7-dehydrocholesterol liposome and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to 7-dehydrocholesterol liposome and application thereof. According to the invention, a biological membrane modified liposome is constructed, and the biological membrane modified liposome comprises an ROS responsive liposome, a neutrophile granulocyte membrane and a renal tubular epithelial cell membrane; the biofilm modified liposome has an active oxygen species (ROS) responsive release function and a dual targeting capability, has the advantages of an integrated drug effect carrier, ROS triggered release, dual bionic targeting, strong target cell uptake and multi-mechanism ferroptosis resistance, and can be used for preparing the biofilm modified liposome. The compound can be used for treating ferroptosis related diseases such as acute kidney injury, myocardial ischemia-reperfusion injury and cerebral ischemia-reperfusion injury, and has wide clinical popularization value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to 7-dehydrocholesterol liposome and application thereof. BACKGROUND

[0002] Acute kidney injury (AKI) is a clinical disease characterized by rapid decline in renal function. Ischemia-reperfusion injury (IRI) is one of the most important triggers of AKI, which often occurs under conditions such as kidney transplantation and cardiovascular surgery. The core pathological mechanism of ischemia-reperfusion injury (IRI) includes the generation of a large amount of ROS during ischemia / reperfusion, the accumulation of free iron caused by iron homeostasis imbalance, and the enhancement of lipid peroxidation reaction, thereby inducing ferroptosis. At present, there is a lack of safe and effective ferroptosis inhibition drug delivery system in clinical practice.

[0003] 7-DHC, as a natural sterol, can inhibit ferroptosis by stabilizing the structure of the cell membrane and interrupting the lipid peroxidation chain reaction. However, it has strong hydrophobicity, is easily metabolized and eliminated in vivo, and lacks tissue or inflammatory lesion targeting, which seriously limits its clinical application.

[0004] Traditional liposomes as drug delivery systems have certain advantages in drug release and targeted delivery due to their good biocompatibility and encapsulation capacity. However, in pathological conditions such as I / R kidney injury, cell membrane structure disorder and endocytosis disorder can significantly reduce the uptake efficiency of ordinary liposomes, limiting their therapeutic effect. In addition, insufficient perfusion of the kidney during I / R often leads to uneven distribution of drugs and low targeting efficiency. Cell membrane biomimetics can prolong the circulation time or provide certain targeting, but lack the ability to respond to key pathological signals of AKI (such as increased ROS levels), making it difficult to achieve on-demand precise release.

[0005] Therefore, the development of a 7-DHC drug delivery carrier that can target the injury site and trigger drug release in response to ROS at the IRI site, and has a dual membrane targeting function, is expected to achieve precise inhibition of ferroptosis and significantly improve the treatment effect of AKI caused by IRI. SUMMARY

[0006] Therefore, the development of a 7-DHC drug delivery carrier that can target the injury site and trigger drug release in response to ROS at the IRI site, and has a dual membrane targeting function, is expected to achieve precise inhibition of ferroptosis and significantly improve the treatment effect of AKI caused by IRI.

[0007] The present application provides a ROS-responsive liposome, which comprises: a 7-DHC liposome and ATP.

[0008] The 7-DHC liposome is prepared from 7-dehydrocholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) and distearoylphosphatidylethanolamine-selenium-selenium-polyethylene glycol (DSPE-SeSe-PEG) in a volume ratio of (10-70):(70-10):15:5; in specific embodiments of the present application, the 7-dehydrocholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) and distearoylphosphatidylethanolamine-selenium-selenium-polyethylene glycol (DSPE-SeSe-PEG) are prepared into ROS-responsive liposomes in a molar ratio of 40:40:15:5, and the performance of the ROS-responsive liposomes is optimal.

[0009] In the ROS-responsive liposome, the concentration of ATP is 0.25 mg / mL to 1.50 mg / mL; preferably 0.75 mg / mL;

[0010] The present application has screened the specific components in the preparation of the ROS-responsive liposome and optimized the component concentration and ratio; in the present application, 7-DHC is selected as the core, then appropriate raw materials with ROS response, increased membrane permeability and triggered drug release are selected to prepare the ROS-responsive liposome; and the ratio and concentration of the raw materials are further optimized, and the experimental results show that the ROS-responsive liposome precursor prepared from DSPC, 7-DHC, DSPE-PEG2000 and DSPE-SeSe-PEG in a molar ratio of 10:70:15:5, 20:60:15:5, 30:50:15:5, 40:40:15:5, 50:30:15:5, 60:20:15:5 and 70:10:15:5, respectively, has the best stability, the particle size distribution is concentrated, and the particle size is about 110 ± 15 nm, which is suitable for drug encapsulation and delivery. On this basis, the ATP encapsulation rate is determined, and the test results show that the ROS-responsive liposome precursor has good ATP encapsulation effect, and the ATP encapsulation concentration is preferably 0.75 mg / mL.

[0011] The present application provides a drug-loaded liposome, which comprises the ROS-responsive liposome and a drug for treating and / or preventing an iron death-related disease.

[0012] The drug for treating and / or preventing an iron death-related disease comprises at least one of an iron metabolism pathway inhibitor, a non-iron chelating agent or an antioxidant.

[0013] Further, the ferroptosis inhibitor includes Deferoxamine (DFO), Vitamin K, Deferiprone (DFP), Deferasirox (DFX) and / or CN128, DFA1;

[0014] The non-iron chelator includes a DMT1 inhibitor.

[0015] The antioxidant includes Ferrostatin-1 (Fer-1), Liproxstatin-1 (Lip-1), N-acetylcysteine and / or Epigallocatechin gallate (EGCG).

[0016] The application provides a biological membrane modified liposome, which comprises a neutrophil membrane, a renal tubular epithelial cell membrane and at least one of the following A) and B):

[0017] A) the ROS-responsive liposome according to the application;

[0018] B) the drug-loaded liposome according to the application.

[0019] Further, the mass ratio of the neutrophil membrane and the renal tubular epithelial cell membrane to the mass of the ROS-responsive liposome according to the application is (4:1):(1:2).

[0020] The mass ratio of the neutrophil membrane and the renal tubular epithelial cell membrane is 1:1.

[0021] The application optimizes the mass ratio of the neutrophil membrane and the renal tubular epithelial cell membrane and the mass ratio of the sum of the mass of the neutrophil membrane and the renal tubular epithelial cell membrane to the mass of the ROS-responsive liposome according to the application. Experimental results show that the NEm / KCm prepared by the mass ratio of the sum of the mass of the neutrophil membrane and the renal tubular epithelial cell membrane to the mass of the ROS-responsive liposome according to the application is 4:1, 3:1 and 2:1 has good stability, and the mass ratio of 2:1 has the best liposome performance.

[0022] The application provides a preparation method of the biological membrane modified liposome, which comprises the following steps:

[0023] Step 1, 7-DHC, DSPC, DSPE-PEG2000 and DSPE-SeSe-PEG are dissolved in chloroform / methanol, and then rotary evaporation is performed to obtain a ROS-responsive liposome precursor;

[0024] Step 2, the ROS-responsive liposome precursor is mixed with ATP to obtain a ROS-responsive liposome.

[0025] Step 3, the ROS-responsive liposome is mixed with the neutrophil membrane and the renal tubular epithelial cell membrane to obtain the biological membrane modified liposome.

[0026] The application provides at least one of the following I)~III) for use in the preparation of a drug for treating and / or preventing an iron death related disease:

[0027] I), the ROS-responsive liposome according to the application;

[0028] II), the drug-loaded liposome according to the application;

[0029] II), the biological membrane modified liposome according to the application.

[0030] Further, the iron death related disease includes acute kidney injury, myocardial ischemia-reperfusion injury and / or cerebral ischemia-reperfusion injury.

[0031] The application provides a drug for treating and / or preventing an iron death related disease, and raw materials of the drug include at least one of the following i)~iii):

[0032] i), the ROS-responsive liposome according to the application;

[0033] ii), the drug-loaded liposome according to the application;

[0034] iii), the biological membrane modified liposome according to the application.

[0035] Further, the drug according to the application further includes a pharmaceutically acceptable auxiliary;

[0036] The pharmaceutically acceptable auxiliary includes at least one of a solvent, a solubilizer, a cosolvent, an emulsifier, a disintegrant, a stabilizer, a plasticizer, a penetration enhancer and / or a sustained release agent or a combination of two or more thereof.

[0037] The pharmaceutically acceptable auxiliary includes at least one of a solvent, a solubilizer, a cosolvent, an emulsifier, a disintegrant, a stabilizer, a plasticizer, a penetration enhancer and / or a sustained release agent or a combination of two or more thereof.

[0038] The solvent includes water or a buffer; the solubilizer includes a surfactant and / or a cyclodextrin;

[0039] The cosolvent includes an organic acid and a sodium salt thereof, an amide compound, an inorganic salt and / or a cyclodextrin and a derivative thereof;

[0040] The emulsifier includes a surfactant, a polysaccharide, a phospholipid, a protein and / or a polyoxyethylene fatty alcohol ether;

[0041] The disintegrants include starches, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, and microcrystalline cellulose.

[0042] The stabilizers include antioxidants, chelating agents, cyclodextrins and their derivatives and / or surfactants;

[0043] The plasticizers include polyols, organic esters, and / or natural polymer derivatives;

[0044] The penetration enhancer includes alcohols, terpenes, and / or fatty acid esters;

[0045] The sustained-release agent includes hydrophilic gel, ethyl cellulose, polyethylene, polyvinyl chloride, ethylene-vinyl acetate copolymer and / or polymethacrylate.

[0046] This invention provides a pharmaceutical combination comprising the pharmaceuticals described herein and other pharmaceuticals for the treatment and / or prevention of ferroptosis-related diseases.

[0047] Other medications used to treat and / or prevent ferroptosis-related diseases include at least one of the following: iron metabolism pathway inhibitors, non-iron chelators, or antioxidants.

[0048] Furthermore, the ferroptosis inhibitors include deferoxamine (DFO), vitamin K, deferiprone (DFP), deferasirox (DFX), and / or CN128 and DFA1.

[0049] The non-ferrous chelating agents include DMT1 inhibitors;

[0050] The antioxidants include Ferrostatin-1 (Fer-1), Liproxstatin-1 (Lip-1), N-acetylcysteine ​​and / or epigallocatechin gallate (EGCG).

[0051] This invention addresses the problems of short half-life, uneven distribution, insufficient targeting, and inability to release on demand in existing 7-DHC drug delivery systems. It provides a biomimetic 7-DHC liposome with ROS responsiveness and dual targeting function to achieve: precise localization and enrichment at IRI lesions in AKI; triggered drug release under high ROS conditions; enhanced uptake of liposomes by damaged renal tubular epithelial cells; and inhibition of ferroptosis and reduction of tissue damage through multi-mechanism synergistic action.

[0052] This invention constructs biomembrane-modified liposomes, which include ROS-responsive liposomes, neutrophil membranes, and renal tubular epithelial cell membranes. These biomembrane-modified liposomes possess both ROS-responsive release function and dual targeting capability, exhibiting advantages such as integrated drug delivery, ROS-triggered release, dual biomimetic targeting, strong target cell uptake, and multi-mechanism anti-ferroptosis. They can be used to treat ferroptosis-related diseases such as acute kidney injury, myocardial ischemia-reperfusion injury, and cerebral ischemia-reperfusion injury, and have broad clinical application value. Attached Figure Description

[0053] Figure 1 The encapsulation efficiency (EE%) of ATP in 7-D@A-Lip is shown.

[0054] Figure 2 The particle size of 7-D@A-mLip was measured over 10 days at different phospholipid to cell membrane protein ratios.

[0055] Figure 3 The particle size distribution and transmission electron microscopy images of 7-D@A-mLip are shown.

[0056] Figure 4 The scavenging rates of 7-D@A-mLip for hydroxyl radicals (•OH) and superoxide anions (O2•⁻) at different concentrations are shown; where A is the scavenging rate of hydroxyl radicals (•OH) and B is the scavenging rate of superoxide anions (O2•⁻).

[0057] Figure 5 The confocal microscopy images show the internalization efficiency of DiD-labeled liposomes in TCMK-1 cells after 3 hours of co-incubation;

[0058] Figure 6 The study demonstrates the antiferroptosis ability of 7-D@A-mLip; where A is a confocal microscopy image of BODIPY 581 / 591 C11 fluorescence (scale bar = 20 μm); B is the quantitative analysis of MFI in each group based on the confocal microscopy image of BODIPY 581 / 591 C11 fluorescence, where Non-Ox, C11 represents unoxidized, and Ox.C11 represents oxidized; C is the cell viability of H / R-damaged TCMK-1 cells treated with 7-D@Lip, 7-D@A-Lip, or 7-D@A-mLip; D is the ROS level in H / R-damaged TCMK-1 cells treated with 7-D@Lip, 7-D@A-Lip, and 7-D@A-mLip, detected using a fluorescent probe and observed by confocal microscopy (scale bar = 50 μm).

[0059] Figure 7 The image shows the renal protective effect of 7-D@A-mLip; where A is a schematic diagram of 7-D@A-mLip treatment for I / R-induced AKI; B is a histological image of each group of kidney tissue stained with H&E (scale bar = 100 μm); C is a longitudinal section of each group of kidneys; D is the renal blood flow analysis of each group by laser Doppler flow method, with the kidneys outlined by black dashed circles.

[0060] Figure 8 H&E staining images of major organs (scale bar = 100 μm) of healthy C57BL / 6 mice after receiving intravenous injections of 7-D@Lip, 7-D@A-Lip, or 7-D@A-mLip every other day for 14 days. Detailed Implementation

[0061] This invention provides 7-dehydrocholesterol liposomes and their applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0062] MFI (mean fluorescence intensity) is an important quantitative indicator widely used in flow cytometry and fluorescence microscopy to assess the expression levels and distribution of fluorescent markers. Quantitative analysis of MFI allows for more accurate comparison of fluorescence signal changes in different samples or under different treatment conditions.

[0063] Nucleus Merge or Nucleus Merge is a merged image of the nucleus, which shows the colocalization of nuclear markers and cytoplasmic markers.

[0064] 7-DHC: 7-Dehydrocholesterol;

[0065] DSPC: 1,2-distearyl-sn-glycerol-3-phosphocholine;

[0066] DSPE-PEG2000: Distearylphosphatidylethanolamine-polyethylene glycol 2000;

[0067] DSPE-SeSe-PEG: Distearylphosphatidylethanolamine-Selenium-Selenium-polyethylene glycol;

[0068] The formulation of this invention, 7-D@A-mLip (biomembrane-modified liposome), can be administered intravenously for ROS-related diseases such as acute kidney injury, myocardial ischemia-reperfusion injury, and cerebral ischemia-reperfusion injury, which are associated with excessive ROS and ferroptosis. In a mouse model of acute kidney injury, the formulation significantly reduced malondialdehyde (MDA) and 4-HNE levels, increased the GSH / GSSG ratio, reduced iron accumulation, and significantly improved renal function indicators (Scr, BUN).

[0069] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0070] Example 1: Preparation of ROS-responsive 7-DHC liposomes (7-D@Lip)

[0071] (I) Preparation of ROS-responsive 7-DHC liposomes (7-D@Lip)

[0072] DSPC, 7-DHC, DSPE-PEG2000, and DSPE-SeSe-PEG were dissolved in a chloroform / methanol (9:1, v:v) mixture at molar ratios of 10:70:15:5, 20:60:15:5, 30:50:15:5, 40:40:15:5, 50:30:15:5, 60:20:15:5, and 70:10:15:5, respectively, to form a homogeneous organic phase. The mixture was then evaporated under reduced pressure in a rotary evaporator to form a lipid membrane. The resulting lipid membrane was hydrated with preheated PBS solution and ultrasonically dispersed to obtain uniformly sized 7-DHC liposomes (7-D@Lip).

[0073] Dynamic light scattering (DLS) results showed that the liposome particle size ranged from 100 to 200 nm. Dynamic monitoring of particle size changes over 10 days revealed significant particle size fluctuations due to excessively low or high 7-DHC ratios, unstable PDI, and uneven dispersion. Liposomes 7-D@Lip prepared from DSPC, 7-DHC, DSPE-PEG2000, and DSPE-SeSe-PEG at a molar ratio of 40:40:15:5 had an average particle size of approximately 110 ± 15 nm and a PDI < 0.25, indicating a concentrated and stable particle size distribution, as shown in Tables 1 and 2. Transmission electron microscopy (TEM) further confirmed that 7-D@Lip exhibited a typical vesicle-like morphology, suitable for drug encapsulation and delivery.

[0074] Table 1

[0075]

[0076] Table 2

[0077]

[0078] (II) Preparation of ATP-loaded 7-DHC liposomes (7-D@A-Lip)

[0079] Liposomes 7-D@Lip (liposome membranes) prepared from DSPC, 7-DHC, DSPE-PEG2000, and DSPE-SeSe-PEG at a molar ratio of 40:40:15:5 were hydrated with PBS solutions containing different concentrations of ATP. The resulting suspensions were then ultrasonically dispersed (frequency 40 kHz, power 100 W, time 5 min) to promote ATP incorporation into the liposomes. Unencapsulated free ATP was removed by dialysis to obtain ATP-loaded ROS-responsive 7-DHC liposomes (7-D@A-Lip).

[0080] The ATP content of 7-D@A-Lip was determined using an ATP assay kit, and the encapsulation efficiency (EE%) was calculated using the following formula:

[0081] EE% = (ATP content in 7-D@A-Lip / Total added ATP) × 100%

[0082] See results Figure 1 Under different initial ATP feed amounts, the encapsulation efficiency of 7-D@A-Lip varied in the range of 30% to 85%, indicating that liposomes have a strong ATP loading capacity. 0.75 mg / mL ATP was selected for the preparation of 7-D@A-Lip.

[0083] (III) Preparation and characterization of dual biomimetic membrane modified liposomes 7-D@A-mLip

[0084] 1. Neutrophil membrane (NEm) extraction: Mouse bone marrow neutrophils were isolated, and the cells were lysed by 5 freeze-thaw cycles to remove the nucleus and organelles. The purified neutrophil membrane (NEm) was obtained by ultracentrifugation (21,0000g, 30min, 4℃).

[0085] 2. Extraction of renal tubular epithelial cell membrane (KCm): Mouse renal tubular epithelial cell line TCMK-1 was cultured in DMEM (10% FBS, 37℃, 5% CO2), collected, and washed with PBS. After sonication lysis, fragments were removed by centrifugation at 12,000g for 15 min, followed by centrifugation at 210,000g for 30 min to obtain the renal tubular cell membrane (KCm).

[0086] 3. Hybrid cell membrane fusion: NEm and KCm are mixed at a mass ratio of 1:1 and a hybrid membrane NEm / KCm is formed by repeated sonication and freeze-thaw cycles.

[0087] 4. Bilayer membrane modification: The prepared 7-D@A-Lip (ATP concentration of 0.75 mg / mL) was mixed with NEm / KCm at different mass ratios (4:1, 3:1, 2:1, 1:1, 1:2), and bilayer membrane modified liposomes 7-D@A-mLip were obtained by extrusion (extruded sequentially through liposome extruders with 400 nm, 200 nm, and 100 nm filter membranes).

[0088] The results are as follows Figure 2 Dynamic light scattering (DLS) detection results and Figure 3 As shown, liposomes prepared with NEm / KCm mass ratios of 4:1, 3:1, and 2:1 had particle sizes between 100 and 150 nm for 7-D@A-mLip. Liposomes prepared with mass ratios of 1:1 and 1:2 showed increased particle sizes, exceeding 200 nm, which is unfavorable for subsequent intravenous administration. Furthermore, during 10-day dynamic monitoring, liposomes prepared with mass ratios of 1:1 and 1:2 showed significant particle size fluctuations and deposition. Liposomes prepared with NEm / KCm at mass ratios of 4:1, 3:1, and 2:1 exhibited good stability. Specifically, 7-D@A-mLip with an NEm / KCm mass ratio of 2:1 showed virtually no increase in particle size (change <10 nm) after 10 days of storage in PBS at 4°C, indicating good colloidal stability. The particle size distribution curve (…) Figure 3 The single peak shape indicates good dispersibility and optimal liposome performance.

[0089] Example 2: ROS Response Release and Clearance Experiment

[0090] The prepared 7-D@A-mLip (all components were prepared under optimal ratios) were placed in PBS solutions containing different concentrations of H2O2 (0, 50, 100, and 200 μM) and incubated at 37°C. The supernatant was collected at 1, 6, 12, and 24 hours. The release of 7-DHC was determined by high-performance liquid chromatography (HPLC), and the release of ATP was detected by fluorescence assay. Simultaneously, the scavenging capacity of different concentrations of 7-D@A-mLip against two types of reactive oxygen species was determined using a hydroxyl radical (•OH) and superoxide anion (O2•⁻) scavenging ability assay kit to evaluate its antioxidant effect.

[0091] The results showed that the release rate of 7-DHC significantly increased with increasing H2O2 concentration. Under 200 μM H2O2 conditions, the cumulative release over 24 hours was approximately 3.5 times that of the control group (0 μM H2O2), indicating that the liposomes exhibited significant ROS-triggered release characteristics. ATP release also showed a similar trend to 7-DHC, with a significant increase in release efficiency at high ROS levels. In free radical scavenging experiments, 7-D@A-mLip demonstrated good scavenging ability against both •OH and O2•⁻. Under equal-dose conditions, its free radical scavenging rate was significantly superior to that of unmodified ordinary liposomes (p<0.01). Figure 4 ).

[0092] This experiment verifies that the 7-D@A-mLip of the present invention can not only achieve on-demand release and improve drug utilization efficiency in a high ROS environment, but also synergistically clear ROS, thereby exerting the dual effect of "responsive release + antioxidant clearance" at the same time, which is of great significance for the treatment of ischemia-reperfusion injury.

[0093] Example 3 Cell uptake experiment

[0094] 7-D@Lip, 7-D@A-Lip, and 7-D@A-mLip liposomes were labeled with the hydrophobic fluorescent dye DiD. TCMK-1 renal tubular epithelial cells were treated under hypoxic-reoxygenation conditions for 12 hours, and then incubated with equal concentrations of the three formulations for another 3 hours. After incubation, the cells were washed with PBS, fixed, and the nuclei were stained with DAPI. The intensity of red fluorescence was observed and recorded using a laser confocal microscope.

[0095] Confocal microscopy revealed that the 7-D@Lip group (without ATP loading) exhibited weak red fluorescence signals within cells, suggesting limited cellular uptake efficiency under ischemic / reoxygenation injury conditions. The 7-D@A-Lip group (ATP-loaded) showed significantly enhanced fluorescence intensity, approximately 1.8 times higher than the 7-D@Lip group, indicating that ATP loading improved the energy-dependent endocytosis of liposomes in damaged cells. The 7-D@A-mLip group showed the strongest fluorescence signal, with an average fluorescence intensity approximately 2 times higher than the 7-D@A-Lip group, indicating that dual membrane modification (NEm+KCm) further enhanced the liposome's inflammatory targeting and homology recognition capabilities. Figure 5 ).

[0096] The experimental results show that the 7-D@A-mLip of the present invention can significantly enhance the uptake efficiency of damaged renal tubular cells under pathological conditions. This is achieved through the combined effects of ATP-improved energy deficiency uptake and dual-membrane modification to provide inflammation and homologous targeting, which helps the liposomes to be delivered efficiently at the lesion site of ischemia-reperfusion injury.

[0097] Example 4: Antiferrodeath Experiment

[0098] TCMK-1 cells were divided into four groups: control group, 7-D@Lip group, 7-D@A-Lip group, and 7-D@A-mLip group. Except for the control group, the other three groups were treated with Erastin (5 μM) for 24 hours to induce ferroptosis. Then, equal concentrations of the three formulations were added and incubated for 12 hours. Intracellular lipid oxidation levels were then measured using the BODIPY 581 / 591 C11 fluorescent probe, intracellular ROS levels were detected using the DCFH-DA fluorescent probe, and cell viability was assessed using the CCK-8 assay.

[0099] The results showed that Erastin treatment significantly increased lipid oxidation levels in model cells, increased ROS content by approximately 3-fold, and reduced cell viability to only about 40%. The 7-D@Lip group showed a decrease in lipid oxidation and ROS levels, with cell viability recovering to approximately 50%; the 7-D@A-Lip group showed further improvement, with a viability approaching 60%; the 7-D@A-mLip group performed best, with the lowest lipid oxidation level, a decrease in ROS content of approximately 70%, and a cell viability exceeding 80%, approaching the level of the normal control group. Figure 6 The above results demonstrate that the 7-D@A-mLip of the present invention exhibits significant anti-ferroptosis activity in vitro, manifested by reduced lipid oxidation, decreased ROS levels, and significantly improved cell viability. Compared to ordinary liposomes and liposomes loaded only with ATP, the dual-membrane modified ROS-responsive system can more effectively achieve drug delivery and therapeutic effects.

[0100] Example 5: Therapeutic effect of 7-D@A-mLip in acute kidney injury

[0101] Healthy adult male C57BL / 6 mice (weighing 22-25g, 6-8 weeks old) were randomly divided into 5 groups.

[0102] The control group underwent only open abdominal dissection of renal vessels, without ischemia.

[0103] AKI model group: A renal ischemia-reperfusion injury-induced AKI model was established, and an equal volume of normal saline was administered.

[0104] Treatment group: An AKI model was established, and 7-D@Lip, 7-D@A-Lip, and 7-D@A-mLip were administered intravenously at a dose of 5 mg / kg (calculated as 7-DHC).

[0105] The method for building the AKI model is as follows:

[0106] An AKI mouse model was established using ischemia-reperfusion injury. Mice were anesthetized with sodium pentobarbital, their back hair was shaved, and routine disinfection was performed. The skin and muscles were incised along both sides of the spine to expose the kidneys and separate the renal arteries. Both renal arteries were clamped with microvascular clamps to occlude blood flow for 30 minutes to induce ischemia. During ischemia, the mice were placed on a 37°C constant-temperature heating pad to maintain body temperature. After 30 minutes, the clamps were removed, and blood flow was restored to achieve reperfusion. The incision was then sutured layer by layer, and preheated saline was injected subcutaneously to prevent dehydration. 30 minutes after reperfusion, mice in the treatment group were injected via tail vein with 7-D@Lip, 7-D@A-Lip, and 7-D@A-mLip at a dose of 5 mg / kg (based on 7-DHC); the control and AKI groups were given saline. All mice were euthanized 24 hours post-surgery, and kidney tissue was collected for subsequent analysis.

[0107] H&E staining showed that the pathological features of the AKI group included renal tubular epithelial cell necrosis, renal tubular lumen dilation, glomerular capillary collapse, and basement membrane rupture. Figure 7 (A in the text). 7-D@A-mLip intervention can significantly alleviate these pathological changes. Furthermore, I / R causes changes in renal hemodynamics and microcirculatory dysfunction. I / R triggers a coagulation cascade, leading to local microthrombus formation, capillary obstruction, and renal medullary vascular congestion (…). Figure 7 (B in the text). Laser Doppler flowmetry showed that blood flow in AKI mice was severely reduced due to congestion. Figure 7 (C in the text). This disruption hinders the kidney's delivery of oxygen and nutrients and increases neutrophil adhesion and infiltration, thereby exacerbating kidney damage in the AKI model. 7-D@A-mLip improved renal congestion and restored blood flow more effectively than other groups, significantly improving renal function.

[0108] Example 6: Safety Assessment of 7-D@A-mLip

[0109] Healthy adult male C57BL / 6 mice were randomly divided into four groups. The experimental groups received intravenous injections of 7-D@Lip, 7-D@A-Lip, and 7-D@A-mLip at a dose of 5 mg / kg (based on 7-DHC), administered every other day for 14 consecutive days. The control group received an equal volume of saline under the same conditions. During the administration period, the mice's general condition, including weight, diet, activity level, and coat condition, was monitored daily. After 14 days, major organs (heart, liver, spleen, lungs, and kidneys) were collected for H&E staining. Compared with the control group, mice in the 7-D@Lip, 7-D@A-Lip, and 7-D@A-mLip experimental groups survived well and did not exhibit significant behavioral abnormalities or weight loss during the administration period. Histological examination showed no significant pathological differences in the major organs of the experimental groups compared to the control group, including inflammatory infiltration or structural abnormalities. Figure 8 The results indicate that the formulation did not induce toxic reactions and has good biocompatibility and in vivo safety, providing important safety evidence for the long-term use of the formulation in subsequent clinical applications.

[0110] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. ROS-responsive liposomes, characterized in that, include: 7-DHC liposomes and ATP; The 7-DHC liposomes include: 7-dehydrocholesterol, 1,2-distearyl-sn-glycerol-3-phosphocholine, distearyl-phosphatidylethanolamine-polyethylene glycol 2000 and distearyl-phosphatidylethanolamine-selenium-selenium-polyethylene glycol; The molar ratio of 7-dehydrocholesterol, 1,2-distearyl-sn-glycerol-3-phosphocholine, distearylphosphatidylethanolamine-polyethylene glycol 2000 and distearylphosphatidylethanolamine-selenium-selenium-polyethylene glycol is (10~70):(70~10):15:

5.

2. The ROS-responsive liposome according to claim 1, characterized in that, The concentration of ATP is 0.25 mg / mL to 1.50 mg / mL.

3. Drug-loaded liposomes, characterized in that, The invention includes the ROS-responsive liposomes of claim 1 or 2 and the medicaments for treating and / or preventing ferroptosis-related diseases; the medicaments for treating and / or preventing ferroptosis-related diseases include at least one of: an iron metabolism pathway inhibitor, a non-iron chelating agent, or an antioxidant.

4. Biomembrane-modified liposomes, characterized in that, include: Neutrophil membrane, renal tubular epithelial cell membrane, and at least one of the following: A) to B) A) The ROS-responsive liposomes according to claim 1 or 2; B) The drug-loaded liposomes according to claim 3.

5. The biomembrane-modified liposome according to claim 4, characterized in that, The ratio of the sum of the masses of the neutrophil membrane and the renal tubular epithelial cell membrane to the mass of the ROS-responsive liposome according to claim 1 or 2 is (4~1):(1~2).

6. The biomembrane-modified liposome according to claim 5, characterized in that, The mass ratio of the neutrophil membrane to the renal tubular epithelial cell membrane is 1:

1.

7. Use of at least one of the following (I) to (III) in the preparation of a medicament for the treatment and / or prevention of ferroptosis-related diseases: I) The ROS-responsive liposomes according to claim 1 or 2; II) The drug-loaded liposomes according to claim 3; II) Biomembrane-modified liposomes according to any one of claims 4 to 6.

8. The application according to claim 7, characterized in that, The iron death-related diseases include acute kidney injury, myocardial ischemia-reperfusion injury, and / or cerebral ischemia-reperfusion injury.

9. A medicine for treating and / or preventing ferroptosis-related diseases, characterized in that, The raw materials include at least one of the following: i) to iii) i) The ROS-responsive liposomes according to claim 1 or 2; ii) The drug-loaded liposomes according to claim 3; iii) The biomembrane-modified liposomes according to any one of claims 4 to 6.

10. A drug combination, characterized in that, The drug of claim 9 and other drugs for treating and / or preventing ferroptosis-related diseases.

Images