Peroxide-containing liposome, preparation method therefor, and use thereof

Abstract

Provided are a peroxide-containing liposome, a preparation method therefor, and use thereof. The peroxide-containing liposome is a spherical vesicle having a double-layer membrane structure and formed by lipid molecules containing one or more peroxy groups, and the lipid molecules contain carbon-carbon double bonds. The peroxide-containing liposome can be obtained by mixing peroxide-containing lipid molecules with an aqueous system, wherein the peroxide-containing lipid molecules are obtained by reacting unsaturated lipid molecules containing -CH=CH- and / or -C≡C- with an additive in the presence of oxygen and a solvent, and the additive is selected from one or more of lipoxygenase, an oxidation catalyst, and an oxidizing agent. By means of in-vitro experiments in cells and in-vivo experiments in animals, it is found that the peroxide-containing liposome can kill tumor cells and can be used as a sensitizing agent in combination with other tumor treatment methods to effectively improve the therapeutic effect on tumors, thus having good application prospects in resisting tumors.

Description

A peroxide-containing liposome, its preparation method and application Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a peroxide-containing liposome, its preparation method, and its application. Background Technology

[0002] Traditional treatments (such as chemotherapy drugs and ionizing radiation) may help control and alleviate local lesions, but they can also lead to the evolution of cancer cells, making them more drug-resistant. The synergistic effect of ferroptosis and tumor therapy offers new treatment opportunities for refractory tumors. Ferroptosis is a type of cell death discovered in recent years, closely related to the metabolism of iron, lipids, and amino acids. It possesses unique morphological, biochemical, and genetic changes that distinguish it from other forms of cell death. Therefore, the potential role of ferroptosis in tumor biology has become a research hotspot in recent years.

[0003] Since Stockwell et al. defined ferroptosis in 2012, eight different regulatory molecules have been identified, including systemic X. c - The ferroptosis indicators include glutathione peroxidase 4 (GPX4), 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), coenzyme Q10 (CoQ10), ferroptosis inhibitory protein 1 (FSP1), lipoxygenases (LOXs), prostatic six-span membrane epithelial antigen 3 (STEAP3), divalent metal ion transporter 1 (DMT1), unstable iron pool, and lipid peroxides (LOOH). These ferroptosis indicators can be integrated into a comprehensive signaling network. This network consists of two braking axes, including system X. c - / GSH / GPX4 and vitamin B5 / acetyl-CoA / HMG-CoA / CoQ10 / FSP1 are used to degrade lipid peroxides, and two driving axes are involved in lipid peroxide generation (PUFA / LOXs / LOOH) and iron metabolism (STEAP3 / DMT1 / Fe). 2+ / LOOH). Among them, GPX4, FSP1 and system X in the brake axle. c - These proteins have been found to play a crucial role in controlling ferroptosis. Chemical inhibitors targeting these proteins have been extensively investigated as candidate ferroptosis initiators. For example, Erastin and sulfasalazine have been identified as systemic X inhibitors. c - Inhibitors of GPX4 include RSL3, FINO2, FIN56, and hexamethylmelamine, which can directly inhibit GPX4 activity; statins (such as atorvastatin) can indirectly reduce GPX4 activity by inhibiting HMG-CoA reductase. However, few of these drugs have been successfully used clinically.

[0004] Traditional cancer treatments suffer from drawbacks such as low sensitivity and easy acquisition of drug resistance; and existing ferroptosis initiators have rarely been successfully applied clinically. Therefore, there is an urgent need for a new type of cancer treatment that is safe, effective, and less likely to induce drug resistance.

[0005] Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a peroxide-containing liposome, its preparation method, and its application. The peroxide-containing liposome is a spherical vesicle with a bilayer membrane structure formed by lipid molecules containing one or more peroxide groups and carbon-carbon double bonds. It can induce ferroptosis in tumor cells, is less prone to drug resistance, and can be used as a drug carrier to encapsulate other anti-tumor drugs for synergistic treatment, exhibiting good synergistic efficacy.

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

[0008] The first aspect of the present invention provides a peroxide-containing liposome, wherein the peroxide-containing liposome is a spherical vesicle with a bilayer membrane structure formed by lipid molecules containing one or more peroxide groups; wherein the lipid molecules contain carbon-carbon double bonds.

[0009] Furthermore, the lipid molecule contains the following groups in its structure:

[0010] A second aspect of the present invention provides a method for preparing peroxide-containing liposomes, comprising the following steps:

[0011] S1. Unsaturated lipid molecules are reacted with additives in the presence of oxygen and solvent to obtain lipid molecules containing peroxy groups; the unsaturated lipid molecules contain -CH=CH- and / or -C≡C-; the additives are selected from one or more of lipoxygenases, oxidation catalysts, and oxidants;

[0012] S2. The lipid molecules containing peroxide groups prepared in S1 are mixed with an aqueous system to obtain the peroxide-containing liposomes.

[0013] Further, in S1, the molar ratio of the unsaturated lipid molecule to the volume of the solvent is (1 μmol - 1 mol): 1 L; in some preferred embodiments, the unsaturated lipid molecule is 1,2-dilinoleoyl-tin-glycero-3-phosphocholine, 1,2-dilinoleoyl-sn-glycero-3-phospho-L-serine, 1,3-linoleic acid diglyceride, or 1,2-dioleoyl lecithin, but is not limited to the types of lipid molecules listed above. Other unsaturated lipid molecules containing divalent groups CH=CH- and / or -C≡C- are also applicable; the solvent is deionized water, physiological saline, PBS buffer solution, or cell culture medium.

[0014] Furthermore, in S1, the oxidation catalyst includes, but is not limited to, one or more of MoS2 nanosheets, GO nanosheets (graphene oxide nanosheets), and FeNGR nanosheets, and any oxidant with catalytic oxidation activity may be used.

[0015] Furthermore, in S1, the oxidant includes, but is not limited to, cumene hydroperoxide and / or hydrogen peroxide.

[0016] Further, in S1, when the additive is lipoxygenase, the reaction concentration of the lipoxygenase is 1×10⁻⁶. 4 -1×10 6 U / mL;

[0017] When the additive is an oxidation catalyst, the reaction concentration of the oxidation catalyst is 0.001-5 mg / mL;

[0018] When the additive is an oxidant, the reaction concentration of the oxidant is 1 μmol-1 mmol.

[0019] Furthermore, in S1, the reaction temperature is 4-300℃, and the reaction time is 0.1 hours-7 days.

[0020] Furthermore, in S1, unsaturated lipid molecules are first dispersed in a solvent to form a mixed solution, and a lipid film is prepared by rotary evaporation. Then, it is reacted with additives in the presence of oxygen and solvent to obtain lipid molecules containing peroxy groups.

[0021] Furthermore, in S2, the aqueous system includes, but is not limited to, deionized water, PBS buffer solution, cell culture medium, such as RPMI 1640 medium.

[0022] Furthermore, in S2, the mixing process is preferably ultrasonic treatment, but it can also be carried out by stirring; the ultrasonic treatment power is preferably 10-100W and the time is 10-60s.

[0023] A third aspect of the present invention provides an antitumor drug comprising peroxide-containing liposomes prepared by the preparation method described in the second aspect.

[0024] Furthermore, the antitumor drug is used to enhance the iron ion transport function of the DMT1 protein, thereby inducing ferroptosis to kill tumor cells.

[0025] Furthermore, the peroxide-containing liposomes serve as carriers to encapsulate the active pharmaceutical ingredient. Preferably, the active pharmaceutical ingredient is used for antitumor purposes and includes, but is not limited to, sulfasalazine, atorvastatin, artesunate, and hexamethylpyrimidine.

[0026] Furthermore, the tumor includes, but is not limited to, human breast tumor cells MCF-7, mouse breast tumor cells 4T1, human or mouse melanoma cells A375, B16, etc.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] This invention provides peroxide-containing liposomes, which are spherical vesicles with a bilayer membrane structure formed by lipid molecules containing one or more peroxide groups and carbon-carbon double bonds. In vitro cell experiments have shown that these peroxide-containing liposomes can enhance the iron transport function of the lysosomal membrane DMT1 protein through post-modification, thereby increasing intracellular iron transport in tumor cells. 2+ The redistribution of the drug triggers ferroptosis, killing tumor cells and making it less likely to develop drug resistance.

[0029] The peroxide-containing liposomes provided by this invention can be used as carriers to encapsulate other anti-tumor drugs. Through in vitro cell experiments and animal experiments, it has been found that the peroxide-containing liposomes can act as sensitizers or synergistically enhance other tumor treatments, showing significant synergistic therapeutic effects and promising application prospects in anti-tumor therapy. Attached Figure Description

[0030] Figure 1 is a flowchart of the preparation process of peroxide-containing liposomes;

[0031] Figure 2 shows the LC-MS chromatograms of the peroxide-containing liposomes prepared in Example 1 and the DLPC standard;

[0032] Figure 3 is a cryo-electron micrograph of liposomes containing peroxides;

[0033] Figure 4 shows the detection results of the BODIPY (581 / 591) fluorescent probe containing peroxide liposomes;

[0034] Figure 5 shows a comparison of cell viability of MCF-7 cells after treatment with different liposomes;

[0035] Figure 6 illustrates the mechanism of action of peroxide-containing liposomes in inducing ferroptosis, where A represents FITC-labeled LIPs. PCPO Fluorescence spectrum of lysosomal DMT1 protein and their co-localization, B represents LIP. PCPO Mass spectrum of DMT1 protein after interaction with DMT1 protein, C represents intracellular Fe after different treatments. 2+ Fluorescence distribution;

[0036] Figure 7 shows docetaxel and peroxide-containing liposomes (LIP). PCPO IC50 in 4T1-R cells 50 Graph showing changes over treatment time;

[0037] Figure 8 shows the changes in tumor volume in breast cancer-bearing mice after treatment with different methods. Detailed Implementation

[0038] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0040] The sources of some of the reagents used in the following examples are as follows:

[0041] 1,2-Dilinoleoyl-tin-glycero-3-phosphocholine (hereinafter referred to as PC, A:B = 18:2), 1,2-dilinoleoyl-sn-glycero-3-phosphate-L-serine (hereinafter referred to as PS, A:B = 18:2), 1,2-dioleoyllecithin (hereinafter referred to as DOPC, A:B = 18:1), and LOX enzyme were all purchased from Sigma-Aldrich Inc.; 1,3-linoleic acid diglyceride (hereinafter referred to as DG, A:B = 18:2) was purchased from Cayman Chemical; and the BODIPY (581 / 591) fluorescent probe was purchased from Thermo Fisher Scientific. In the above, A represents the number of carbon atoms in a single carbon chain, and B represents the number of unsaturated double bonds in a single carbon chain.

[0042] Example 1

[0043] This embodiment involves peroxide-containing liposomes (LIPs). PCPO The synthesis process is as follows:

[0044] 2.5 μL of MoS2 nanosheet material dispersed in aqueous phase (5 mg / mL, concentration determined by ICP-OES) was added to 0.5 mL of 0.25 mg / mL 1,2-dilinoleoyl-tin-glycero-3-phosphocholine (PC) dispersion (chloroform solvent). The mixture was incubated at 37 °C with oxygen for 2 h. After the reaction was completed, the mixture was centrifuged at 20000 g for 10 min. The supernatant was collected and the reaction product was identified by triple quadrupole liquid chromatography-mass spectrometry (LC-MS). As shown in Figure 2, a peroxy group -OOH was successfully attached to PC, and the product PC-OOH was prepared. The product eluted at 10.2 min and had a molecular weight of 814.5.

[0045] PC-OOH was dispersed in water and sonicated in a water bath for 30 seconds to obtain peroxide-containing liposomes. The cryo-electron microscopy image of the prepared peroxide-containing liposomes is shown in Figure 3. As can be seen from the figure, spherical vesicles with a bilayer membrane structure are formed.

[0046] Example 2

[0047] This embodiment involves peroxide-containing liposomes (LIPs). PCPO LIP DGPO LIP PSPO The synthesis process is shown in Figure 1, and the specific steps are as follows:

[0048] Measure 0.5 mL of a 1 mg / mL PC, DG, or PS solution (all in chloroform) into a 5 mL round-bottom flask. Place the flask on a rotary evaporator and set the pressure, rotation speed, and temperature to 400 mbar, 80 rpm, and 25 °C, respectively. Evacuate the flask to remove the chloroform solvent. At this point, a uniform thin film of PC, DG, or PS will form on the inner wall of the flask. Add 1 mL of a solution containing 10 mg / mL PC, DG, or PS into the flask. 5 LOX enzyme was prepared in RPMI 1640 solution. Oxygen was slowly introduced into the solution, and the mixture was heated in an oil bath at 37°C with magnetic stirring at 300 rpm for 4 hours to catalyze the PC reaction. Then, the mixture was sonicated in a water bath round-bottom flask for 20 seconds, and the reaction solution was collected into a 2 mL EP tube. Finally, the EP tube was centrifuged at 4°C and 20000 g for 10 minutes to remove the LOX enzyme. The supernatant in the EP tube was the LIP. PCPO LIP DGPO or LIP PSPO Prepared LIP, for later use. PCPO LIP DGPO and LIP PSPO All of them have a double-membrane spherical vesicle structure.

[0049] Peroxide-containing liposomes were analyzed using a liquid chromatography system combined with an LTQXL mass spectrometer and an electrospray ionization (ESI) source. MS data were collected and analyzed using Xcalibur software (Thermo Xcalibur 2.2). As shown in Table 1, a peroxide group -OOH was successfully linked to PC, DG, or PS.

[0050] Table 1

[0051] Example 3

[0052] This embodiment involves peroxide-containing liposomes (LIPs). DOPCThe synthesis of lipid peroxides was performed, and the formation of lipid peroxides was detected using the BODIPY (581 / 591) fluorescent probe, as shown in Figure 1. The specific procedures are as follows:

[0053] Measure 0.5 mL of 1 mg / mL DOPC solution (chloroform solvent) into a 5 mL round-bottom flask. Place the flask on a rotary evaporator and set the pressure, rotation speed, and temperature to 400 mbar, 80 rpm, and 25 °C, respectively. Remove the chloroform solvent by vacuuming. At this point, DOPC forms a uniform film on the inner wall of the round-bottom flask. Add 1 mL of PBS buffer to the flask and sonicate it in a water bath for 20 seconds. This liquid is a peroxide-free DOPC liposome solution. Collect the reaction solution in a 1.5 mL EP tube for later use. Take 500 μL of the above DOPC liposome solution into a 1.5 mL EP tube, add 8 M H2O2, and incubate at room temperature for 2 hours. Then add 2 μL of 10 mM C11-BODIPY and continue incubating in the dark for 30 minutes. 100 μL of each sample was added to a 96-well black plate. The samples were then placed under a fluorescence microscope to observe the formation of lipid peroxides at an excitation wavelength of 488 nm and an emission wavelength of 520 nm. As shown in Figure 4, DOPC liposomes can be oxidized by H2O2 to form peroxide-containing liposomes (LIPs). DOPC .

[0054] Example 4

[0055] Taking the peroxide-containing liposomes prepared in Example 2 as an example, this example studies the killing effect of peroxide-containing liposomes on tumor cells. Unmodified liposomes prepared from PC, DG, and PS, respectively, and peroxide-containing liposomes prepared in Examples 2-4 were used to treat human breast cancer cell line MCF-7 (MCF-7R) cells. After treatment for 24 hours, cell viability was detected by MTS assay. The specific operation is as follows:

[0056] Will contain 5×10 3 100 μL of a suspension of drug-resistant breast cancer cells (MCF-7R) was added to a 96-well cell culture plate and incubated overnight. After removing the supernatant, 100 μL of liposomes (LIPs) containing 200 μM drug were added. PC LIP DG LIP PS ) or liposomes containing peroxides (LIP) PCPO LIP DGPO LIP PSPOAdd RPMI 1640 medium or an equal volume of fresh medium to a 96-well plate. After incubation for 24 hours, remove the supernatant, add 120 μL MTS working solution to each well, and incubate in the dark for 2 hours. Transfer 100 μL MTS reaction solution from each well to another 96-well plate, and measure the optical density (OD) of the MTS reaction solution at 490 nm using a microplate reader. 490 Finally, cell viability for each group was calculated using the following formula (1).

[0057] Among them, OD T OD C and OD B These represent the OD values ​​of the treatment group, the control group, and the MTS working solution itself, respectively. 490 Values. The detection results are shown in Figure 5. All unmodified liposomes had no effect on cell viability, while the three types of liposomes containing peroxides significantly inhibited the proliferation of MCF-7R cells. Among them, the liposomes formed by PC-OOH significantly inhibited the proliferation of MCF-7R cells. PCPO The killing effect was strongest in MCF-7R cells, reaching 80.33%.

[0058] Example 5

[0059] Peroxide-containing liposomes LIP prepared in Example 2 PCPO For example, this embodiment studies the mechanism of action of peroxide-containing liposomes on tumor cell killing, as detailed below:

[0060] (1) The colocalization of lysosomal membrane DMT1 protein and FITC-labeled liposomes that have entered the cell was observed by fluorescence microscopy. The specific operation is as follows:

[0061] MCF-7R cell suspension (20×10) 4 Add 100 μL of the solution to an eight-well confocal microplate and incubate overnight. Discard the supernatant and add 100 μL of the solution containing 100 μM FITC-labeled LIP. PCPOThe solution was added to the experimental group. After 12 hours of incubation, the cells were washed three times with PBS. Then, 100 μL of 4% paraformaldehyde was added to each group of samples, and the cells were fixed at room temperature for 20 min. The paraformaldehyde fixative was removed, and the cells were washed three times with PBS. 100 μL of 0.3% Triton X-100 PBS solution was added, and the cells were incubated permeabilized for 10 min. Then, 3% BSA saline solution was added to the permeabilized cells, and the cells were incubated at room temperature for 30 min. The supernatant was removed, and 100 μL of DMT1 antibody solution diluted 1:100 was added. The cells were incubated overnight at 4°C. After removing the primary antibody solution, the cells were washed three times with PBS. Then, 100 μL of Cy3-labeled secondary antibody dilution (1:500) and Hoechst 33342 solution (10 μg / mL) were added, and the cells were incubated in the dark for 1 hour. After rinsing with PBS, the distribution of lysosomal membrane DMT1 protein (red) and liposomes (green) and their co-localization were observed by scanning confocal microscopy at excitation wavelengths of 594 nm and 488 nm, respectively.

[0062] The test results are shown in Figure 6A. After 12 hours of incubation, FITC-labeled LIPs... PCPO It is co-localized with the lysosomal DMT1 protein.

[0063] (2) DMT1 protein was extracted from lysosomal membranes for gel electrophoresis separation, and gel bands containing DMT1 protein were obtained for proteolytic digestion and proteomics analysis to further validate LIP. PCPO The specific mechanism of action of the DMT1 protein is as follows:

[0064] Add 2 mL of MCF-7R cell suspension (5 × 10⁻⁶) to a 6-well plate. 5 (Number of wells), incubate the plate overnight in an incubator. Discard the supernatant, and add 2 mL of fresh RPMI 1640 medium, or medium containing 100 μL of RPMI. PCPOAn equal volume of RPMI 1640 medium solution was added to each 6-well plate. After culturing the cells for 12 hours, the medium was removed from the wells, and the cells were scraped off with a cell scraper and collected into EP tubes by centrifugation. 150 μL of RIPA cell lysis buffer containing 1% protease inhibitor was added to the EP tubes, and the tubes were frozen in liquid nitrogen for 5 minutes. The cells were then thawed by sonication in a water bath, and this process was repeated 5 times to fully lyse the cells. The EP tubes were then centrifuged at 20,000 g for 10 minutes. The supernatant lysate was collected, and protein concentration was determined using a BCA kit. Then, using a Mini-PROTEAN Tetra system, protein samples were added to a 10% SDS-PAGE gel. The concentration voltage was set to 80 V, and the separation voltage to 120 V. The protein samples were separated using Tris-glycine-SDS electrophoresis buffer. Finally, the gel containing the target band was cut off for protease digestion, and the digestion supernatant was collected for proteomics analysis.

[0065] The test results are shown in Figure 6B, LIP PCPO After interacting with the DMT1 protein, it chemically modified the DMT1 protein, specifically causing the amino acids in the DMT1 protein to form disulfide bonds at positions 245 and 248 and positions 334 and 365, as shown in the figure.

[0066] (3) The LIP was further investigated using FeRhoNox-1 fluorescent reagent. PCPO For intracellular Fe 2+ The impact of distribution is explained in the following steps:

[0067] Inoculate 0.1 mL of a solution containing 20 × 10⁻⁶ mg / L in an eight-well dish. 4 Prepare one MCF-7R suspension and incubate overnight in an incubator. Remove the culture medium and add 100 μM LIP solution to each well according to the grouping. PCPO Add 100 μL of 25 μM RSL3 medium solution and incubate for 12 h. Wash each experimental group of cells three times with PBS buffer, 1 min each time. Finally, add 200 μL of RPMI 1640 medium containing 10 μg / mL FeRhoNox-1 and 10 μg / mL Hoechst 33342, and incubate in the dark for one hour. After removing the supernatant and washing with PBS, observe the intracellular ferrous ions (Fe2+) in each experimental group of cells using a confocal scanning microscope at excitation wavelengths of 405 nm and 559 nm. 2+ The distribution of ) was studied. Ferrous ammonium sulfate (Fe(NH4)2(SO4)2) was used as a positive control in the experiment.

[0068] The experimental results are shown in Figure 6C. When Fe in the control group (group Ctrl in the figure) was... 2+When distributed in a dotted pattern, peroxide-containing liposomes LIP PCPO Make Fe 2+ It is diffusely distributed within cells, and its distribution pattern is similar to that of the positive control group (FAS group in the figure).

[0069] In summary, peroxide-containing liposomes (LIPs) PCPO By post-modifying the lysosomal membrane DMT1 protein to enhance its iron transport function, intracellular iron in tumor cells can be increased. 2+ Redistribution triggers ferroptosis, killing tumor cells.

[0070] Example 6

[0071] Peroxide-containing liposomes LIP prepared in Example 2 PCPO For example, this embodiment studies the drug resistance of peroxide-containing liposomes to tumor cells. The specific operation is as follows:

[0072] A suspension of doxorubicin-resistant mouse breast cancer cells 4T1-R cells (1×10⁻⁶) was prepared. 5 Equal amounts of docetaxel (2 mL / well) or liposomes (0.5 mL / well) were added to each well of a 6-well plate and incubated at 37°C for 24 hours. Cells were then exposed to 0.25 × IC50 doses of docetaxel and liposomes containing peroxides, respectively. After 24 hours, cell density was assessed using an inverted microscope. When cells adapted to the test concentration and showed >80% confluence, the exposure dose of docetaxel or liposomes was increased. Otherwise, the exposure concentration was maintained for another 24 hours. This continuous exposure for 60 days yielded cells that had evolved resistance.

[0073] The test results are shown in Figure 7. During long-term treatment, liposomes containing peroxides showed sustained antitumor effects and did not trigger the development of drug resistance in doxorubicin-resistant mouse breast cancer cells.

[0074] Example 7

[0075] Peroxide-containing liposomes LIP prepared in Example 2 PCPO For example, this embodiment studies the encapsulation effect of peroxide-containing liposomes on different drugs (sulfasalazine (SSZ), atorvastatin (AT), artesunate (AS), and hexamethylpyrimethamine (HMM)) and their synergistic effect on tumor killing. Details are as follows:

[0076] Sulfasalazine, atorvastatin, artesunate, and hexamethylpyrimidine were dissolved in dimethyl sulfoxide at a drug concentration of 200 μM, respectively. These solutions were then added to 500 μL chloroform solutions containing 120 μM unmodified liposomes and 80 μM peroxide-containing liposomes, respectively. The solvent was evaporated using a rotary evaporator (120 rpm) at room temperature to obtain thin films in round-bottom flasks, which were then hydrated with 1 mL of deionized water. The films were sonicated in a water bath (40 W) for 30 seconds and extruded through an Avanti extruder equipped with a 0.1 μm polycarbonate membrane. Free hydrophobic drugs were removed by centrifugation at 20000 g for 10 min, yielding drug-encapsulated peroxide-containing liposomes. The drug-encapsulated peroxide-containing liposomes were then lyophilized using a vacuum centrifuge and dissolved in dimethyl sulfoxide or methanol for UV-Vis spectroscopy detection. Encapsulation efficiency (EE) was calculated using the following formula:

[0077] Among them, C L and C T These are the concentrations of hydrophobic drugs encapsulated in liposomes and the total concentration of drugs used in liposome encapsulation, respectively.

[0078] Human breast cancer cell line MCF-7 (MCF-7R) was treated with a single drug, drugs containing peroxide liposomes, and drugs encapsulated in peroxide liposomes, respectively. After 24 h of treatment, cell viability was assessed using the MTS assay. The combination index (CI) of peroxide liposomes and different drugs was calculated as follows: CI = AB / (A×B), where AB is the survival rate of tumor cells after treatment with drugs encapsulated in peroxide liposomes, A is the survival rate of tumor cells after treatment with a single drug, and B is the survival rate of tumor cells after treatment with peroxide liposomes. CI > 1 indicates antagonistic effect, CI = 1 indicates additive effect, and CI < 1 indicates synergistic effect.

[0079] The calculation results of the above packaging efficiency and joint index are shown in Table 2 below:

[0080] Table 2

[0081] Table 2 shows that peroxide-containing liposomes can encapsulate different active pharmaceutical ingredients, achieving an encapsulation rate of 68.38% for atorvastatin. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) PCPO It showed synergistic effects with different drugs, among which the peroxide-containing liposomes loaded with artesunate showed the best synergistic killing effect on tumor cells.

[0082] Example 8

[0083] This embodiment uses artesunate and peroxide-containing liposomes (LIPs). PCPO and the peroxide-containing liposomes LIPs loaded with artesunate prepared in Example 6PCPO These were used to treat breast cancer-bearing mice, and the specific procedures were as follows:

[0084] Construction of a breast cancer animal model: 1) BALB / c female mice were anesthetized by intraperitoneal injection of 100 μL of 5% chloral hydrate saline solution; 2) 80 μL of cell suspension (4T1-R, 5×10⁻⁶ cells) was injected into the fourth pair of mammary pads of the mice. 5 3) Resuscitate anesthetized mice on a 37°C constant temperature heating pad and return them to their cages for rearing.

[0085] Administration: Tail vein injection, three times a week. Among them, the control group received 100 μL of normal saline (n=5); the artesunate group (11.7 mg / kg, n=5); the peroxide-containing liposome group (100 mg / kg, n=5); and the peroxide-containing liposome group loaded with artesunate (AS) (111.7 mg / kg, n=5).

[0086] After receiving appropriate treatment, the length (L) and width (W) of the tumor were measured every 3 days using calipers, and the tumor volume was calculated using the following formula:

[0087] The tumor growth curves of mice treated with different methods are shown in Figure 8. As can be seen from the figure, peroxide-containing liposomes and peroxide-containing liposomes loaded with artesunate can significantly inhibit tumor growth. The combined treatment resulted in a tumor size 2.5 times smaller than that of the unmodified liposome control group (AS), showing a significant synergistic therapeutic effect.

[0088] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A peroxide-containing liposome, characterized in that, Spherical vesicles with a bilayer membrane structure formed by lipid molecules containing one or more peroxy groups; the lipid molecules contain carbon-carbon double bonds.

2. The peroxide-containing liposome according to claim 1, characterized in that, The lipid molecule contains the following groups in its structure:

3. A method for preparing peroxide-containing liposomes as described in claim 1 or 2, characterized in that, Includes the following steps: S1. Unsaturated lipid molecules are reacted with additives in the presence of oxygen and solvent to obtain lipid molecules containing peroxy groups; the unsaturated lipid molecules contain -CH=CH- and / or -C≡C-; the additives are selected from one or more of lipoxygenases, oxidation catalysts, and oxidants; S2. The lipid molecules containing peroxide groups prepared in S1 are mixed with an aqueous system to obtain the peroxide-containing liposomes.

4. The preparation method according to claim 3, characterized in that, In S1, the ratio of the molar amount of the unsaturated lipid molecules to the volume of the solvent is (1 μmol - 1 mol): 1 L; The unsaturated lipid molecules are 1,2-dilinoleoyl-tin-glycero-3-phosphocholine, 1,2-dilinoleoyl-sn-glycero-3-phospho-L-serine, 1,3-linoleic acid diglyceride, or 1,2-dioleoyl lecithin. The solvent is deionized water, physiological saline, PBS buffer solution, or cell culture medium.

5. The preparation method according to claim 3, characterized in that, In S1, the oxidation catalyst is selected from one or more of MoS2 nanosheets, GO nanosheets, and Fe / N co-doped graphene nanosheets; The oxidant is cumene hydroperoxide and / or hydrogen peroxide.

6. The preparation method according to claim 3, characterized in that, In S1, when the additive is lipoxygenase, the reaction concentration of the lipoxygenase is 1×10⁻⁶. 4 -1×10 6 U / mL; When the additive is an oxidation catalyst, the reaction concentration of the oxidation catalyst is 0.001-5 mg / mL; When the additive is an oxidant, the reaction concentration of the oxidant is 1 μM-1 mM.

7. The preparation method according to claim 3, characterized in that, In S1, the reaction temperature is 4-300℃ and the reaction time is 0.1 hours-7 days.

8. The preparation method according to claim 3, characterized in that, In S2, the mixed processing method is ultrasonic processing; the power of ultrasonic processing is 10-100W, and the time is 10-60s.

9. An antitumor drug, characterized in that, It includes the peroxide-containing liposomes as described in claim 1 or 2, or the peroxide-containing liposomes prepared by the preparation method described in any one of claims 3-8.

10. The antitumor drug according to claim 9, characterized in that, The peroxide-containing liposomes serve as carriers to encapsulate the active pharmaceutical ingredient.

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