to illustrate the present invention, which is only used to help understand the present invention and is not intended to limit the present invention. For those skilled in the art, according to the concept of the present invention, some simple deductions, modifications or substitutions can be made.

Claims

1. A nanoformulation for enhancing the therapeutic effect of ferroptosis, characterized in that: The invention comprises an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is a ferroptosis inducer and atovaquone; The ferroptosis inducing agent is sorafenib; The carrier is albumin, and the albumin is selected from human serum albumin, bovine serum albumin, ovalbumin or mouse serum albumin.

2. The method for preparing the nanoformulation according to claim 1, characterized in that: The following steps are involved: Step 1, preparing an organic solvent solution of atovaquone; Step 2, preparing an organic solvent solution of a ferroptosis inducing agent; Step 3, preparing a phosphate buffer solution of the carrier; Step 4, mixing the organic solvent solution of atovaquone with the organic solvent solution of the ferroptosis inducer, and adding the mixed solution dropwise into the phosphate buffer solution of the carrier under stirring conditions to obtain a nanoformulation.

3. The preparation method according to claim 2, characterized in that: The concentration of atovaquone in step 1 is 1 to 25 mg / mL.

4. The preparation method according to claim 2, characterized in that: In step 2, the ferroptosis inducing agent is sorafenib, and the concentration of sorafenib is 1 to 40 mg / mL.

5. The preparation method according to claim 2, characterized in that: In step 3, the carrier is bovine serum albumin, and the concentration of bovine serum albumin is 0.5-30 mg / mL.

6. The preparation method according to claim 2, characterized in that: In step 4, the molar ratio of atovaquone to the ferroptosis inducer is 0.25:1 to 4:1, and the volume ratio of the mixed solution to the phosphate buffer solution of the carrier is 1:5 to 1:

30.

7. Use of the nanoformulation according to claim 1 in the preparation of tumor therapeutic drugs.

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