Use of pfoi in preparation of a drug for inducing ferroptosis of tumor cells

Abstract

The application relates to application of PFOI in preparation of a tumor cell ferroptosis-inducing drug and belongs to the technical field of medicines. The application provides application of PFOI in preparation of a tumor cell ferroptosis-inducing drug. The application verifies the correlation between PFOI and tumor cell ferroptosis through experiments, proves that PFOI can cause mitochondrial atrophy, membrane density increase and mitochondrial cristae reduction of cancer cells, cause ROS increase of the cancer cells, regulate GSH metabolism and fatty acid biosynthesis of the cancer cells, cause GSH exhaustion and lipid deposition, and promote the cancer cells to occur ferroptosis by inhibiting the expression of SLC7A11. Based on this, the application provides a new use of PFOI for inducing tumor cell ferroptosis, provides experimental basis and a new treatment strategy for clinical treatment of cancer, and provides new technical support for targeted treatment of cancer.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of PFOI in preparation of a drug for inducing tumor cell ferroptosis. BACKGROUND

[0002] Ferroptosis is an iron-dependent, non-apoptotic form of cell death, which is characterized by GSH depletion, GPX4 inactivation and accumulation of cytotoxic lipid reactive oxygen species (ROS), ultimately leading to fatal plasma membrane damage and membrane perforation. Morphologically, ferroptosis is characterized by mitochondrial atrophy, increased membrane density and reduced mitochondrial cristae, without showing typical morphological features of traditional apoptosis. Ferroptosis is regulated by multiple pathways, including System Xc-, the mevalonate pathway and the mitochondrial DHODH pathway. System Xc- is a dimer embedded in the cell membrane, composed of SLC3A2 and SLC7A11, of which SLC7A11 is the main functional subunit and is highly expressed in various human cancers. Recent studies have shown that GSH biosynthesis induced by SLC7A11 overexpression can inhibit lipid peroxidation and ferroptosis, thereby promoting tumor growth.

[0003] As a unique cell death mechanism, ferroptosis has attracted great interest in the cancer research community, as targeting ferroptosis may provide new treatment opportunities for cancers that are difficult to cure by traditional therapy. Ferroptosis agents are compounds that induce cell death through iron-dependent substrates. The main types currently include iron chelators, ferroptosis inducers, iron overload agents, and mitochondrial-targeted ferroptosis agents. Ferroptosis agents can also be used in combination with other compounds to enhance their cytotoxic effects.

[0004] Perfluoroctyl iodide (PFOI) is an organic synthesis intermediate and a pharmaceutical intermediate, which plays an important role in the fields of organic synthesis and medicine, and can be used as a key intermediate to participate in various chemical reactions and drug preparation processes. Studies have shown that PFOI has an impact on cell migration ability and may be involved in regulating the expression of long non-coding RNA MALAT-1. However, the existing technology has not reported the efficacy of PFOI in inducing tumor cell ferroptosis. SUMMARY

[0005] The application provides a new use of PFOI for inducing tumor cell ferroptosis, specifically the application of PFOI in preparation of a drug for inducing tumor cell ferroptosis.

[0006] Technical solutions of the application:

[0007] Application of PFOI in preparation of a drug for inducing tumor cell ferroptosis.

[0008] Further, the tumor cell iron death-inducing drug has PFOI as the only active ingredient or one of the active ingredients.

[0009] Further, the tumor cell iron death-inducing drug has PFOI in an amount of 0.1wt%-99wt%.

[0010] Further, the tumor cell iron death-inducing drug further comprises a pharmaceutically acceptable excipient and / or carrier.

[0011] Further, the tumor cell iron death-inducing drug contains PFOI liposomes.

[0012] Further, the tumor cell is a lung cancer cell.

[0013] Further, the tumor cell iron death-inducing drug can cause mitochondrial atrophy, increased membrane density, and reduced mitochondrial cristae in cancer cells.

[0014] Further, the tumor cell iron death-inducing drug can cause increased ROS in cancer cells.

[0015] Further, the tumor cell iron death-inducing drug can regulate GSH metabolism and fatty acid biosynthesis in cancer cells, leading to GSH depletion and lipid deposition.

[0016] Further, the tumor cell iron death-inducing drug promotes iron death in cancer cells by inhibiting the expression of SLC7A11.

[0017] Advantages of the present application:

[0018] The present application has been tested and verified that the PFOI liposome nanoparticles prepared in Example 1 are related to tumor cell iron death, and it is proved that the PFOI liposome nanoparticles prepared in Example 1 can cause mitochondrial atrophy, increased membrane density, and reduced mitochondrial cristae in cancer cells; cause increased ROS in cancer cells; regulate GSH metabolism and fatty acid biosynthesis in cancer cells, leading to GSH depletion and lipid deposition; promote iron death in cancer cells by inhibiting the expression of SLC7A11. Based on this, the present application provides a new use of PFOI for inducing tumor cell iron death, specifically the application of PFOI in the preparation of a drug for inducing tumor cell iron death, which provides experimental basis and new treatment strategy for the clinical treatment of lung cancer, and provides new technical support for cancer targeted therapy. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Transmission electron microscopy of the PFOI liposome nanoparticles prepared in Example 1;

[0020] Figure 2The transmission electron microscope image of H460 cells after PFOI liposome nanoparticle treatment in Example 2;

[0021] Figure 3 The ROS fluorescence intensity comparison chart in A549 cells after PFOI liposome nanoparticle treatment in Example 3;

[0022] Figure 4 The reverse verification chart of PFOI liposome nanoparticle activating ferroptosis of lung cancer cells in Example 4;

[0023] Figure 5 The non-targeted metabolomics result analysis chart of LLC cells treated with PFOI liposome nanoparticles in Example 5-OPLS-DA score chart of PFOI group and blank control group;

[0024] Figure 6 The non-targeted metabolomics result analysis chart of LLC cells treated with PFOI liposome nanoparticles in Example 5-differential metabolite volcano chart of PFOI group and blank control group;

[0025] Figure 7 The non-targeted metabolomics result analysis chart of LLC cells treated with PFOI liposome nanoparticles in Example 5-differential metabolite heat map of PFOI group and blank control group;

[0026] Figure 8 The non-targeted metabolomics result analysis chart of LLC cells treated with PFOI liposome nanoparticles in Example 5-KEGG-based metabolic pathway chart of PFOI group and blank control group;

[0027] Figure 9 The result chart of PFOI liposome nanoparticle promoting ferroptosis-related protein expression in Example 6. DETAILED DESCRIPTION

[0028] The technical solutions of the present application are further described below in conjunction with the examples, but are not limited thereto. Any modification or equivalent replacement to the technical solutions of the present application without departing from the spirit and scope of the present application shall be covered in the protection scope of the present application. The process equipment or device in the following examples is not specifically noted, and the raw materials used in the examples of the present application are commercially available. If not specifically indicated, the technical means used in the examples of the present application are conventional means known to those skilled in the art.

[0029] Example 1

[0030] The present embodiment provides a PFOI liposome nanoparticle and a preparation method.

[0031] The preparation method of PFOI liposome nanoparticles in the embodiment is as follows:

[0032] Step one, according to the molar ratio of dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylglycerol (DPPG) and cholesterol = 83:2:15, each substance was weighed and uniformly mixed, dissolved with chloroform, the dissolved surfactant was evaporated by a rotary evaporator, then dried in a vacuum oven at 40℃ overnight, and finally dispersed in a certain amount of water by ultrasonic oscillation to obtain a mixture of surfactants for standby;

[0033] Step two, perfluorooctyl iodide PFOI, the mixture of surfactants obtained in step one, glycerol and water were mixed in a mass ratio of 20:2:2:76, and then treated with an ultrasonic water bath probe for 4 min, mixed uniformly, and extruded by an extruder (Avanti mini extruder) to prepare PFOI nanoemulsion;

[0034] Step three, the PFOI nanoemulsion obtained in step two was removed by dialysis to remove the components not effectively coated to obtain PFOI liposome nanoparticles with an average particle size of 120±10 nm.

[0035] Figure 1 The transmission electron microscope image of the PFOI liposome nanoparticles prepared in Example 1; it can be seen that the PFOI liposome nanoparticles have good dispersibility in water, are spherical in shape, and have a diameter of about 50 nm.

[0036] Example 2

[0037] In this embodiment, the effect of the PFOI liposome nanoparticles prepared in Example 1 on the ultrastructure of lung cancer cells was observed by transmission electron microscopy.

[0038] The PFOI liposome nanoparticles prepared in Example 1 were co-incubated with lung cancer cells, and the steps were as follows:

[0039] (1) Take the LLC cells in the logarithmic growth phase to prepare a cell suspension, count, and then plate 5×10 5 / well in a 6-well plate, and add 2 ml of medium to each well, and then incubate in a cell culture incubator. Add the PFOI liposome nanoparticle and high-glucose medium mixture (the concentration of PFOI liposome nanoparticles is 10 μl / ml) to the well plate, and then continue to incubate in the cell culture incubator for 24 hours;

[0040] (2) Discard the supernatant, add 2 ml of PBS solution to each well, and wash twice; collect the cells in a tube, centrifuge at 1000 rpm for 5 minutes, and carefully discard the liquid;

[0041] (3) Add 1 ml of 2.5% glutaraldehyde in 0.1M PBS (PH 7.4) to fix the sample at room temperature for 2 hours, rinse with 0.1M PBS (PH 7.4) for 15 minutes, and rinse a total of 3 times;

[0042] (4) Prepare a mixture of acetone and embedding agent at a ratio of 1:1, and immerse the sample in the mixture. The next day, immerse the sample in pure embedding agent for 8-10 hours. Adjust the temperature of the incubator to 60°C, and after the temperature reaches the set value, place the sample in the incubator and react for 2 days. Then, place the sample in the embedding agent, and finally cut thin sections. Place the sections in a saturated aqueous solution of 2% uranyl acetate for 16 minutes. Then, place the sections in lead citrate for the same amount of time, and place the sections in the incubator for 8-10 hours. Observe the internal structure of the treated cells under an electron microscope, as shown in FIG. 2. Figure 2

[0043] Compared with the control group, the mitochondria of the PFOI liposome nanoparticle treatment group prepared in Example 1 were reduced in volume and increased in membrane density, indicating that the PFOI liposome nanoparticles prepared in Example 1 can change the morphology of cell mitochondria, which is a characteristic feature of ferroptosis that is different from other cell apoptosis methods.

[0044] Example 3

[0045] In this example, the effect of the PFOI liposome nanoparticles prepared in Example 1 on reactive oxygen species (ROS) in lung cancer cells was detected by a DCFH-DA fluorescent probe.

[0046] In this example, the method for detecting reactive oxygen species by PFOI liposome nanoparticles is as follows:

[0047] (1) After the lung cancer LLC cells were plated, the experiment was divided into four groups: PFOI liposome nanoparticle + radiotherapy group (PFOI + RT group), PFOI liposome nanoparticle group (PFOI group), radiotherapy group (RT group), and control group (Control group). The PFOI + RT group: After the PFOI liposome nanoparticles were oxygenated for 30 minutes, the PFOI liposome nanoparticles were mixed in the culture medium at a concentration of 10 μl / ml and added to a confocal dish, which was then incubated in a 37°C incubator for 2 hours. After washing twice with PBS, the sample was irradiated with a handheld radiotherapy device at a frequency of 50 kV and a current of 75 μA at a distance of 3 cm for 10 minutes. The PFOI group: The PFOI liposome nanoparticles were mixed in the culture medium at a concentration of 10 μl / ml and added to a confocal dish, which was then incubated in a 37°C incubator for 2 hours. After washing twice with PBS, the sample was irradiated with a handheld radiotherapy device at a frequency of 50 kV and a current of 75 μA at a distance of 3 cm for 10 minutes. The RT group: The sample was irradiated with a handheld radiotherapy device at a frequency of 50 kV and a current of 75 μA at a distance of 3 cm for 10 minutes. The Control group: The Control group was not treated, and was washed twice with PBS and replaced with fresh culture medium.

[0048] ​(2) Dilute the lipid peroxidation reagent to 1 μl / ml and DCFH-DA to 20 μmol / L with serum-free medium. Discard the culture medium, add the diluted lipid peroxidation reagent and DCFH-DA, and place in the incubator for 30 minutes. Wash 3 times.

[0049] (3) Add 4', 6-diamidino-2-phenylindole (DAPI) dropwise.

[0050] (4) Wash 3 times and mount the sample.

[0051] (5) Observe and photograph under a laser confocal microscope. The results are shown in FIG. 1. Figure 3

[0052] Compared with the control group, the fluorescence intensity of the PFOI liposome nanoparticle treatment group prepared in Example 1 was significantly enhanced, indicating that the PFOI liposome nanoparticle prepared in Example 1 can cause accumulation of reactive oxygen species in cells.

[0053] Example 4

[0054] This example detects whether Ferrostatin-1 can rescue the ferroptosis induced by the PFOI liposome nanoparticle prepared in Example 1.

[0055] In this example, the steps of the PFOI liposome nanoparticle detection ferroptosis method are as follows:

[0056] 1. LLC lung cancer cells were inoculated in culture bottles and divided into three groups: a control group (untreated), a PFOI liposome nanoparticle treatment group, and a Ferrostatin-1 pretreatment + PFOI liposome nanoparticle treatment group.

[0057] 2. The cells were inoculated in culture bottles at a density of 2 x 10 5 cells / well, and cultured for 24 hours to adhere. The cells were treated with a culture medium containing 10 μM Ferrostatin-1 for 6 hours. After 6 hours, the culture supernatant was gently aspirated, and the cells were washed twice with PBS buffer to remove residual Ferrostatin-1.

[0058] 3. Prepare the PFOI liposome nanoparticle solution prepared in Example 1, with a concentration of 10 μl / ml. Add the PFOI liposome nanoparticle solution to the cells, and continue to incubate the cells in a 37°C, 5% CO2 incubator for 24 hours.

[0059] ​4. After the treatment, the cells, including suspended cells and adherent cells, were collected and washed twice with PBS. The apoptosis detection was performed using Annexin V-FITC / PI double staining kit according to the kit instructions. The stained cell samples were detected on the machine (flow cytometer) for analysis of the proportion of apoptotic cells, and the results are shown in Figure 4 .

[0060] As shown in Figure 4 , the cells in the PFOI group were significantly apoptotic, and the apoptosis of the cells in the PFOI+FER-1 group was significantly reduced. The ferroptosis inhibitor can significantly inhibit the apoptosis rate caused by the PFOI liposome nanoparticles prepared in Example 1, which indicates that the PFOI liposome nanoparticles prepared in Example 1 can activate the ferroptosis pathway of cancer cells.

[0061] Example 5

[0062] This example verifies the specific pathway of ferroptosis induced by the PFOI liposome nanoparticles prepared in Example 1 in lung cancer cells by non-targeted metabolomics detection experiment.

[0063] PFOI was used to treat LLC cells, and 10 μl / ml concentration of PFOI liposome nanoparticles was incubated with 2×10 5 LLC cells in a 37°C incubator for 24 hours. The cells were washed twice with 4°C pre-cooled PBS, scraped and resuspended in 1 ml PBS, and then centrifuged to remove the supernatant. Next, the cells were quenched with 1 ml of pre-cooled methanol at -80°C for 30 minutes. Then, the suspension was vortexed at 4°C for 3 min and ultrasonicated in an ice bath for 30 min. After centrifugation at 13300 rpm for 10 min, the supernatant was transferred to a new tube for GC-MS detection.

[0064] The specific pathway of ferroptosis induced by the PFOI liposome nanoparticles prepared in Example 1 in lung cancer cells was analyzed by GC-MS non-targeted metabolomics. The metabolomics results obtained after GC-MS detection of LLC cells showed that the concentration of 40 metabolites in LLC cells changed significantly after treatment with PFOI NPs, of which 19 showed a downward trend and 21 showed an upward trend, as shown in Figures 5-8 , which are OPLS-DA score plots, differential metabolite volcano plots, differential metabolite heat maps and KEGG-based metabolic pathway maps of PFOI group-P and control group-C, respectively.

[0065] The above four figures collectively show the significant metabolic changes obtained by GC-MS non-targeted metabolomics analysis after LLC cells were treated with PFOI liposome nanoparticles (PFOI NPs) prepared in Example 1. These results show that the PFOI NP treatment group and the control group are significantly distinguished in the metabolic profile, with significant changes in metabolite concentration, involving multiple key metabolic pathways, further supporting the hypothesis that PFOI NPs induce ferroptosis by affecting cellular metabolic pathways.

[0066] Example 6

[0067] This example verifies the specific pathway of ferroptosis induced by PFOI liposome nanoparticles prepared in Example 1 in lung cancer cells by Western blot experiment.

[0068] The method steps for detecting the specific pathway of ferroptosis by PFOI liposome nanoparticles in this example are as follows:

[0069] 1. 2 x 10 5 H460 cells were seeded in culture bottles and placed in a culture incubator at 37°C, 5% CO2, and cultured for 24 hours to adhere. The experiment was divided into three groups: control group, 6-hour treatment group (incubated with PFOI liposome nanoparticles at a concentration of 10 μl / ml for 6 hours), and 24-hour treatment group (incubated with PFOI liposome nanoparticles at a concentration of 10 μl / ml for 24 hours).

[0070] 2. After treatment, the cells were washed twice with PBS, scraped off and collected into a centrifuge tube. Ice-cold RIPA lysis buffer was added and lysed on ice for 30 minutes. The lysate was centrifuged at 12000 rpm for 10 minutes at 4°C, and the supernatant was collected as the total protein sample.

[0071] 3. The protein concentration was determined using a BCA protein quantification kit.

[0072] 4. SDS-PAGE and membrane transfer: 30 μl of protein sample was loaded per well and electrophoretically separated in a 10% SDS-PAGE gel. The separated proteins were transferred from the gel to a PVDF membrane under the following conditions: 100V, 90 minutes.

[0073] 5. Blocking and antibody incubation: PVDF membrane was blocked with 5% skim milk or BSA in TBST buffer for 1 hour at room temperature. SLC7A11 primary antibody was diluted at a ratio of 1:100 and incubated overnight at 4°C. The PVDF membrane was washed with TBST buffer for 10 minutes three times. The corresponding HRP-labeled secondary antibody was diluted at a ratio of 1:1000 and incubated at room temperature for 1 hour. The PVDF membrane was washed with TBST buffer for 10 minutes three times.

[0074] 6. Detection: The membrane was treated with ECL chromogenic reagent, and protein signals were detected using a chemiluminescence imaging system. Images of the target protein bands on the membrane were acquired, and quantitative analysis was performed using image analysis software.

[0075] like Figure 9 As shown, the expression of SLC7A11 in the 24-hour treatment group was significantly lower than that in the 6-hour treatment group and the control group, indicating that PFOI liposome nanoparticles may induce ferroptosis by inhibiting SLC7A11. Inhibition of SLC7A11 leads to a decrease in glutathione levels, increased lipid peroxidation, and induces ferroptosis.

Claims

1. Application of perfluorooctyl iodide in the preparation of drugs that induce ferroptosis in lung cancer cells.

2. The use of perfluorooctyl iodide according to claim 1 in the preparation of a drug for inducing ferroptosis in lung cancer cells, characterized in that, The drug that induces ferroptosis in lung cancer cells has perfluorooctyl iodide as its sole active ingredient or one of its active ingredients.

3. The use of perfluorooctyl iodide according to claim 2 in the preparation of a drug for inducing ferroptosis in lung cancer cells, characterized in that, The perfluorooctyl iodide content in the drug that induces ferroptosis in lung cancer cells is 0.1 wt% to 99 wt%.

4. The use of perfluorooctyl iodide according to claim 3 in the preparation of a drug for inducing ferroptosis in lung cancer cells, characterized in that, The drug for inducing ferroptosis in lung cancer cells also includes pharmaceutically acceptable excipients and / or carriers.

5. The use of perfluorooctyl iodide according to any one of claims 1-4 in the preparation of a drug for inducing ferroptosis in lung cancer cells, characterized in that, The drug that induces ferroptosis in lung cancer cells contains lipid nanoparticles of perfluorooctyl iodide.

6. The use of perfluorooctyl iodide according to claim 5 in the preparation of a drug for inducing ferroptosis in lung cancer cells, characterized in that, The drug that induces ferroptosis in lung cancer cells can cause mitochondrial atrophy, increased membrane density, and reduced mitochondrial cristae in lung cancer cells.

7. The use of perfluorooctyl iodide according to claim 6 in the preparation of a drug for inducing ferroptosis in lung cancer cells, characterized in that, The drug that induces ferroptosis in lung cancer cells can lead to an increase in ROS in lung cancer cells.

8. The use of perfluorooctyl iodide according to claim 7 in the preparation of a drug for inducing ferroptosis in lung cancer cells, characterized in that, The drug that induces ferroptosis in lung cancer cells can regulate GSH metabolism and fatty acid biosynthesis in lung cancer cells, leading to GSH depletion and lipid deposition.

9. The use of perfluorooctyl iodide according to claim 8 in the preparation of a drug for inducing ferroptosis in lung cancer cells, characterized in that, The drug that induces ferroptosis in lung cancer cells promotes ferroptosis in lung cancer cells by inhibiting the expression of SLC7A11.

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