Free radical capture type phospholipid-like material and liposome and application thereof

Abstract

The invention relates to the technical field of biological medicines, and discloses a free radical capture type phospholipid-like material, which has the following structural general formula: sn-1 and sn-2 positions are ester bonds; the hydrophilic group at the head of the phospholipid is phosphatidylcholine, R1 is an aliphatic chain and R2 is a free radical capture group, or R1 is a free radical capture group and R2 is an aliphatic chain. The invention also discloses a free radical capture type liposome for reducing the cardiotoxicity of doxorubicin. The free radical capture type liposome comprises the free radical capture type phospholipid-like material, phospholipid, cholesterol, phospholipid-polyethylene glycol, a pH regulating molecule, an auxiliary drug-loading molecule, an isotonic regulating molecule and an active drug. The liposome can be used for preparing medicines for treating tumors such as bladder cancer, breast cancer and ovarian cancer, the cardiotoxicity of adriamycin is remarkably reduced while the anticancer efficacy of adriamycin is ensured, and the liposome has good clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to free radical scavenging phospholipid materials, their liposomes, and applications. Background Technology

[0002] Doxorubicin, a representative anthracycline chemotherapy drug, has become one of the core drugs for the clinical treatment of solid tumors (such as breast cancer and ovarian cancer) and hematological malignancies (such as leukemia and lymphoma) due to its broad-spectrum anticancer activity. Its anticancer mechanism is clear: on the one hand, it directly blocks DNA replication and cancer cell division by embedding itself into the DNA double helix and inhibiting topoisomerase II activity; on the other hand, it generates free radicals through pathways such as chelating iron ions and reducing quinone groups, damaging the DNA, proteins, and cell membrane structure of cancer cells, thereby efficiently killing tumor cells. However, the clinical application of doxorubicin is significantly limited by its severe dose-dependent cardiotoxicity—the cumulative dose that patients can tolerate is only 550 mg / m². 2 Exceeding this dosage significantly increases the risk of myocardial damage and may even lead to irreversible heart failure, which becomes a key bottleneck restricting its efficacy and the improvement of patients' quality of life.

[0003] The core mechanism of doxorubicin cardiotoxicity has been confirmed to be closely related to cardiomyocyte ferroptosis. A 2019 study explicitly proposed that doxorubicin-induced cardiomyocyte ferroptosis is the core driving factor of its cardiotoxicity. Specifically, the process involves: the SLC22A3 transporter on the surface of cardiomyocytes specifically promoting doxorubicin uptake, leading to an abnormally high intracellular drug concentration; doxorubicin further upregulates the expression of heme oxygenase-1 in cardiomyocytes, which generates a large amount of free iron ions by degrading heme; these free iron ions induce a lipid peroxidation chain reaction via the Fenton reaction, ultimately leading to cardiomyocyte ferroptosis and irreversible cardiac damage. Elucidating this mechanism provides a clear targeted intervention direction for alleviating doxorubicin cardiotoxicity—inhibiting cardiomyocyte ferroptosis.

[0004] Current interventions for doxorubicin cardiotoxicity have significant limitations and fail to meet clinical needs: ① Iron chelating agents: Represented by dextromethorphan (a clinically approved drug) and deferoxone, these block the initiation of ferroptosis by chelating free iron ions. While dextromethorphan can reduce cardiotoxicity, it antagonizes the anticancer efficacy of doxorubicin; deferoxone, although it can improve myocardial function, has insufficient targeting of cardiomyocytes, and systemic exposure easily leads to side effects such as iron metabolism disorders. ② Enzyme activators: Such as selenium-enriched Cordyceps militaris, which inhibits lipid peroxidation by activating glutathione peroxidase 4. However, these natural products have complex compositions, poor batch-to-batch stability, and activation efficiency is significantly affected by individual metabolic differences, making clinical translation difficult. ③ Free radical scavengers: Represented by Fer-1, which can directly inhibit the lipid peroxidation chain reaction. However, its small molecular structure leads to poor metabolic stability, limiting its in vivo application. Therefore, in order to address the clinical challenge of doxorubicin cardiotoxicity, there is an urgent need to develop a novel intervention formulation that combines targeted inhibition of cardiomyocyte ferroptosis, high metabolic stability, and no antagonistic effect against anticancer drugs, in order to overcome existing technological bottlenecks and expand the clinical application value of doxorubicin. Summary of the Invention

[0005] The purpose of this invention is to provide free radical scavenging phospholipid materials, their liposomes, and applications. These liposomes possess free radical scavenging and ferroptosis inhibition capabilities, efficient doxorubicin loading capacity, high stability, and long cycling properties.

[0006] To achieve the above objectives, the present invention provides a free radical scavenging phospholipid-like material having the following general structural formula:

[0007]

[0008] Both sn-1 and sn-2 positions are ester bonds, and both sn-1 and sn-2 positions are R1 and R2 connecting bonds to glycerol;

[0009] The hydrophilic group at the head of the phospholipid is phosphatidylcholine, R1 is an aliphatic chain, and R2 is a free radical scavenging group, or R1 is a free radical scavenging group and R2 is an aliphatic chain.

[0010] Furthermore, in the free-capture phospholipid material, the aliphatic chain is a saturated aliphatic chain with 6-26 carbon atoms, preferably 14-22 carbon atoms, more preferably 16-18 carbon atoms, and most preferably 18 carbon atoms (the specific structure is shown in the figure below):

[0011]

[0012] Furthermore, in the free radical scavenging phospholipid material, the free radical scavenging groups are Ferrostatin-1 / Fer-1, SRS11-92 (AA9), phenoxazine Pnx and its derivatives, preferably Pnx and its derivatives, more preferably Pnx derivatives (18:0-Pnx-C2 PC, 18:0-Pnx-C4 PC, 18:0-Pnx-C6 PC), and most preferably Pnx derivative (18:0-Pnx-C6 PC). The specific structure of the phospholipid material is shown in the figure below:

[0013]

[0014] The present invention also provides the application of a free radical scavenging phospholipid material in the preparation of a liposomal formulation for reducing the cardiotoxicity of doxorubicin, wherein the free radical scavenging phospholipid material is the aforementioned free radical scavenging phospholipid material.

[0015] The present invention also provides a free radical scavenging liposome for reducing the cardiotoxicity of doxorubicin, comprising the following components: the above-mentioned free radical scavenging phospholipid material, phospholipid, cholesterol, phospholipid-polyethylene glycol, pH adjusting molecule, drug-carrying molecule, isotropic adjusting molecule, and active drug.

[0016] Furthermore, in the liposomes, the molar ratio of free radical scavenging phospholipid material is 0.5-30%, preferably 1-20%, more preferably 1-10%, and most preferably 2-5%; the molar ratio of phospholipid is 25-60%, preferably 35-56%, more preferably 45-56%, and more preferably 53-56%; the molar ratio of cholesterol is 33-40%, preferably 35-40%, and more preferably 37-40%; and the molar ratio of DSPE-mPEG 2000 is 3-10%, preferably 4-8%, and more preferably 5-6%.

[0017] Furthermore, the phospholipid is one of dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearyl phosphatidylcholine (DSPC), hydrogenated phosphatidylcholine (HSPC), dioleoyl phosphatidylcholine (DOPC), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), and dioleoyl phosphatidylethanolamine (DOPE), preferably DSPC and HSPC, more preferably HSPC.

[0018] Furthermore, the phospholipid-polyethylene glycol is distearate phosphatidylethanolamine-polyethylene glycol 2000, the pH adjusting molecule is hydrochloric acid, sodium hydroxide and histidine, the auxiliary drug-carrying molecule is ammonium sulfate, and the isotonic adjusting molecule is sucrose.

[0019] Furthermore, the free radical scavenging liposome for reducing the cardiotoxicity of doxorubicin is loaded with anthracycline chemotherapy drugs, preferably doxorubicin and its salts, more preferably doxorubicin hydrochloride, and the drug loading method is active drug loading or passive drug loading, preferably active drug loading, more preferably ammonium sulfate gradient method.

[0020] The above-mentioned method for preparing free radical scavenging liposomes for reducing doxorubicin cardiotoxicity includes thin film hydration-extrusion, microfluidic method, solvent injection method, reverse evaporation method, or double emulsion method.

[0021] Furthermore, the present invention also provides the application of free radical scavenging liposomes for reducing the cardiotoxicity of doxorubicin in the preparation of a medicament for treating tumors, wherein the medicament is administered by intravenous infusion; the tumors include bladder cancer, breast cancer, ovarian cancer, thyroid cancer, Kaposi's sarcoma, leukemia, lymphoma, osteosarcoma, pediatric tumors, preferably metastatic breast cancer, advanced ovarian cancer, multiple myeloma, and Kaposi's sarcoma.

[0022] Therefore, the advantages and positive effects of the free radical scavenging liposome for mitigating doxorubicin cardiotoxicity provided by this invention are:

[0023] 1. The free radical scavenging liposomes provided by this invention for reducing doxorubicin cardiotoxicity contain free radical scavenging phospholipid materials. Their free radical scavenging active groups can effectively inhibit lipid peroxidation and ferrodeation, thereby reducing doxorubicin cardiotoxicity from a mechanistic perspective. At the same time, the material has a phospholipid-like structure and has a high affinity for phospholipids and cholesterol in traditional liposome formulations, enabling it to be efficiently loaded into liposomes.

[0024] 2. This liposome possesses free radical scavenging and ferroptosis inhibition capabilities, highly efficient doxorubicin loading capacity, high stability, and long circulation characteristics. It can be used to prepare drugs for treating tumors such as bladder cancer, breast cancer, and ovarian cancer, significantly reducing cardiotoxicity while maintaining the anticancer efficacy of doxorubicin, demonstrating good clinical application value.

[0025] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1The hydrodynamic particle size stability (n=3) of the three free radical capturing blank liposomes in Example 7 is shown. A is a blank liposome loaded with 18:0-Fer PC: Fer LP (20%), B is a blank liposome loaded with 18:0-AA9 PC: AA9 LP (20%), and C is a blank liposome loaded with 18:0-Pnx PC: Pnx LP (2%).

[0028] Figure 2 The efficacy analysis of three free radical scavenging phospholipids in Example 8 for inhibiting lipid peroxidation is as follows: A is blank liposomes loaded with 18:0-Fer PC: Fer LP (20%), B is blank liposomes loaded with 18:0-AA9 PC: AA9 LP (20%), and C is blank liposomes loaded with 18:0-Pnx PC: Pnx LP (2%). The data are expressed as mean ± standard error (n=3).

[0029] Figure 3 For the performance analysis of three free radical scavenging blank liposomes in Example 9 in reducing doxorubicin hydrochloride (10 μM)-induced H9c2 ferroptosis in rat cardiomyocytes, A is blank liposome loaded with 18:0-Fer PC: Fer LP (20%), B is blank liposome loaded with 18:0-AA9 PC: AA9 LP (20%), and C is blank liposome loaded with 18:0-Pnx PC: Pnx LP (2%). Data are expressed as mean ± standard error (n = 6), ns indicates no statistical difference, ** indicates p < 0.01, and *** indicates p < 0.001.

[0030] Figure 4This document describes the qualitative and quantitative analysis of ferroptosis markers in rat cardiomyocytes H9c2 after treatment with various doxorubicin preparations in Example 10. A represents the relative content of (reduced) glutathione, B represents the relative content of reduced thioredoxin, C represents the relative content of total thiols, D represents the relative content of malondialdehyde, E represents the relative content of 4-hydroxynonanal, F represents the quantitative fluorescence analysis of lipid peroxides, G represents the imaging of lipid peroxides by the fluorescent probe Liperfluo (excitation / emission wavelength: 488nm / 515-650nm, scale bar: 40μm), Ctl represents the negative control group (no drug / preparation group), Dox·HCl represents doxorubicin hydrochloride, Doxil represents commercially available doxorubicin hydrochloride liposomes, Doxil+Fer LP (20%) represents the mixed administration group of Doxil and blank liposome Fer LP (20%), and Doxil+AA9 represents the mixed administration group. LP (20%): Doxil mixed with blank liposome AAP9LP (20%), Dox@Fer LP (5%), Dox@AAP9 LP (5%) and Dox@Pnx LP (2%): Three radical scavenging liposomes loaded with doxorubicin hydrochloride, Fer LP (20%), AA9 LP (20%), Dox@Fer LP (5%), Dox@AAP9 LP (5%) and Dox@Pnx LP (2%). Detailed information is shown in Tables 1, 2, 4, 5 and 6, respectively. The doxorubicin hydrochloride dose was fixed at 10 μM. Data are expressed as mean ± standard error (n = 3-6). * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.

[0031] Figure 5This is a qualitative and quantitative analysis of cardiac ultrasound parameters in BALB / c mice after 14 days of administration of various doxorubicin preparations in Example 11. A represents the echocardiogram of BALB / c mice after treatment; B represents the left ventricular ejection fraction after administration of various preparations; C represents the fractional shortening; D represents the left ventricular end-diastolic diameter; E represents the left ventricular end-diastolic diameter; F represents the left ventricular end-diastolic volume; G represents the left ventricular end-diastolic volume; Saline represents the saline group (negative control group); Dox·HCl represents doxorubicin hydrochloride; Doxil represents commercially available doxorubicin hydrochloride liposomes; Doxil+Fer LP (20%) represents the mixed administration group of Doxil and blank liposome Fer LP (20%); Doxil+AA9 LP (20%) represents the mixed administration group of Doxil and blank liposome AAP9LP (20%); Dox@Pnx represents the mixed administration group of Doxil and blank liposome AAP9LP (20%). LP (2%): Detailed information on free radical scavenging liposomes loaded with doxorubicin hydrochloride, FerLP (20%), AA9 LP (20%) and Dox@Pnx LP (2%) are shown in Tables 1, 2 and 6, respectively. The doxorubicin hydrochloride dose was fixed at 20 mg / kg. Data are expressed as mean ± standard error (n = 3-6), ns indicates no statistical difference, and *** indicates p < 0.001.

[0032] Figure 6 This study aimed to quantitatively analyze cardiac injury markers in the serum of BALB / c mice 14 days after administration of various doxorubicin preparations. In the table, A represents the relative content of aspartate aminotransferase (AST) in the serum of BALB / c mice after administration of various preparations; B represents the relative content of cardiac natriuretic peptide (CAT); C represents the relative content of brain natriuretic peptide (BNP); and D represents the relative content of lactate dehydrogenase (LD). Saline was the saline group (negative control group). Dox·HCl was doxorubicin hydrochloride. Doxil was a commercially available doxorubicin hydrochloride liposome. Doxil+FerLP (20%) was a mixture of Doxil and blank liposome Fer LP (20%). Doxil+AA9 LP (20%) was a mixture of Doxil and blank liposome AAP9 LP (20%). Dox@Pnx LP (2%) was a free radical scavenging liposome loaded with doxorubicin hydrochloride. Fer LP (20%), AA9 LP (20%), and Dox@Pnx were also included in the drug description. Detailed information on LP (2%) is shown in Tables 1, 2 and 6. The dose of doxorubicin hydrochloride was fixed at 20 mg / kg. Data are expressed as mean ± standard error (n = 3-6), and *** indicates p < 0.001.

[0033] Figure 7This study aimed to quantitatively analyze ferroptosis-related indicators in the heart tissue of BALB / c mice 14 days after administration of various doxorubicin preparations (n=3). In this study, A represents the relative content of reduced glutathione in the heart tissue of BALB / c mice after administration of various preparations; B represents the relative content of malondialdehyde (MDA); C represents the relative content of prostaglandin intraperoxidase 2 (PI2); D represents the relative content of heme oxygenase-1 (HIO2); E represents the relative content of heme; F represents the relative content of non-heme iron; Salin represents the saline group (negative control group); Dox·HCl represents doxorubicin hydrochloride; Doxil represents commercially available doxorubicin hydrochloride liposomes; Doxil+Fer LP (20%) represents the mixed administration group of Doxil and blank liposome Fer LP (20%); Doxil+AA9LP (20%) represents the mixed administration group of Doxil and blank liposome AAP9 LP (20%); and Dox@Pnx LP (2%) represents free radical scavenging liposomes loaded with doxorubicin hydrochloride. Detailed information on LP (20%), AA9 LP (20%), and Dox@Pnx LP (2%) is shown in Tables 1, 2, and 6, respectively. The doxorubicin hydrochloride dose was fixed at 20 mg / kg. Data are expressed as mean ± standard error (n = 3-6), ns indicates no statistical difference, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0034] Figure 8 For the morphological analysis of BALB / c mouse heart tissue 14 days after administration of various doxorubicin preparations, representative H&E staining and Masson staining images of BALB / c mouse heart tissue after administration of various preparations are shown (scale bar: 100 μm). Saline: saline group (negative control group), Dox·HCl: doxorubicin hydrochloride, Doxil: commercial doxorubicin hydrochloride liposomes, Doxil+Fer LP (20%): mixed administration group of Doxil and blank liposome Fer LP (20%), Doxil+AA9LP (20%): mixed administration group of Doxil and blank liposome AAP9 LP (20%), Dox@Pnx LP (2%): free radical scavenging liposomes loaded with doxorubicin hydrochloride. Detailed information on Fer LP (20%), AA9 LP (20%) and Dox@Pnx LP (2%) is shown in Tables 1, 2 and 6, respectively. The doxorubicin hydrochloride dose was fixed at 20 mg / kg. Detailed Implementation

[0035] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0036] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0037] The instruments, equipment, reagents and materials used in the embodiments were all obtained through commercial means; the methods and steps not described in detail in the embodiments are all conventional techniques in the art.

[0038] Example 1

[0039] This embodiment illustrates how to prepare a free radical scavenging blank liposome loaded with a phospholipid-like material (18:0-Fer PC), and characterizes its particle size, drug loading, and encapsulation efficiency.

[0040] Weigh 2.03 mg of 18:0-Fer PC, 9.86 mg of HSPC, 4.00 mg of CHOL (cholesterol), and 4.00 mg of DSPE-mPEG2000 (molar ratio 18:0-Fer PC:HSPC:CHOL:DSPE-mPEG2000 = 10:47.2:37.5:5.3) into a 25 mL round-bottom flask. Add 5 mL of anhydrous ethanol and heat in a 60 °C water bath until dissolved. Remove the anhydrous ethanol by rotary evaporation under reduced pressure at 60 °C for 30 min to form a film. Add 5 mL of ultrapure water and hydrate in a 60 °C water bath for 20 min, followed by intermittent sonication for 5 min. Then, extrude the film using a liposome extruder (200 nm, 100 nm) and finally purify it using a dextran gel column (NWDexG-25F) to obtain blank liposomes FerLP (10%).

[0041] Weigh 4.06 mg of 18:0-Fer PC, 7.77 mg of HSPC, 4.00 mg of CHOL, and 4.00 mg of DSPE-mPEG2000 (molar ratio 18:0-Fer PC:HSPC:CHOL:DSPE-mPEG2000 = 20:37.2:37.5:5.3) into a 25 mL round-bottom flask. Add 5 mL of anhydrous ethanol and heat in a 60 °C water bath until dissolved. Remove the anhydrous ethanol by rotary evaporation under reduced pressure at 60 °C for 30 min to form a film. Add 5 mL of ultrapure water and hydrate in a 60 °C water bath for 20 min, followed by intermittent sonication for 5 min. Then, extrude the film using a liposome extruder (200 nm, 100 nm) and finally purify it using a dextran gel column (NWDexG-25F) to obtain blank liposomes FerLP (20%).

[0042] Weigh 6.09 mg of 18:0-Fer PC, 5.68 mg of HSPC, 4.00 mg of CHOL, and 4.00 mg of DSPE-mPEG2000 (molar ratio 18:0-FerPC:HSPC:CHOL:DSPE-mPEG2000 = 30:27.2:37.5:5.3) into a 25 mL round-bottom flask. Add 5 mL of anhydrous ethanol and heat in a 60 °C water bath until dissolved. Remove the anhydrous ethanol by rotary evaporation under reduced pressure at 60 °C for 30 min to form a film. Add 5 mL of ultrapure water and hydrate in a 60 °C water bath for 20 min, followed by intermittent sonication for 5 min. Then, extrude the film using a liposome extruder (200 nm, 100 nm) and finally purify it using a dextran gel column (NWDexG-25F) to obtain blank liposome Fer LP (30%).

[0043] The blank liposomes were diluted 10-fold, and their hydrodynamic particle size was measured at room temperature using dynamic light scattering. After freeze-drying, the drug loading and encapsulation efficiency of the 18:0-Fer PC were measured by high-performance liquid chromatography. The results are shown in Table 1.

[0044] Table 1.18: 0-Fer PC drug loading, encapsulation efficiency, hydrodynamic particle size and polydispersity index

[0045]

[0046] Experimental results show that as the molar ratio of 18:0-Fer PC increases from 10% to 30%, the amount of 18:0-Fer PC loaded in the resulting liposomes increases, the encapsulation efficiency decreases, and the hydrodynamic particle size increases, but the polydispersity index of the liposomes does not change significantly.

[0047] Example 2

[0048] This embodiment illustrates how to prepare a free radical scavenging blank liposome loaded with a phospholipid-like material (18:0-AA9 PC), and characterizes its particle size, drug loading, and encapsulation efficiency.

[0049] Weigh 2.28 mg of 18:0-AA9 PC, 9.86 mg of HSPC, 4.00 mg of CHOL, and 4.00 mg of DSPE-mPEG2000 (molar ratio 18:0-AA9 PC:HSPC:CHOL:DSPE-mPEG2000 = 10:47.2:37.5:5.3) into a 25 mL round-bottom flask. Add 5 mL of anhydrous ethanol and heat in a 60 °C water bath until dissolved. Remove the anhydrous ethanol by rotary evaporation under reduced pressure at 60 °C for 30 min to form a film. Add 5 mL of ultrapure water and hydrate in a 60 °C water bath for 20 min, followed by intermittent sonication for 5 min. Then, extrude the film using a liposome extruder (200 nm, 100 nm) and finally purify it using a dextran gel column (NWDexG-25F) to obtain blank AA9 LP liposomes (10%).

[0050] Weigh 4.55 mg of 18:0-AA9 PC, 7.77 mg of HSPC, 4.00 mg of CHOL, and 4.00 mg of DSPE-mPEG2000 (molar ratio 18:0-AA9 PC:HSPC:CHOL:DSPE-mPEG2000 = 20:37.2:37.5:5.3) into a 25 mL round-bottom flask. Add 5 mL of anhydrous ethanol and heat in a 60 °C water bath until dissolved. Remove the anhydrous ethanol by rotary evaporation under reduced pressure at 60 °C for 30 min to form a film. Add 5 mL of ultrapure water and hydrate in a 60 °C water bath for 20 min, followed by intermittent sonication for 5 min. Then, extrude the film using a liposome extruder (200 nm, 100 nm) and finally purify it using a dextran gel column (NWDexG-25F) to obtain blank AA9 LP (20%) liposomes.

[0051] Weigh 6.83 mg of 18:0-AA9 PC, 5.68 mg of HSPC, 4.00 mg of CHOL, and 4.00 mg of DSPE-mPEG2000 (molar ratio 18:0-AA9PC:HSPC:CHOL:DSPE-mPEG2000 = 30:27.2:37.5:5.3) into a 25 mL round-bottom flask. Add 5 mL of anhydrous ethanol and heat in a 60 °C water bath until dissolved. Remove the anhydrous ethanol by rotary evaporation under reduced pressure at 60 °C for 30 min to form a film. Add 5 mL of ultrapure water and hydrate in a 60 °C water bath for 20 min, followed by intermittent sonication for 5 min. Then, extrude the film using a liposome extruder (200 nm, 100 nm) and finally purify it using a dextran gel column (NWDexG-25F) to obtain blank AA9 LP (30%).

[0052] The blank liposomes were diluted 10-fold and their hydrodynamic particle size was measured at room temperature. After freeze-drying, the drug loading and encapsulation efficiency of 18:0-AA9 PC were measured by high-performance liquid chromatography. The results are shown in Table 2.

[0053] Table 2.18: Drug loading, encapsulation efficiency, hydrodynamic particle size and polydispersity index of 0-AA9 PC

[0054]

[0055] Experimental results showed that as the molar ratio of 18:0-AA9 PC increased from 10% to 30%, the amount of 18:0-AA9 PC loaded in the resulting liposomes increased, the encapsulation efficiency decreased, and the hydrodynamic particle size and polydispersity index did not change significantly.

[0056] Example 3

[0057] This embodiment illustrates how to prepare a free radical scavenging blank liposome loaded with a phospholipid-like material (18:0-Pnx PC), and characterizes its particle size, drug loading, and encapsulation efficiency.

[0058] Weigh 0.40 mg of 18:0-Pnx PC, 11.60 mg of HSPC, 4.00 mg of CHOL, and 4.00 mg of DSPE-mPEG2000 (molar ratio 18:0-Pnx PC:HSPC:CHOL:DSPE-mPEG2000 = 2:56:37:5) into a 25 mL round-bottom flask. Add 5 mL of anhydrous ethanol and heat in a 60 °C water bath until dissolved. Remove the anhydrous ethanol by rotary evaporation under reduced pressure at 60 °C for 30 min to form a film. Add 5 mL of ultrapure water and hydrate in a 60 °C water bath for 20 min, followed by intermittent sonication for 5 min. Then, extrude the film using a liposome extruder (200 nm, 100 nm) and finally purify it using a dextran gel column (NWDex G-25F) to obtain blank liposome Pnx LP (2%).

[0059] Weigh 1.00 mg of 18:0-Pnx PC, 11.00 mg of HSPC, 4.00 mg of CHOL, and 4.00 mg of DSPE-mPEG2000 (molar ratio 18:0-Pnx PC:HSPC:CHOL:DSPE-mPEG2000 = 5:53:37:5) into a 25 mL round-bottom flask. Add 5 mL of anhydrous ethanol and heat in a 60 °C water bath until dissolved. Remove the anhydrous ethanol by rotary evaporation under reduced pressure at 60 °C for 30 min to form a film. Add 5 mL of ultrapure water and hydrate in a 60 °C water bath for 20 min, followed by intermittent sonication for 5 min. Then, extrude the film using a liposome extruder (200 nm, 100 nm) and finally purify it using a dextran gel column (NWDex G-25F) to obtain blank liposome Pnx LP (5%).

[0060] Weigh 2.00 mg of 18:0-Pnx PC, 10.00 mg of HSPC, 4.00 mg of CHOL, and 4.00 mg of DSPE-mPEG2000 (molar ratio 18:0-Pnx PC:HSPC:CHOL:DSPE-mPEG2000 = 10:48:37:5) into a 25 mL round-bottom flask. Add 5 mL of anhydrous ethanol and heat in a 60 °C water bath until dissolved. Remove the anhydrous ethanol by rotary evaporation under reduced pressure at 60 °C for 30 min to form a film. Add 5 mL of ultrapure water and hydrate in a 60 °C water bath for 20 min, followed by intermittent sonication for 5 min. Then, extrude the film using a liposome extruder (200 nm, 100 nm) and finally purify it using a dextran gel column (NWDex G-25F) to obtain blank liposome Pnx LP (10%).

[0061] The blank liposomes were diluted 10-fold and their hydrodynamic particle size was measured at room temperature. After freeze-drying, the drug loading and encapsulation efficiency of 18:0-Pnx PC were measured by high-performance liquid chromatography. The results are shown in Table 3.

[0062] Table 3.18: 0-Pnx PC drug loading, encapsulation efficiency, hydrodynamic particle size and polydispersity index

[0063]

[0064] Experimental results show that as the molar ratio of 18:0-Pnx PC increases from 2% to 10%, the amount of 18:0-Pnx PC loaded in the resulting liposomes increases, the encapsulation efficiency is high, and the hydrodynamic particle size and polydispersity index do not change significantly.

[0065] Example 4

[0066] This embodiment illustrates how to prepare free radical scavenging doxorubicin liposomes containing the phospholipid-like material 18:0-Pnx PC, and characterizes their particle size, drug loading, and encapsulation efficiency.

[0067] First, 1.01 mg of 18:0-Fer PC, 10.91 mg of HSPC, 4.00 mg of CHOL, and 4.00 mg of DSPE-mPEG2000 (molar ratio 18:0-Fer PC:HSPC:CHOL:DSPE-mPEG2000 = 5:52.2:37.5:5.3) were weighed into a 25 mL round-bottom flask. 5 mL of anhydrous ethanol was added, and the mixture was heated in a 60 °C water bath until dissolved. The anhydrous ethanol was removed by rotary evaporation under reduced pressure at 60 °C for 30 min to form a film. 5 mL of 33 mg / mL ammonium sulfate buffer solution (pH 5.0) was added, and the mixture was hydrated in a 60 °C water bath and incubated for 20 min. The mixture was then intermittently sonicated for 5 min. The liposomes were extruded using a liposome extruder (200 nm, 100 nm) and passed through a dextran gel column (NWDexG-25F) to remove the external aqueous phase ammonium sulfate, yielding blank liposomes. 10 mmol / L histidine and 10% w / v sucrose (pH 6.8) were added to the blank liposomes at a 1:1 (v / v) ratio. An 8 mg / mL Dox·HCl aqueous solution was then added dropwise at a drug-liposome ratio of 1:8 (w / w) for active drug loading. The pH of the in vitro aqueous phase of the liposomes was adjusted to 7.0 with hydrochloric acid and sodium hydroxide solution. After incubation at room temperature for 20 min, the free Dox·HCl was removed by passing the solution through a dextran gel column (Sephadex LH-20) to obtain doxorubicin liposomes Dox@FerLP (5%).

[0068] Similarly, 2.03 mg of 18:0-Fer PC, 9.86 mg of HSPC, 4.00 mg of CHOL, and 4.00 mg of DSPE-mPEG2000 (molar ratio 18:0-FerPC:HSPC:CHOL:DSPE-mPEG2000 = 10:47.2:37.5:5.3) were weighed into a 25 mL round-bottom flask, 5 mL of anhydrous ethanol was added, and the mixture was heated in a 60 °C water bath until dissolved. The anhydrous ethanol was removed by rotary evaporation under reduced pressure at 60 °C for 30 min to form a film. Subsequent steps were the same as above to obtain doxorubicin liposomes Dox@FerLP (10%). Similarly, by changing the excipient ratio, weigh 3.04 mg of 18:0-Fer PC, 8.82 mg of HSPC, 4.00 mg of CHOL, and 4.00 mg of DSPE-mPEG2000 (molar ratio 18:0-FerPC:HSPC:CHOL:DSPE-mPEG2000 = 15:42.2:37.5:5.3), and follow the same steps to obtain doxorubicin liposomes Dox@FerLP (15%).

[0069] The doxorubicin liposomes were diluted 10-fold and their hydrodynamic particle size was measured at room temperature. The blank liposomes were freeze-dried and their drug loading and encapsulation efficiency (18:0-Fer PC and doxorubicin) were measured by high performance liquid chromatography. The results are shown in Table 4.

[0070] Table 4 18:0-Fer PC drug loading, encapsulation efficiency, hydrodynamic particle size and polydispersity index

[0071]

[0072]

[0073] Experimental results showed that as the molar ratio of 18:0-Fer PC increased from 5% to 15%, the amount of 18:0-Fer PC loaded in the resulting doxorubicin-loaded liposomes increased, but the doxorubicin loading and encapsulation efficiency both decreased. This indicates that as the content of 18:0-Fer PC increases, the stability of the liposomes decreases, resulting in leakage of the encapsulated doxorubicin and a decrease in the amount of drug loaded.

[0074] Example 5

[0075] This embodiment illustrates how to prepare free radical scavenging doxorubicin liposomes containing phospholipid-like material 18:0-AA9 PC, and characterizes their particle size, drug loading, and encapsulation efficiency.

[0076] First, weigh 1.14 mg of 18:0-AA9 PC, 10.91 mg of HSPC, 4.00 mg of CHOL, and 4.00 mg of DSPE-mPEG2000 (molar ratio 18:0-AA9 PC:HSPC:CHOL:DSPE-mPEG2000 = 5:52.2:37.5:5.3) into a 25 mL round-bottom flask. Add 5 mL of anhydrous ethanol and heat in a 60 °C water bath until dissolved. Remove the anhydrous ethanol by rotary evaporation under reduced pressure at 60 °C for 30 min to form a film. Add 5 mL of 33 mg / mL ammonium sulfate buffer solution (pH 5.0) and hydrate in a 60 °C water bath for 20 min. Incubate intermittently sonicate for 5 min. Extrude using a liposome extruder (200 nm, 100 nm) and pass through a dextran gel column (NWDexG-25F) to remove the external aqueous phase ammonium sulfate, obtaining blank liposomes. 10 mmol / L histidine and 10% w / v sucrose (pH 6.8) were added to the blank liposomes at a 1:1 (v / v) ratio. Dox·HCl aqueous solution with a concentration of 8 mg / mL was then added dropwise at a drug-liposome ratio of 1:8 (w / w) for active drug loading. The pH of the in vitro aqueous phase of the liposomes was adjusted to 7.0 with hydrochloric acid and sodium hydroxide solution. After incubation at room temperature for 20 min, the free Dox·HCl was removed by passing the solution through a dextran gel column (Sephadex LH-20) to obtain doxorubicin liposomes Dox@AA9LP (5%).

[0077] Similarly, by changing the excipient ratio, weigh 2.28 mg of 18:0-AA9 PC, 9.86 mg of HSPC, 4.00 mg of CHOL, and 4.00 mg of DSPE-mPEG2000 (molar ratio 18:0-AA9PC:HSPC:CHOL:DSPE-mPEG2000 = 10:47.2:37.5:5.3), and obtain doxorubicin liposomes Dox@AA9 LP (10%) by following the same operating steps.

[0078] The doxorubicin liposomes were diluted 10-fold and their hydrodynamic particle size was measured at room temperature. The blank liposomes were freeze-dried and their drug loading and encapsulation efficiency (18:0-AA9 PC and doxorubicin) were measured by high performance liquid chromatography. The results are shown in Table 5.

[0079] Table 5.18: Drug loading, encapsulation efficiency, hydrodynamic particle size and polydispersity index of 0-AA9 PC

[0080]

[0081] Experimental results showed that as the molar ratio of 18:0-AA9 PC increased from 5% to 10%, the drug loading of 18:0-AA9 PC in the resulting doxorubicin-loaded liposomes increased, but the drug loading and encapsulation efficiency of doxorubicin decreased. This indicates that as the content of 18:0-AA9 PC increases, the stability of the liposomes decreases, resulting in leakage of encapsulated doxorubicin and a decrease in drug loading.

[0082] Example 6

[0083] This embodiment illustrates how to prepare free radical scavenging doxorubicin liposomes containing the phospholipid-like material 18:0-Pnx PC, and characterizes their particle size, drug loading, and encapsulation efficiency.

[0084] First, weigh 0.40 mg of 18:0-Pnx PC, 11.60 mg of HSPC, 4.00 mg of CHOL, and 4.00 mg of DSPE-mPEG2000 (molar ratio 18:0-Pnx PC:HSPC:CHOL:DSPE-mPEG2000 = 2:56:37:5) into a 25 mL round-bottom flask. Add 5 mL of anhydrous ethanol and heat in a 60 °C water bath until dissolved. Remove the anhydrous ethanol by rotary evaporation under reduced pressure at 60 °C for 30 min to form a film. Add 5 mL of 33 mg / mL ammonium sulfate buffer solution (pH 5.0) and hydrate in a 60 °C water bath for 20 min. Incubate intermittently sonicate for 5 min. Extrude the liposomes using a liposome extruder (200 nm, 100 nm) and remove the ammonium sulfate from the aqueous phase using a dextran gel column (NWDexG-25F) to obtain blank liposomes. 10 mmol / L histidine and 10% w / v sucrose (pH 6.8) were added to the blank liposomes at a 1:1 (v / v) ratio. Dox·HCl aqueous solution with a concentration of 8 mg / mL was then added dropwise at a drug-liposome ratio of 1:8 (w / w) for active drug loading. The pH of the in vitro aqueous phase of the liposomes was adjusted to 7.0 with hydrochloric acid and sodium hydroxide solution. After incubation at room temperature for 20 min, the free Dox·HCl was removed by passing the solution through a dextran gel column (Sephadex LH-20) to obtain doxorubicin liposomes Dox@Pnx LP (2%).

[0085] Based on this, the excipient ratio was changed, and 1.00 mg of 18:0-Pnx PC, 11.00 mg of HSPC, 4.00 mg of CHOL and 4.00 mg of DSPE-mPEG2000 (molar ratio 18:0-PnxPC:HSPC:CHOL:DSPE-mPEG2000 = 5:53:37:5) were weighed into a 25 mL round-bottom flask. 5 mL of anhydrous ethanol was added and heated in a 60 °C water bath until dissolved. The anhydrous ethanol was removed by rotary evaporation under reduced pressure at 60 °C for 30 min to form a film. The subsequent steps were the same as above to obtain doxorubicin liposomes Dox@PnxLP (5%). Similarly, the proportion of phospholipid excipients was further increased. 2.00 mg of 18:0-Pnx PC, 10.00 mg of HSPC, 4.00 mg of CHOL, and 4.00 mg of DSPE-mPEG2000 (molar ratio 18:0-PnxPC:HSPC:CHOL:DSPE-mPEG2000 = 10:48:37:5) were weighed into a 25 mL round-bottom flask. The same subsequent steps were used to obtain doxorubicin liposomes Dox@Pnx LP (10%).

[0086] The doxorubicin liposomes were diluted 10-fold and their hydrodynamic particle size was measured at room temperature. The blank liposomes were then freeze-dried and their drug loading and encapsulation efficiency (18:0-Pnx PC and doxorubicin) were measured by high-performance liquid chromatography. The results are shown in Table 6.

[0087] Table 6.18: 0-Pnx PC drug loading, encapsulation efficiency, hydrodynamic particle size and polydispersity index

[0088]

[0089] Experimental results showed that as the molar ratio of 18:0-Pnx PC increased from 2% to 5% and 10%, the drug loading of 18:0-Pnx PC in the resulting doxorubicin-loaded liposomes increased the most, but the drug loading and encapsulation efficiency of doxorubicin decreased. This indicates that as the content of 18:0-Pnx PC increases, the stability of the liposomes decreases, resulting in leakage of encapsulated doxorubicin and a decrease in drug loading.

[0090] Example 7

[0091] This embodiment uses hydrodynamic particle size as an indicator to examine the stability of three types of free radical scavenging liposomes prepared in Examples 1-3 over 48 hours.

[0092] Three types of free radical scavenging liposomes, Fer LP (20%), AA9 LP (20%), and Pnx LP (2%), were diluted 10-fold, stored at room temperature, and their hydrodynamic particle size was measured at different time points. The results are as follows: Figure 1 As shown.

[0093] The experimental results showed that the three liposomes, Fer LP (20%), AA9 LP (20%) and Pnx LP (2%), all had good stability.

[0094] Example 8

[0095] Lipid peroxidation is a free radical-mediated chain reaction that converts lipids into hydroperoxides and other oxidation products, and is a hallmark of ferroptosis. The fluorescence-inhibited autooxidation (FENIX) assay can quantitatively determine the efficacy of compounds in inhibiting lipid peroxidation. In this example, liposomes containing the oxidation-sensitive fluorescent probe STY-BODIPY were used to investigate the free radical scavenging ability of three ferroptosis inhibitors: 18:0-Fer PC, 18:0-AA9 PC, and 18:0-Pnx PC.

[0096] The specific steps are as follows: Liposomes containing only lecithin were prepared in PBS (10 mM, pH 7.4) using a conventional membrane hydration method and extruded through a 100 nm porous membrane. The liposomes were mixed with STY-BODIPY (1 mM) in a black 96-well plate and diluted to 225 μL with PBS buffer. After shaking and mixing for 1 minute, the plate was incubated at 37°C for 5 minutes using a microplate reader. Subsequently, 25 μL of DTUN (2 mM) was added to the plate. Therefore, the final concentrations of the reagents in the reaction system were as follows: 1 mM liposomes, 1 μM STY-BODIPY, and 200 μM DTUN. The fluorescence intensity (λ) of the mixture was recorded every 2 minutes. ex / λ em =488nm / 518nm). Similarly, to determine the free radical scavenging ability of 18:0-Fer PC, 18:0-AA9 PC, and 18:0-Pnx PC, liposomes, STY-BODIPY, and different concentrations of 18:0-Fer PC, 18:0-AA9 PC, and 18:0-Pnx PC were mixed in 96-well plates and diluted to 225 μL with PBS buffer (10 mM, pH 7.4). After incubation at 37°C for 5 min, 25 μL of DTUN was added to the wells; the final concentrations in the wells were: 1.0 mM liposomes, 1.0 μM STY-BODIPY, (4, 6, or 8 μM) ferroptosis inhibitors (18:0-Fer PC, 18:0-AA9 PC, and 18:0-Pnx PC), and 200 μM DTUN. The fluorescence intensity of the mixture was recorded every 2 minutes.

[0097] The inhibition rate constant (K) is calculated using the following formula. inh ) and stoichiometric coefficient (n).

[0098] 1) Convert the experimentally measured fluorescence data (RFU) to obtain [ox-STY-BODIPY]:

[0099]

[0100] In the formula, [STY-BODIPY] is the concentration (M) of the fluorescent probe at time t=0.

[0101] 2) Using PMC as the standard to calculate the initiation rate (PMC stoichiometric coefficient n = 2):

[0102]

[0103] In the formula, n is the stoichiometric coefficient, R i [RTA] represents the initiation rate (M / s), [RTA] represents the antioxidant concentration (M), and t inh The suppression time is in seconds.

[0104] 3) Calculate the stoichiometric coefficient n using Formula 2:

[0105]

[0106] In the formula, Ri is the initiation rate (M / s), [RTA] is the antioxidant concentration (M), and t inh The suppression time is in seconds.

[0107] 4) Determine R by the intersection time and slope of the tangent lines of the inhibition and non-inhibition period curves. inh With t inh :

[0108]

[0109] In the formula, [STY-BODIPY] is the fluorescent probe concentration (M) at time t=0, Ri is the initiation rate (M / s), [RTA] is the antioxidant concentration (M), and t inh The suppression time is in seconds.

[0110] The results are as follows Figure 2 As shown in Table 7, Table 7 summarizes the experimental parameters for fluorescence-inhibited auto-oxidation (FENIX). The experimental results show that with the inhibition rate constant (K... inh Using 18:0-Pnx PC as an indicator, the free radical capture efficiencies of the three biomimetic ferroptosis inhibitors are as follows: 18:0-Pnx PC > 18:0-AA9 PC > 18:0-Fer PC.

[0111] Table 7. Summary of experimental parameters for fluorescence-inhibited auto-oxidation (FENIX)

[0112] 18:0-Fer PC 18:0-AA9 PC 18:0-Pnx PC <![CDATA[T inh (×10 4 S)]]> 1.96±0.04 1.12±0.16 1.59±0.06 n 0.87±0.02 0.41±0.06 1.34±0.43 <![CDATA[K inh (×10 4 M -1 S -1 )]]> 2.28±0.05 4.06±0.63 7.41±2.04

[0113] Example 9

[0114] In this embodiment, rat cardiomyocytes H9c2 were selected. Using cell viability as an indicator, the MTT assay was used to investigate the effect of the three types of free radical scavenging liposomes prepared in Examples 1-3 on inhibiting doxorubicin-induced H9c2 cell death, as well as the dose-dependent relationship.

[0115] The specific steps are as follows: H9c2 cells in the logarithmic growth phase were seeded into 96-well plates at a density of 4500 cells per well and cultured adherently (5% CO2, 37℃) for 24 hours. Subsequently, three types of free radical scavenging liposomes were added to the 96-well plates: Fer LP (20%), AAP9 LP (20%), and Pnx LP (2%). Specifically, the concentration of free radical scavenging phospholipid 18:0-FerPC in Fer LP (20%) was 0-10 μM; the concentration of free radical scavenging phospholipid 18:0-AA9 PC in AAP9 LP (20%) was 0-10 μM; and the concentration of free radical scavenging phospholipid 18:0-Pnx PC in Pnx LP (2%) was 0-5 μM. After drug addition, adherent culture continued. After 24 hours, the drug-containing culture medium was aspirated from each well, and 100 μL of 0.5 mg / mL MTT medium solution was added to each well. The cells were incubated for another 4 hours, then the culture medium was aspirated from each well, and 100 μL of DMSO was added to each well. The cells were then incubated in the dark at room temperature with a shaker for 20 minutes until all the purple crystals dissolved. The absorbance of the samples at 490 nm was then measured using a microplate reader. Cell viability was calculated using the following formula: Viability = (OD value of experimental group / OD value of blank control group) × 100%. Cell viability was plotted against drug concentration. The results are shown below. Figure 3 As shown.

[0116] Experimental results show that the efficacy of the three biomimetic ferroptosis inhibitors is concentration-dependent, with 18:0-PnxPC exhibiting the highest ferroptosis inhibition efficiency.

[0117] Example 10

[0118] Doxorubicin-induced cardiomyocyte iron overload and ferroptosis are important mechanisms of doxorubicin cardiotoxicity, and inhibiting cardiomyocyte ferroptosis can significantly reduce doxorubicin-induced cardiac damage. This example illustrates the efficacy of various free radical-scavenging doxorubicin liposomes in reducing doxorubicin-induced lipid peroxidation and ferroptosis in cardiomyocytes at the cellular level.

[0119] The specific steps are as follows: H9c2 cells in the logarithmic growth phase are seeded into 10cm culture dishes, with 3 × 10 cells per dish. 6Cells were cultured adherently (5% CO2, 37℃) for 24 hours. Subsequently, various doxorubicin formulations were added to the culture dishes, including Dox·HCl: doxorubicin hydrochloride; Doxil: commercial doxorubicin liposomes; Doxil+Fer LP (20%): a physical mixture of commercial doxorubicin liposomes and blank liposomes Fer LP (20%) loaded with 18:0-Fer PC; Dox@Fer LP (5%): free radical scavenging (18:0-Fer PC) liposomes loaded with doxorubicin hydrochloride; Doxil+AA9LP (20%): a physical mixture of commercial doxorubicin liposomes and blank liposomes AA9 LP (20%) loaded with 18:0-AA9 PC; Dox@AA9LP (5%): free radical scavenging (18:0-AA9 PC) liposomes loaded with doxorubicin hydrochloride; and Dox@Pnx LP (2%): free radical scavenging (18:0-Pnx) liposomes loaded with doxorubicin hydrochloride. PC liposomes were used, with doxorubicin concentration fixed at 10 μM. Ctl served as the negative control group (no drug / formulation group). The 18:0-Fer PC concentration was maintained consistent between the Doxil+Fer LP (20%) and Dox@Fer LP (5%) groups; the 18:0-AA9 PC concentration was also maintained consistent between the Doxil+AA9 LP (20%) and Dox@AA9 LP (5%) groups. Cells were collected after 24 hours and washed three times with pre-cooled PBS (0.01 M, pH 7.4). Cells were sonicated on ice for 5 minutes, followed by centrifugation (12,000 g, 10 min) to obtain the cell lysate supernatant. Glutathione, reduced thioredoxin, and total thiols were determined using the 5,5'-dithio-bis-(2-nitrobenzoic acid) colorimetric method. Malondialdehyde (MDA) content was determined using the thiobarbiturate colorimetric method, and 4-hydroxy-2-nonenal (4-HNE) content was determined using a competitive ELISA kit. H9c2 cells (100,000 cells / plate) were cultured in confocal microplates, and the aforementioned doxorubicin preparations (doxorubicin concentration fixed at 10 μM) were added to the culture plates. After 24 hours of incubation, the drugs were washed off with PBS, and then the fluorescent probe Liperfluo (5 μM) was added. After 0.5 hours of incubation, the cells were washed three times with PBS and observed under a confocal laser scanning microscope (E). x =488nm, E m =515-650nm), and finally the fluorescence intensity was quantified using ImageJ software (n=3), the results are as follows. Figure 4 As shown.

[0120] Experimental results showed that different types of free radical scavenging liposomes were effective in inhibiting Dox-induced lipid peroxidation and ferroptosis in H9c2 cells, among which 18:0-Pnx PC in Dox@Pnx LP (2%) liposomes showed superior efficacy.

[0121] Example 11

[0122] This example illustrates the efficacy of various types of doxorubicin liposomes with free radical scavenging properties in reducing doxorubicin cardiotoxicity in male BALB / c mice.

[0123] Male BALB / c mice aged 8-10 weeks were randomly divided into 6 groups, including saline (Saline), free doxorubicin (Dox·HCl), control liposome Doxil, a mixture of Doxil and blank liposome Fer1 LP (20%) (Doxil+Fer LP (20%)), a mixture of Doxil and blank liposome AA9 LP (20%) (Doxil+AA9 LP (20%)), and doxorubicin liposome Dox@Pnx LP (2%), with 6 mice in each group. Mice in each group were administered doxorubicin via tail vein every other day, with a dose of 5 mg / kg per administration, for a total of four administrations, resulting in a cumulative dose of 20 mg / kg. In the mixed liposomes, the single dose of 18:0-Fer PC in Fer LP (20%) was 4 mg / kg, administered four times, for a cumulative dose of 16 mg / kg. Similarly, in AAP9 LP (20%), the single dose of 18:0-AA9 PC was 4 mg / kg, administered four times for a cumulative dose of 16 mg / kg.

[0124] (1) Using mouse echocardiography as an indicator, the ability of various free radical-scavenging doxorubicin liposomes to reduce doxorubicin cardiotoxicity was evaluated. The specific steps were as follows: On day 14 after administration, echocardiographic data were collected using two-dimensional targeted M-mode imaging. Left ventricular ejection fraction, fractional shortening, left ventricular end-diastolic and end-systolic diameters, as well as left ventricular end-diastolic and end-systolic volumes, were quantitatively measured from the echocardiographic images. Results are as follows: Figure 5 As shown.

[0125] (2) The ability of various free radical-scavenging doxorubicin liposomes to reduce doxorubicin cardiotoxicity was evaluated using cardiac injury markers in mouse serum. The specific steps were as follows: On day 14 after administration, blood was collected from mice via orbital sampling. After centrifugation at room temperature, serum was obtained. The levels of aspartate aminotransferase, cardiac natriuretic peptide, brain natriuretic peptide, and lactate dehydrogenase in mouse serum were measured using a double-antibody sandwich ELISA kit. Results are as follows: Figure 6 As shown.

[0126] (3) The ability of various free radical-scavenging doxorubicin liposomes to reduce doxorubicin cardiotoxicity was evaluated using ferroptosis markers in mouse heart tissue. The specific steps were as follows: On day 14 after administration, mice were sacrificed and dissected. Fresh mouse hearts were ground and centrifuged to obtain the tissue homogenate supernatant. The contents of glutathione, reduced thioredoxin, and total thiols were determined using the 5,5'-dithio-bis-(2-nitrobenzoic acid) colorimetric method, and the MDA content was determined using the thiobarbiturate colorimetric method. The contents of prostaglandin peroxidase 2 and heme oxygenase-1 were determined using a double-antibody sandwich ELISA kit. The contents of heme and non-heme iron were determined using a colorimetric method. The results are as follows: Figure 7 As shown.

[0127] (4) The ability of various free radical-scavenging doxorubicin liposomes to reduce doxorubicin cardiotoxicity was evaluated using mouse heart tissue morphology staining as an indicator. The specific steps were as follows: On day 14 after administration, mice were sacrificed and dissected. The hearts were fixed in neutral 4% paraformaldehyde for 24 hours, dehydrated, embedded in paraffin, and serially sectioned to a thickness of 5 μm. Sections were stained with hematoxylin-eosin (HE) and subjected to routine histological examination under an optical microscope. To measure collagen deposition, selected sections were stained with Masson's staining method and subjected to routine histological examination under an optical microscope. The results are as follows: Figure 8 As shown.

[0128] Experimental results showed that different types of free radical scavenging liposomes were effective in inhibiting Dox-induced lipid peroxidation and ferroptosis in mouse models, and had the effect of reducing Dox cardiotoxicity.

[0129] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A free radical scavenging phospholipid-like material, characterized in that, It has the following general structural formula: Both sn-1 and sn-2 positions are ester bonds, and both sn-1 and sn-2 positions are R1 and R2 connecting bonds to glycerol; The hydrophilic group at the head of the phospholipid is phosphatidylcholine PC, R1 is an aliphatic chain, and R2 is a free radical scavenging group, or R1 is a free radical scavenging group and R2 is an aliphatic chain.

2. The free radical scavenging phospholipid-like material according to claim 1, characterized in that, The aliphatic chain is a saturated aliphatic chain with 6-26 carbon atoms; the free radical scavenging groups are Ferrostatin-1, SRS11-92, phenoxazine and its derivatives.

3. The application of a free radical scavenging phospholipid material in the preparation of liposomal formulations for mitigating doxorubicin cardiotoxicity, characterized in that, The free radical scavenging phospholipid material is the free radical scavenging phospholipid material as described in claim 1.

4. A free radical scavenging liposome for reducing doxorubicin cardiotoxicity, characterized in that, It comprises the following components: the free radical scavenging phospholipid material as described in claim 1, phospholipids, cholesterol, phospholipid-polyethylene glycol, pH adjusting molecules, drug-carrying auxiliary molecules, isotropic adjusting molecules, and active pharmaceutical ingredients.

5. A free radical scavenging liposome for mitigating doxorubicin cardiotoxicity according to claim 4, characterized in that, In liposomes, the molar ratio of free radical scavenging phospholipids is 0.5-30%, the molar ratio of phospholipids is 25-60%, the molar ratio of cholesterol is 33-40%, and the molar ratio of phospholipids to polyethylene glycol is 3-10%.

6. A free radical scavenging liposome for mitigating doxorubicin cardiotoxicity according to claim 5, characterized in that, In liposomes, the molar ratio of free radical scavenging phospholipids is 1-20%, the molar ratio of phospholipids is 35-56%, the molar ratio of cholesterol is 35-40%, and the molar ratio of DSPE-mPEG 2000 is 4-8%.

7. A free radical scavenging liposome for mitigating doxorubicin cardiotoxicity according to claim 4, characterized in that, The phospholipid is one of the following: dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearyl phosphatidylcholine, hydrogenated phosphatidylcholine, dioleoyl phosphatidylcholine, dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, and dioleoyl phosphatidylethanolamine.

8. A free radical scavenging liposome for mitigating doxorubicin cardiotoxicity according to claim 4, characterized in that, The phospholipid-polyethylene glycol is distearate phosphatidylethanolamine-polyethylene glycol 2000, the pH adjusting molecule is hydrochloric acid, sodium hydroxide and histidine, the auxiliary drug-carrying molecule is ammonium sulfate, and the isotonic adjusting molecule is sucrose.

9. A free radical scavenging liposome for mitigating doxorubicin cardiotoxicity according to claim 4, characterized in that, The loaded drug is an anthracycline chemotherapy drug, and the loading method is either active or passive loading.

10. The use of a free radical scavenging liposome for mitigating doxorubicin cardiotoxicity as described in any one of claims 4-9 in the preparation of a drug for treating tumors, characterized in that, The drug is administered via intravenous infusion; the tumors include bladder cancer, breast cancer, ovarian cancer, thyroid cancer, Kaposi's sarcoma, leukemia, lymphoma, osteosarcoma, and pediatric tumors.

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