Application of PFOI in preparation of medicine for inducing tumor cell ferroptosis

Abstract

The invention relates to application of PFOI in preparation of a medicine for inducing tumor cell ferroptosis, and belongs to the technical field of medicine. The invention provides application of PFOI in preparation of a medicine for inducing tumor cell ferroptosis. The invention verifies the correlation between PFOI and tumor cell ferroptosis through experiments, and proves that PFOI can cause cancer cell mitochondrial atrophy, membrane density increase and mitochondrial ridge reduction; the ROS of cancer cells is increased; regulating cancer cell GSH metabolism and fatty acid biosynthesis, resulting in GSH depletion and lipid deposition; the ferroptosis of cancer cells is promoted by inhibiting the expression of the SLC7A11. On the basis, the invention provides a new application of PFOI in inducing tumor cell ferroptosis, an experimental basis and a new treatment strategy are provided for clinical treatment of cancers, and a new technical support is provided for targeted treatment of cancers.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to the application of PFOI in the preparation of drugs for inducing ferroptosis of tumor cells. Background Art

[0002] Ferroptosis is an iron-dependent, non-apoptotic form of cell death characterized by GSH depletion, GPX4 inactivation, and accumulation of cytotoxic lipid reactive oxygen species (ROS), ultimately leading to lethal plasma membrane damage and membrane perforation. Morphologically, ferroptosis is characterized by mitochondrial atrophy, increased membrane density, and reduced mitochondrial cristae, rather than the typical morphological features of traditional apoptosis. Ferroptosis is regulated by multiple pathways, including the cystine-glutamate antiporter 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 subunit that performs functions and is highly expressed in a variety of human cancers. Recent studies have shown that increased GSH biosynthesis caused by overexpression of SLC7A11 inhibits lipid peroxidation and ferroptosis, thereby promoting tumor growth.

[0003] Ferroptosis, as a unique cell death mechanism, has aroused great interest in the cancer research community, because targeting ferroptosis may provide new therapeutic opportunities for treating cancers that are difficult to cure with traditional therapies. Ferroptosis agents are compounds that induce cell death through iron-dependent substrates. The main types include iron chelators, ferroptosis inducers, iron overloading agents, and mitochondria-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 pharmaceutical intermediate. It plays an important role in the field of organic synthesis and medicine. It can be used as a key intermediate to participate in a variety of chemical reactions and drug preparation processes. Studies have shown that PFOI affects cell migration ability and may be involved in regulating the expression of long non-coding RNA MALAT-1. However, the prior art has not reported the efficacy of PFOI in inducing ferroptosis of tumor cells. Summary of the invention

[0005] The present invention provides a new use of PFOI for inducing ferroptosis of tumor cells, specifically, use of PFOI in preparing a drug for inducing ferroptosis of tumor cells.

[0006] The technical solution of the present invention:

[0007] Application of PFOI in the preparation of drugs that induce ferroptosis of tumor cells.

[0008] Furthermore, the drug for inducing tumor cell ferroptosis contains PFOI as the only active ingredient or one of the active ingredients.

[0009] Furthermore, the content of PFOI in the drug for inducing tumor cell ferroptosis is 0.1wt% to 99wt%.

[0010] Furthermore, the drug for inducing tumor cell ferroptosis also includes pharmaceutically acceptable excipients and / or carriers.

[0011] Furthermore, the drug for inducing tumor cell ferroptosis contains lipid nanoparticles of PFOI.

[0012] Furthermore, the tumor cells are lung cancer cells.

[0013] Furthermore, the drug that induces tumor cell ferroptosis can cause cancer cell mitochondrial atrophy, increased membrane density, and reduced mitochondrial cristae.

[0014] Furthermore, the drug that induces tumor cell ferroptosis can lead to an increase in ROS in cancer cells.

[0015] Furthermore, the drug that induces tumor cell ferroptosis can regulate cancer cell GSH metabolism and fatty acid biosynthesis, leading to GSH depletion and lipid deposition.

[0016] Furthermore, the drug that induces tumor cell ferroptosis promotes ferroptosis of cancer cells by inhibiting the expression of SLC7A11.

[0017] Beneficial effects of the present invention:

[0018] The present invention verifies the correlation between the PFOI liposome nanoparticles prepared in Example 1 and the ferroptosis of tumor cells through experiments, and proves that the PFOI liposome nanoparticles prepared in Example 1 can cause mitochondrial atrophy, increased membrane density, and reduced mitochondrial cristae in cancer cells; increase ROS in cancer cells; regulate GSH metabolism and fatty acid biosynthesis in cancer cells, leading to GSH depletion and lipid deposition; and promote ferroptosis in cancer cells by inhibiting the expression of SLC7A11. Based on this, the present invention provides a new use of PFOI for inducing ferroptosis of tumor cells, specifically the use of PFOI in the preparation of drugs for inducing ferroptosis of tumor cells, which provides experimental basis and new treatment strategies for the clinical treatment of lung cancer, and provides new technical support for targeted cancer therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a transmission electron microscopy image of the PFOI liposome nanoparticles prepared in Example 1;

[0020] Figure 2This is a transmission electron micrograph of H460 cells after being acted upon by PFOI liposome nanoparticles in Example 2;

[0021] Figure 3 This is a comparison diagram of the ROS fluorescence intensity in A549 cells after being acted upon by PFOI liposome nanoparticles in Example 3;

[0022] Figure 4 This is a reverse verification diagram of the ability of PFOI liposome nanoparticles to activate ferroptosis in lung cancer cells in Example 4;

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

[0024] Figure 6 This is the non-targeted metabolomics analysis result diagram of LLC cells treated with PFOI liposome nanoparticles in Example 5 - the volcano diagram of differential metabolites between the PFOI group and the blank control group;

[0025] Figure 7 This is the non-targeted metabolomics analysis result of LLC cells treated with PFOI liposome nanoparticles in Example 5 - heat map of differential metabolites between the PFOI group and the blank control group;

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

[0027] Fig. 9 This is a graph showing the results of PFOI liposome nanoparticles promoting the expression of ferroptosis-related proteins in Example 6. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described below in conjunction with the embodiments, but it is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be included in the protection scope of the present invention. The process equipment or devices not specifically noted in the following embodiments are all conventional equipment or devices in the art. If not specifically specified, the raw materials used in the embodiments of the present invention can be obtained commercially; if not specifically specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0029] Example 1

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

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

[0032] Step 1, according to the molar ratio of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG) and cholesterol = 83:2:15, each substance is weighed, uniformly mixed, dissolved with chloroform, the dissolved surfactant is evaporated to dryness by a rotary evaporator, then dried in a vacuum oven at 40°C overnight, and finally dispersed in a certain amount of water by ultrasonic oscillation to obtain a surfactant mixture for standby use;

[0033] Step 2: Perfluorooctyl iodide PFOI, the mixture of the surfactant obtained in step 1, glycerol, and water are mixed in a mass ratio of 20:2:2:76, and subjected to 85% ultrasonic treatment with a water bath probe for 4 minutes, mixed evenly, and extruded by an extruder (Avanti mini extruder) to prepare a PFOI nanoemulsion;

[0034] Step 3: The PFOI nanoemulsion obtained in step 2 is dialyzed to remove the components that are not effectively coated, thereby obtaining PFOI liposome nanoparticles with an average particle size of 120±10 nm.

[0035] Figure 1 This is a transmission electron micrograph 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 example, transmission electron microscopy was used to observe the effect of the PFOI liposome nanoparticles prepared in Example 1 on the ultrastructure of lung cancer cells.

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

[0039] (1) Take LLC cells in the logarithmic growth phase to prepare a cell suspension, count them, and plate 5×10 5 / well, each well was filled with culture medium to 2 ml, cultured in a cell culture incubator, a mixture of PFOI liposome nanoparticles and high-glucose culture medium was added to the well plate (the concentration of PFOI liposome nanoparticles was 10 μl / ml), and cultured in a 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 into a tube, centrifuge at 1000 rpm for 5 minutes, and carefully discard the liquid;

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

[0042] (4) Prepare a mixture of acetone and embedding agent in a ratio of 1:1, place the specimen in the mixture, soak it fully, and soak it in pure embedding agent for 8-10 hours the next day. Adjust the incubator temperature to 60°C. After the temperature is reached, place the specimen in the incubator, react for 2 days, then place it in the embedding agent, and finally cut it into thin slices. Place it in a 2% saturated aqueous solution of uranyl acetate for 16 minutes. Then place it in lead citrate for the same time and place it in the incubator for 8-10 hours. Observe the internal structure of the treated cells under an electron microscope, such as Figure 2 shown.

[0043] Compared with the control group, the mitochondrial volume of the group treated with PFOI liposome nanoparticles prepared in Example 1 was reduced and the membrane density was increased, indicating that the PFOI liposome nanoparticles prepared in Example 1 can change the morphology of cell mitochondria, which is also a feature that distinguishes ferroptosis from other cell apoptosis modes.

[0044] Example 3

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

[0046] In this embodiment, the steps of the PFOI liposome nanoparticle detection method for reactive oxygen species are as follows:

[0047] (1) After LLC cells were plated, the experiments were divided into four groups: PFOI liposome nanoparticles + radiotherapy group (PFOI+RT group), PFOI liposome nanoparticles group (PFOI group), radiotherapy group (RT group), and control group (Control group). 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 10μl / ml, added to the confocal dish, incubated in a 37℃ incubator for 2 hours, washed twice with PBS, and irradiated with a handheld radiotherapy device at an operating frequency of 50kV and 75μA for 10 minutes at a distance of 3cm for irradiation radiotherapy; PFOI group: PFOI liposome nanoparticles were mixed in the culture medium at a concentration of 10μl / ml, added to the confocal dish, incubated in a 37℃ incubator for 2 hours, washed twice with PBS; RT group: Irradiated with a handheld radiotherapy device at an operating frequency of 50kV and 75μA for 10 minutes at a distance of 3cm for irradiation radiotherapy; Control group: The Control group without special treatment was also washed twice with PBS and replaced with fresh culture medium to unify the objective conditions.

[0048] (2) Dilute the lipid peroxidation reagent to 1 μl / ml and DCFH-DA to 20 μmol / L in serum-free culture medium. Discard the culture medium, add the lipid peroxidation reagent dilution solution and DCFH-DA, place in an incubator and incubate for 30 minutes, then rinse three times.

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

[0050] (4) Rinse three times and seal the slides.

[0051] (5) Observe and take pictures using a laser confocal microscope. The results are as follows Figure 3 shown.

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

[0053] Example 4

[0054] In this example, the ferroptosis inhibitor Ferrostatin-1 was used to detect whether it could rescue the ferroptosis of lung cancer cells induced by the PFOI liposome nanoparticles prepared in Example 1.

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

[0056] 1. Lung cancer cells LLC were inoculated into culture flasks and divided into three groups: control group (untreated), PFOI liposome nanoparticles treatment group, Ferrostatin-1 pretreatment + PFOI liposome nanoparticles treatment group;

[0057] 2. Cells were plated at 2 x 10 5 Cells were seeded at a density of 10 cells / well in a culture flask and cultured for 24 hours to allow them to adhere to the wall. The cells were treated with a 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 at a concentration of 10 μl / ml. Add the PFOI liposome nanoparticle solution to the cells and continue to incubate the cells in an incubator at 37° C. and 5% CO 2 for 24 hours.

[0059] 4. After the treatment, collect the cells, including suspended cells and adherent cells, and wash them twice with PBS. Use the Annexin V-FITC / PI double staining kit to detect cell apoptosis according to the kit instructions. The stained cell samples are tested on a flow cytometer to analyze the proportion of apoptotic cells. The results are as follows: Figure 4 shown.

[0060] like Figure 4 As shown, the cells in the PFOI group showed significant apoptosis, and the apoptosis of 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 by which the PFOI liposome nanoparticles prepared in Example 1 induce ferroptosis in lung cancer cells through non-targeted metabolomics detection experiments.

[0063] LLC cells were treated with PFOI. PFOI liposome nanoparticles at a concentration of 10 μl / ml were mixed with 2×10 5 LLC cells were incubated in a 37°C incubator for 24 hours, 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. After that, the suspension was vortexed at 4°C for 3 minutes and sonicated in an ice bath for 30 minutes. After centrifugation at 13300 rpm for 10 minutes, the supernatant was transferred to a new tube for GC-MS detection.

[0064] The specific pathway of ferroptosis induced by 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 concentrations of 40 metabolites in LLC cells changed significantly after being treated with PFOI NPs, of which 19 showed a downward trend and 21 showed an upward trend, such as Figure 5-Figure 8 Shown are the OPLS-DA score map, differential metabolite volcano map, differential metabolite heat map and KEGG-based metabolic pathway map of the PFOI group-P and the control group-C, respectively.

[0065] The above four figures comprehensively 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 there is a clear distinction between the metabolite spectrum of the PFOI NPs-treated group and the control group, and the metabolite concentrations change significantly, 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 uses Western blot experiments to verify the specific pathway by which the PFOI liposome nanoparticles prepared in Example 1 induce ferroptosis in lung cancer cells.

[0068] In this embodiment, the specific method steps of the PFOI liposome nanoparticle detection of ferroptosis are as follows:

[0069] 1. 2×10 5 H460 cells were inoculated into culture flasks and placed in an incubator at 37°C, 5% CO2 for 24 hours to allow them to adhere to the wall. The experiment was divided into three groups: control group, 6-hour treatment group (10 μl / ml PFOI liposome nanoparticles were co-incubated with cells for 6 hours), and 24-hour treatment group (10 μl / ml PFOI liposome nanoparticles were co-incubated with cells for 24 hours).

[0070] 2. After treatment, wash the cells twice with PBS, scrape the cells and collect them in a centrifuge tube. Add ice-cold RIPA lysis buffer and lyse on ice for 30 minutes. Centrifuge the lysate at 12000rpm for 10 minutes at 4℃ and collect the supernatant as the total protein sample.

[0071] 3. Determine the protein concentration using the BCA protein quantification kit.

[0072] 4. SDS-PAGE and membrane transfer: Load 30 μl of protein sample per well and separate by electrophoresis in 10% SDS-PAGE gel. Transfer the separated protein from the gel to a PVDF membrane at 100 V for 90 minutes.

[0073] 5. Blocking and antibody incubation: Block the PVDF membrane with 5% skim milk powder or BSA in TBST buffer and incubate at room temperature for 1 hour. Incubate with SLC7A11 primary antibody at a dilution ratio of 1:100 at 4°C overnight. Wash the PVDF membrane 3 times with TBST buffer for 10 minutes each. Incubate with the corresponding HRP-labeled secondary antibody at a dilution of 1:1000 at room temperature for 1 hour. Wash the PVDF membrane 3 times with TBST buffer for 10 minutes each.

[0074] 6. Detection: Treat the membrane with ECL colorimetric reagent and detect protein signals in a chemiluminescent imaging system. Obtain images of the target protein bands on the membrane and perform quantitative analysis using image analysis software.

[0075] like Fig. 9 As shown in the figure, 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 cell ferroptosis by inhibiting the pathway of SLC7A11. The inhibition of SLC7A11 will lead to a decrease in glutathione levels, increase lipid peroxidation, and induce ferroptosis.

Claims

1. Application of PFOI in the preparation of drugs that induce ferroptosis of tumor cells.

2. The use of PFOI according to claim 1 in the preparation of a drug for inducing ferroptosis of tumor cells, characterized in that: The drug for inducing tumor cell ferroptosis has PFOI as the only active ingredient or one of the active ingredients.

3. The use of PFOI according to claim 2 in the preparation of a drug for inducing ferroptosis of tumor cells, characterized in that: The content of PFOI in the drug for inducing tumor cell ferroptosis is 0.1 wt% to 99 wt%.

4. The use of PFOI in the preparation of a drug for inducing ferroptosis of tumor cells according to claim 3, characterized in that: The drug for inducing tumor cell ferroptosis also includes pharmaceutically acceptable excipients and / or carriers.

5. The use of PFOI according to any one of claims 1 to 4 in the preparation of a drug for inducing ferroptosis of tumor cells, characterized in that: The drug for inducing tumor cell ferroptosis contains lipid nanoparticles of PFOI.

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

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

8. The use of PFOI in the preparation of a drug for inducing ferroptosis of tumor cells according to claim 7, characterized in that: The tumor cell ferroptosis-inducing drug can regulate cancer cell GSH metabolism and fatty acid biosynthesis, leading to GSH depletion and lipid deposition.

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

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