ROS (reactive oxygen species) response type liposome composite material as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to an ROS response type liposome composite material and a preparation method and application thereof.The ROS response type liposome composite material is prepared by jointly loading alkannin and rosuvastatin to liposome modified by an ROS sensitive connecting substance DSPE-TK-PEG2000, further wrapping a biomimetic membrane on the outer layer, and preparing the ROS response type liposome composite material. The ROS response type liposome composite material is obtained. According to the present invention, by using the ROS response design characteristic, the specific aggregation and the response type release of the drug at the diseased region are achieved, the blood circulation half-life period of the drug is effectively prolonged, and the anti-atherosclerosis effects of the shikonin and the rosuvastatin with two different mechanisms are combined so as to achieve the effect of effective treatment of the homocysteine induced atherosclerosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and more specifically, relates to a ROS-responsive liposome composite material, its preparation method, and its application. Background Technology

[0002] Atherosclerosis, or AS for short, is a chronic inflammatory vascular disease associated with lipid metabolism disorders and is considered a major pathological basis for cardiovascular and cerebrovascular diseases. In recent years, increasing research has shown a close correlation between elevated homocysteine ​​levels and cardiovascular and cerebrovascular diseases; it has even been called another "cardiovascular killer" after hypertension, hyperlipidemia, and hyperglycemia. As an independent risk factor for atherosclerosis, homocysteine ​​can self-oxidize to produce reactive oxygen species, oxidizing low-density lipoprotein and stimulating macrophages to transform into a pro-inflammatory phenotype driven by glycolytic metabolic reprogramming, leading to increased lipid uptake and accelerated foam cell formation. These dysfunctional macrophages secrete large amounts of pro-inflammatory factors such as TNF-α and IL-6, chemokines, proteases, and TGF-β, promoting the migration of smooth muscle cells from the media to the intima and their proliferation in the intima, accelerating the progression of atherosclerotic plaques. Therefore, inhibiting macrophage glycolytic metabolic reprogramming, improving the inflammatory microenvironment, and alleviating plaque formation may become an effective strategy for treating atherosclerosis.

[0003] Shikonin, or SKN for short, is a natural naphthoquinone with anti-inflammatory activity. It has been shown to have cardioprotective effects. Studies have found that SKN can not only target pyruvate kinase isoenzyme 2 to inhibit glycolysis, thereby inhibiting macrophage polarization and smooth muscle cell proliferation and migration, but also activate CPT1α to promote fatty acid β-oxidation. Rosuvastatin, on the other hand, can competitively inhibit HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis, blocking the production of mevalonate, thus significantly reducing endogenous cholesterol production, increasing the expression of LDL receptors on the surface of hepatocytes, and accelerating the clearance of low-density lipoprotein from the blood. In addition to directly lowering LDL-C, it can also slightly increase high-density lipoprotein cholesterol by 5%–10% and improve the lipid profile by reducing the synthesis of very low-density lipoprotein, achieving a "three-in-one" lipid-regulating effect. Furthermore, statins such as rosuvastatin also have anti-inflammatory and antioxidant properties, which help in their anti-atherosclerotic effects.

[0004] However, some inherent properties of shikonin, such as high toxicity, low bioavailability, and poor solubility, limit its application in biomedicine. Rosuvastatin, as a first-line clinical treatment, has a significant lipid-lowering effect, but its rapid clearance after oral administration, low accumulation in plaque tissue, and limited peripheral tissue penetration due to its hydrophilicity greatly reduce its therapeutic efficacy. Therefore, improving the bioavailability and targeted distribution of shikonin and rosuvastatin is a key issue in the treatment of atherosclerosis. Summary of the Invention

[0005] This invention provides a ROS-responsive liposome composite material to address the problems existing in the prior art.

[0006] The present invention first provides a ROS-responsive liposome composite material, wherein the ROS-responsive liposome composite material is obtained by loading shikonin and rosuvastatin onto liposomes modified with ROS-sensitive linker DSPE-TK-PEG2000, and further encapsulating the outer layer with a biomimetic membrane.

[0007] A second aspect of this invention provides a method for preparing the aforementioned ROS-responsive liposome composite material, comprising the following steps: Shikonin, DSPE-TK-PEG2000, lecithin and cholesterol were dissolved together in an organic solvent, and the organic solvent was removed by vacuum distillation to obtain a liposome membrane loaded with shikonin. The liposome membrane loaded with rosuvastatin and shikonin was hydrated to obtain a liposome dispersion co-loaded with shikonin and rosuvastatin. Preparation of biomimetic membrane dispersion; The biomimetic membrane dispersion was mixed with a liposome dispersion co-loaded with shikonin and rosuvastatin, and the mixture was reacted to obtain the ROS-responsive liposome composite material.

[0008] Preferably, the mass ratio of shikonin, DSPE-TK-PEG2000, lecithin and cholesterol is 0.069~0.08:0.25:5:2.5.

[0009] Preferably, the pressure during vacuum distillation is controlled at 0.07 MPa to 0.1 MPa.

[0010] Preferably, the hydration conditions are: hydration at 35℃~40℃ for 1 hour.

[0011] Preferably, the mass ratio of the biomimetic membrane dispersion to the liposome dispersion co-loaded with shikonin and rosuvastatin is 2.5~5:0.25~0.5.

[0012] Preferably, the reaction conditions are as follows: Ultrasonication for 10 minutes, followed by stirring at 37°C for 60 minutes.

[0013] A third aspect of the present invention provides an application of the aforementioned ROS-responsive liposome composite material, which is used to prepare a drug for treating atherosclerosis.

[0014] The present invention has the following beneficial effects: This invention provides a ROS-responsive liposome composite material, which is obtained by co-loading shikonin and rosuvastatin onto liposomes modified with the ROS-sensitive linker DSPE-TK-PEG2000, and further encapsulating the outer layer with a biomimetic membrane. This invention also designs a biocompatible ROS-responsive biomimetic nanocomposite. The nanocomposite loads the lipophilic drug shikonin and the water-soluble drug rosuvastatin onto the hydrophobic and hydrophilic layers of liposomes, respectively, modifies them with the ROS-sensitive linker DSPE-TK-PEG2000, and then encapsulates the liposomes with a biomimetic macrophage membrane. This biomimetic nanocomposite can target ROS-rich plaque regions, achieving precise drug release and effectively solving the problem of poor solubility of lipophilic drugs. The biomimetic membrane encapsulation, through the inherent immune escape and recruitment characteristics of macrophages, can prolong the circulating half-life of the drug in vivo, thereby improving the bioavailability of the drug to target cells.

[0015] Furthermore, the ROS-responsive liposome composite material described in this invention has the following advantages: (1) The present invention uses liposomes, a nanodelivery material approved by the FDA, which has the characteristics of improving the solubility of encapsulated substances, reducing the adverse reactions and toxicity of free drugs, flexible charge and size control, and surface modification.

[0016] (2) This invention simultaneously loads the traditional Chinese medicine shikonin and the Western medicine rosuvastatin, actively realizing the idea of ​​"integration of traditional Chinese and Western medicine". The combined use of the drugs is an innovative strategy to solve the differences in their physicochemical properties and reduce toxicity. This combined use strategy provides a new approach to the treatment of atherosclerosis from the three dimensions of "metabolism-lipid-inflammation".

[0017] (3) The biomimetic liposome composite material with co-loaded drug provided by the present invention can replace different types of biomimetic membranes and drugs, creating broader prospects in other aspects of the pharmaceutical field, such as anti-cancer, anti-inflammatory, and vaccine delivery. Attached Figure Description

[0018] Figure 1Transmission electron microscopy (TEM) images of TK-ML@(S+R)NPs and their responsive release in a ROS environment are shown. In the TEM images, A is a TK-ML@(S+R)NPs image and B is a TK-ML@(S+R)NPs image of ROS-responsive release.

[0019] Figure 2 The images show Coomassie Brilliant Blue staining and Western blot images of the outer macrophage membrane of TK-ML@(S+R)NPs. In the images, A is the Coomassie Brilliant Blue staining image and B is the Western blot image.

[0020] Figure 3 This is an experimental diagram of the fusion of macrophage membranes with liposomes TK-L@(S+R)NPs.

[0021] Figure 4 The graphs show the biosafety of TK-ML@(S+R)NPs, where A is the hemolysis rate graph and B is the erythrocyte morphology graph.

[0022] Figure 5 Fluorescent images of TK-ML@(S+R)NPs taken up by macrophages.

[0023] Figure 6 The figure shows the results of TK-ML@(S+R)NPs inhibiting lipid deposition in macrophages.

[0024] Figure 7 The figure shows the results of TK-ML@(S+R)NPs inhibiting oxidative stress in macrophages.

[0025] Figure 8 The figure shows the results of TK-ML@(S+R)NPs inhibiting macrophage glycolysis. In this figure, A represents the expression level of PKM2, and B represents the amount of ATP produced by the cell.

[0026] Figure 9 The diagram illustrates the effects of the atherosclerotic microenvironment on smooth muscle cells. Figure A shows the results of TK-ML@(S+R)NPs inhibiting smooth muscle cell proliferation under co-culture conditions; Figure B shows the results of TK-ML@(S+R)NPs inhibiting smooth muscle cell migration under co-culture conditions.

[0027] Figure 10 The results show the blood half-life and in vivo targeting assays of TK-ML@(S+R)NPs. In this figure, A represents the blood half-life in mice; B is a statistical graph of A; C represents the targeting results of atherosclerotic plaque sites in mice; and D represents the targeting results of TK-ML@(S+R)NPs in different organs of mice.

[0028] Figure 11 The effect of HE treatment on Hcy-induced advanced atherosclerosis.

[0029] Figure 12 The images show the ORO staining effects of treating Hcy-induced advanced atherosclerosis. In this image, A shows the targeting of TK-ML@(S+R)NPs to atherosclerotic plaques in mice; and B shows Oil Red O staining images of the aortic root after treatment with different drug groups. Detailed Implementation

[0030] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0031] The inventive concept of this invention is as follows: This invention provides a ROS-responsive liposome composite material. The liposomes in this composite material, due to their cell membrane-like structure and excellent biocompatibility, offer a highly adaptable platform for targeted drug delivery. They can enhance drug solubility, stability, and targeted delivery while minimizing systemic side effects, effectively addressing the problems of poor solubility and low bioavailability of shikonin, a hydrophobic drug, and further prolonging the blood circulation time of rosuvastatin. The outer layer is modified with DSPE-TK-PEG2000 to respond to ROS-enriched plaque regions, thereby achieving accurate and rapid drug release.

[0032] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0033] The list of abbreviations for this invention is shown in Table 1.

[0034] Table 1. List of abbreviations for this invention Example 1 A method for preparing a ROS-responsive liposome composite material is as follows: S1. Shikonin, DSPE-TK-PEG2000, lecithin and cholesterol are dissolved together in an organic solvent. After removing the organic solvent by vacuum distillation, a liposome membrane loaded with shikonin is obtained.

[0035] 5 mg of lecithin, 2.5 mg of cholesterol, 0.25 mg of DSPE-TK-PEG2000, and 0.069 mg of SKN were dissolved in 5 mL of chloroform. The mixture was then added to a round-bottom flask. The solution was sonicated in a water bath at 90 W for 10 min to ensure thorough mixing and reaction of the components. The solvent was then removed by vacuum distillation at 45 °C, with the pressure maintained at 0.1 MPa, to allow the solution to form a uniform film on the inner wall of the flask, thus obtaining a liposome membrane loaded with shikonin.

[0036] S2. The liposome membrane loaded with rosuvastatin and shikonin is hydrated to obtain a liposome dispersion co-loaded with shikonin and rosuvastatin.

[0037] Dissolve 0.66 mg of RSV in 1 mL of DMSO, add it to the round-bottom flask of S1, sonicate in a water bath for 10 min, and hydrate with PBS at 37 °C for 1 h to obtain a liposome dispersion co-loaded with shikonin and rosuvastatin, denoted as TK-L@(S+R)NPs.

[0038] S3. Prepare a biomimetic membrane dispersion.

[0039] RAW264.7 macrophages were cultured in 15cm culture dishes until the cell density reached 90%. After washing three times with PBS, the cells were scraped off and collected into centrifuge tubes. The cells were centrifuged at 800g for 10 min at 4°C. The cells were resuspended in 1 mL of 1mM PMSF pre-cooled RIPA lysis buffer and lysed on ice for 1 h. The cells were then sonicated at 80W for 10 min at 4°C. The cells were then subjected to five freeze-thaw cycles at -80°C and 37°C, each for 30 min. Finally, the cells were centrifuged at 13000 rpm for 30 min at 4°C, and the precipitate was collected as the macrophage membrane. The macrophage membrane was resuspended in PBS and sonicated to obtain a biomimetic membrane dispersion, denoted as MØm.

[0040] S4. Mix the biomimetic membrane dispersion with the liposome dispersion co-loaded with shikonin and rosuvastatin, and react to obtain the ROS-responsive liposome composite material.

[0041] 1 mL of 5 mg / mL TK-L@(S+R)NPs solution and 12.5 μL of 20 mg / mL biomimetic membrane dispersion were mixed, sonicated in a water bath for 10 min, and stirred in a water bath at 37 °C for 60 min to obtain a biomimetic liposome nanocomposite material co-loaded with SKN and RVS, denoted as TK-ML@(S+R)NPs.

[0042] Example 2 The application of a ROS-responsive liposome composite material is as follows: 1. First, the TK-ML@(S+R)NPs prepared in Example 1 were analyzed, as follows: Detection methods: The prepared nanoparticles TK-ML@(S+R)NPs and the TK-ML@(S+R)NPs samples that were released in response to the ROS environment were observed under a transmission electron microscope. The morphology of the samples was recorded and analyzed. Cell membrane proteins were detected by Coomassie Brilliant Blue and Western blotting. Cell fusion experiments were used to verify membrane function.

[0043] Test results: such as Figure 1 As shown, transmission electron microscopy revealed that TK-ML@(S+R)NPs consisted of uniformly dispersed spherical liposome nanoparticles with a particle size of approximately 100 nm. In a high-level ROS environment, the phospholipid bilayer of the TK-ML@(S+R)NPs liposomes was disrupted, exhibiting a distinct membrane fragmentation structure. Figure 2 As shown, the membrane characteristic protein analysis of the prepared TK-ML@(S+R)NPs showed that the biomimetic membrane was successfully wrapped on the surface of TK-L@(S+R)NPs and retained the cell membrane surface characteristic protein CD11b. Figure 2 In this context, MØm represents the biomimetic membrane dispersion, and M@LP represents TK-L@(S+R)NPs. For example... Figure 3 As shown, the macrophage membrane fused with the liposome, indicating that the macrophage membrane and the liposome TK-L@(S+R)NPs hybridized successfully.

[0044] 2. Biosafety verification.

[0045] (1) Experimental methods.

[0046] Six groups were set up by co-incubating a certain volume of drug solutions of different concentrations with an equal volume of 4% (v / v) red blood cell suspension, as shown in Table 2.

[0047] Table 2 Dosage of different drugs Different concentrations of the drug were incubated with red blood cells at 37°C for 6 hours. The hemolysis rate and red blood cell morphology of different groups were calculated. The specific procedure was as follows:

[0048] After co-incubation, the red blood cells were centrifuged at 3500 rpm, 4°C, and 5 min to collect the supernatant. The absorbance at 562 nm was measured using a microplate reader. To further observe the effects of different drugs on red blood cell morphology, images of samples after different treatments were captured under an inverted fluorescence microscope.

[0049] (2) Experimental results.

[0050] Figure 4 The biosafety of the ROS-responsive liposome composite material TK-ML@(S+R)NPs prepared in Example 1 of this invention was investigated. Figure 4As shown in A, in the hemolysis experiment, the hemolysis rate in the drug group was less than 5% compared to the H2O treatment group; Figure 4 As shown in Figure B, no atypical red blood cells were found in the red blood cell morphology image, indicating that the TK-ML@(S+R)NPs prepared in this invention have good biocompatibility at the cellular level.

[0051] 3. In vitro targeted assay.

[0052] Lip and TK-ML@(S+R)NPs were labeled with dihydroporphyrin ce6 to prepare L@ce6 and TKML@ce6, respectively. RAW264.7 cells were seeded in 24-well plates and cultured at 37°C and 5% CO2 for 24 h. After incubation with 100 μM Hcy for 24 h, different of the above-mentioned nanomedicines were added to the RAW264.7 wells for 4 h. The cells were then washed three times with PBS, fixed with 4% paraformaldehyde for 10 min, stained with DAPI for 5 min, and photographed using a laser confocal microscope.

[0053] (1) Preparation method of L@ce6 NPs.

[0054] 6.6 mg lecithin, 3.3 mg cholesterol, and 20 mg / mL ce6 were dissolved in DMSO and then dissolved together in 5 mL of chloroform solution. The mixture was thoroughly mixed and the organic solvent was removed by vacuum evaporation to obtain a liposome membrane. 1 mL of PBS was added for hydration for 1 h, and the membrane was sonicated in a 60 W water bath for 5 min to obtain a liposome dispersion loaded with ce6.

[0055] (2) Preparation method of TK-ML@ce6NPs.

[0056] 6.6 mg lecithin, 3.3 mg cholesterol, 0.33 mg DSPE-TK-PEG2000, and 20 mg / ml ce6 were dissolved in DMSO and then dissolved together in 5 mL of chloroform solution. The mixture was thoroughly mixed, and the organic solvent was removed by vacuum evaporation to obtain liposome membranes. 1 mL of PBS was added for hydration for 1 h, and the mixture was sonicated in a 60 W water bath for 5 min to obtain a ce6-loaded liposome dispersion. Then, 0.33 mg of macrophage membrane was added, and the mixture was stirred at 37 °C for 60 min, centrifuged at 13600 rpm for 10 min, and the precipitate was reconstituted with PBS to obtain TK-ML@ce6NPs.

[0057] (3) Experimental results.

[0058] Figure 5 Fluorescent images of macrophages uptake of the ROS-responsive biomimetic liposome composite material TK-ML@(S+R)NPs prepared for this invention. Figure 5As shown, the uptake rate of the TK-ML@ce6NPs group was significantly higher than that of Lip@ce6, indicating that the modification of DSPE-TK-PEG2000 and the encapsulation of macrophage membranes can effectively target nanomaterials to atherosclerotic plaque sites.

[0059] 4. The therapeutic effect of treating atherosclerosis at the cellular level.

[0060] (1) TK-ML@(S+R)NPs effectively inhibit lipid deposition in macrophages, including the following steps: RAW264.7 macrophages were seeded in 12-well plates. Once the cell density reached 70%, macrophages were induced with 100 μM hemolytic anhydrase (Hcy) for 24 h. Then, 30 μg / mL oxidized low-density lipoprotein and different drug concentrations as shown in Table 2 were added, and the cells were cultured in serum-free medium for 24 h. Residual medium was washed with PBS, with the control group not undergoing Hcy induction. Next, the cells were incubated with 0.3% Oil Red O working solution for 30 min, followed by washing with 60% isopropanol for 3 s. Finally, lipid droplet formation in the cells was observed under an inverted microscope.

[0061] Figure 6 Fluorescence images showing the inhibition of macrophage lipid deposition by the ROS-responsive liposome composite material TK-ML@(S+R)NPs prepared in this invention. Figure 6 As shown, compared to the model group, the TK-ML@(S+R)NPs treatment group showed an inhibition rate of approximately 50% in macrophage lipid deposition.

[0062] (2) TK-ML@(S+R)NPs effectively inhibited the level of oxidative stress in macrophages, including the following steps: RAW264.7 cells were fed at a rate of 1×10⁻⁶. 4 Cells were stored at a density of cells / mL in 24-well plates. After cell adhesion, different concentrations of the drug shown in Table 2 were added to the cells for 2 hours, followed by incubation with 5 times the normal modeling dose of Hcy to induce ROS production for 4 hours. The control group did not undergo Hcy induction. After washing the cells three times with PBS, ROS were labeled with the fluorescent probe DCFH-DA. Subsequently, the green fluorescence signal in the cells was observed using a Leica laser confocal microscope.

[0063] Figure 7 Fluorescent images of the ROS-responsive liposome composite material TK-ML@(S+R)NPs prepared in Example 1 of this invention, used to clear ROS from macrophages. Figure 7 As shown, compared to the model group, TK-ML@(S+R)NPs treatment can reduce the level of ROS in macrophages by about 60%.

[0064] 5. The effect of inhibiting macrophage glycolysis at the cellular level.

[0065] (1) TK-ML@(S+R)NPs effectively inhibited the expression of PKM2, a key enzyme in glycolysis, at the cellular level. The specific steps are as follows: RAW264.7 cells were fed at a rate of 1×10⁻⁶. 4 Cells were stored at a density of cells / mL in 24-well plates. After cell adhesion, different concentrations of drugs shown in Table 2 were added to the cells for 2 hours, followed by the addition of 100 μM Hcy to induce an atherosclerosis model for 24 hours. The control group did not undergo Hcy induction. After washing the cells three times with PBS, they were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton-100 for 10 minutes, blocked with 20% goat serum for 1 hour, incubated with PKM2 primary antibody at 4°C overnight, incubated with fluorescent secondary antibody at room temperature for 2 hours, stained with DAPI for 5 minutes, and photographed using a laser confocal microscope.

[0066] Figure 8 Image A shows the fluorescence image of TK-ML@(S+R)NPs effectively inhibiting the expression of PKM2, a key enzyme in glycolysis, at the cellular level. Atherosclerosis is a chronic inflammatory disease. Inflammatory stimulation induces the expression of a large number of glycolytic enzymes in macrophages. High glycolysis promotes the synthesis of inflammatory factors, causing inflammatory macrophages to excessively take up oxidized low-density lipoprotein, transforming them into foam cells and accelerating the progression of atherosclerosis. PKM2 is a key enzyme in the glycolysis process, and SKN, as a specific inhibitor of PKM2, effectively inhibits glycolysis and alleviates atherosclerosis.

[0067] (2) TK-ML@(S+R)NPs effectively restore ATP production at the cellular level. The specific steps are as follows: RAW264.7 cells were fed at a rate of 1×10⁻⁶. 4 Cells were stored at a density of cells / mL in 24-well plates. After cell attachment, different concentrations of the drug shown in Table 2 were added to the cells for 2 hours, followed by the induction of an atherosclerosis model with 100 μM Hcy for 24 hours. The control group did not undergo Hcy induction. The ATP production of the cells was measured after complete cell lysis using an ATP assay kit.

[0068] Figure 8 B represents a statistical graph showing the effective restoration of ATP production at the cellular level by TK-ML@(S+R)NPs. During atherosclerosis, cellular energy metabolism is primarily characterized by excessive activation of glycolysis, leading to insufficient ATP production, macrophage dysfunction, excessive secretion of pro-inflammatory factors, and impaired cholesterol transport, thus exacerbating the development and progression of atherosclerosis. Figure 8As shown in Figure B, compared to the model group, TK-ML@(S+R)NPs treatment can increase the level of ATP in macrophages by about 1.5 times.

[0069] 6. Investigate the effects of the atherosclerotic microenvironment on smooth muscle cells.

[0070] (1) Under co-culture conditions, TK-ML@(S+R)NPs effectively inhibited the proliferation of smooth muscle cells at the cellular level. The specific steps are as follows: RAW264.7 cells were incubated in 6 cm cell culture dishes. After cell attachment, different concentrations of drugs (as shown in Table 2) were added to the cells for 2 hours, followed by the addition of 100 μM Hcy. An atherosclerosis model was induced after 24 hours, with the control group not receiving Hcy induction. After stimulation, the supernatant from the culture media of different groups was collected for smooth muscle cell culture. The proliferative capacity of smooth muscle cells was assessed using an EDU kit after 24 hours.

[0071] Figure 9 Image A shows the fluorescence image of TK-ML@(S+R)NPs effectively inhibiting smooth muscle cell proliferation at the cellular level. Homocysteine ​​can self-oxidize to produce reactive oxygen species, oxidizing low-density lipoprotein and stimulating macrophages to transform into a pro-inflammatory phenotype driven by glycolytic metabolic reprogramming, taking up more lipids and accelerating foam cell formation. These dysfunctional macrophages secrete large amounts of pro-inflammatory factors, chemokines, proteases, and TGF-β, promoting the migration of smooth muscle cells from the media to the intima, where they proliferate extensively, accelerating the progression of atherosclerotic plaques. Figure 9 As shown in Figure A, compared to the model group, TK-ML@(S+R)NPs treatment of macrophages can effectively improve the inflammatory microenvironment, reduce the proliferation capacity of smooth muscle cells by about 50%, and inhibit the formation of "fibrous caps".

[0072] (2) Under co-culture conditions, TK-ML@(S+R)NPs effectively inhibited the migration ability of smooth muscle cells at the cellular level. The specific steps are as follows: RAW264.7 cells were fed at a rate of 1×10⁻⁶. 4 Cells were stored at a density of cells / mL in 24-well plates. After cell attachment, different concentrations of the drugs shown in Table 2 were added to the cells for 2 hours, followed by the addition of 100 μM Hcy. An atherosclerosis model was induced after 24 hours, with the control group not receiving Hcy induction. After stimulation, the supernatant from different groups was collected for smooth muscle culture. A scratch assay was performed at 0 hours, and images were taken under an inverted microscope. After 24 hours, images were taken again to compare the scratch area at different time points.

[0073] Figure 9Image B shows how TK-ML@(S+R)NPs effectively inhibit smooth muscle cell migration at the cellular level. Figure 9 As shown in Figure B, compared to the model group, TK-ML@(S+R)NPs treatment of macrophages can effectively improve the inflammatory microenvironment and reduce the migration ability of smooth muscle cells by about 30%.

[0074] Figure 9 Among them, 1: Control; 2: Model; 3: S+R; 4: TK-ML@(S+R)NPs.

[0075] 7. A semi-quantitative method for detecting fluorescence intensity was used to determine the blood half-life and in vivo targeting of TK-ML@(S+R)NPs.

[0076] (1) TK-ML@ce6 was prepared by labeling TK-ML@(S+R)NPs with dihydroporphyrin ce6. After C57 mice were injected with 200 μL of ce6 and TK-ML@ce6 at a dose of 5 mg / kg via the tail vein, blood samples were collected at different time points of 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, and 24 h for fluorescence intensity measurement.

[0077] The preparation method of TK-ML@ce6NPs is as follows: 6.6 mg lecithin, 3.3 mg cholesterol, 0.33 mg DSPE-TK-PEG2000, and 20 mg / ml ce6 were dissolved in DMSO and then dissolved together in 5 mL of chloroform solution. The mixture was thoroughly mixed, and the organic solvent was removed by vacuum evaporation to obtain liposome membranes. 1 mL of PBS was added for hydration for 1 hour, and the mixture was sonicated in a 60 W water bath for 5 min to obtain a ce6-loaded liposome dispersion. Then, 0.33 mg of macrophage membrane was added, and the mixture was stirred at 37 °C for 60 min, centrifuged at 13600 rpm for 10 min, and the precipitate was reconstituted with PBS to obtain TK-ML@ce6NPs.

[0078] Figure 10 A represents the blood half-life of the TK-ML@(S+R)NPs of this invention in C57 mice. Figure 10 B is a statistical graph. For example... Figure 10 As shown in Figure B, the blood circulation half-lives of ce6 and TK-ML@ce6 are 0.29 h and 1.43 h, respectively. This indicates that the biomimetic membrane-masked nanoformulation is beneficial for prolonging blood circulation time.

[0079] (2) L@ce6 and TK-ML@ce6 were prepared by labeling Lip and TK-ML@(S+R) with dihydroporphyrin ce6. ApoE - / -After feeding mice a high-fat, high-protein diet for 3 months, 200 μL of ce6, L@ce6NPs, and TK-ML@ce6 at a dose of 5 mg / kg was injected into the tail vein. Twelve hours later, the mouse aorta was harvested for fluorescence imaging.

[0080] The preparation method of L@ce6NPs is as follows: 6.6 mg lecithin, 3.3 mg cholesterol, and 20 mg / ml ce6 were dissolved in DMSO and then dissolved together in 5 mL of chloroform solution. The mixture was thoroughly mixed and the organic solvent was removed by vacuum evaporation to obtain a liposome membrane. 1 mL of PBS was added for hydration for 1 hour, and the membrane was sonicated in a 60 W water bath for 5 min to obtain a liposome dispersion loaded with ce6.

[0081] Figure 10 The C in this invention represents the targeting of TK-ML@(S+R)NPs to atherosclerotic plaque sites in mice. For example... Figure 10 As shown in Figure C, compared with the ce6 and L@ce6 groups, the TK-ML@ce6 group showed significant aggregation at the aortic plaque, indicating that the prepared TK-ML@(S+R)NPs biomimetic nano-formulation can effectively target the atherosclerotic plaque site.

[0082] Figure 10 The value of D represents the targeting of the TK-ML@(S+R)NPs of this invention to different organs in mice. Since the liver and kidneys are the main organs responsible for drug metabolism and elimination, small NPs accumulate in these two organs.

[0083] 8. ROS-responsive nanocomposite TK-ML@(S+R)NPs is used to treat Hcy-induced atherosclerosis.

[0084] Randomly select 8-week-old ApoE - / - Mice were divided into 6 groups for drug administration, as shown in Table 3. Except for the Control group, which was fed a normal diet, the other groups were fed a high-fat, high-protein diet. After 4 weeks of feeding, the drugs were administered via tail vein injection once every 3 days for 8 consecutive weeks. After that, animal tissues were collected, and the heart, liver, spleen, lungs, and kidneys of the mice were dissected and sectioned for H&E staining to examine the in vivo biosafety of different treatment groups.

[0085] High-fat, high-egg diet: 2% methionine + 15% fat + 1.5% cholesterol + basic maintenance feed.

[0086] Table 3 Grouping of Atherosclerosis Treatment Note: In Table 3, "-" indicates no medication was administered.

[0087] Figure 11The results show HE staining. The results indicate that no obvious organic lesions were observed in the organs of mice in different treatment groups, demonstrating that the biomimetic nanomedicine constructed in this invention has good biosafety.

[0088] 9. ROS-responsive biomimetic nanocomposite TK-ML@(S+R)NPs was used to evaluate the in vivo biocompatibility of different treatment groups.

[0089] Using the administration method described in section 8 above, after obtaining animal samples, the aorta was dissected and stained with Oil Red O to examine the treatment effects of different treatment groups.

[0090] Figure 12 Image A shows gross stained images of blood vessels affected by atherosclerosis after treatment with different drug groups. As shown in the figure, the white area within the upper circle represents plaque. Observations in the model group revealed that atherosclerotic plaques were distributed throughout the aorta, particularly concentrated at the aortic root, thoracic aorta, and abdominal aortic branches. Compared to the model group, mice treated with TK-ML@(S+R)NPs showed a significant reduction in aortic plaques.

[0091] Figure 12 Image B shows Oil Red O staining images of the aortic root after treatment with different drug groups. As shown in the figure, TKML@(S+R)NPs significantly reduced plaque lipid droplets after treatment, indicating a significant therapeutic effect.

[0092] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0093] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0094] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A ROS-responsive liposome composite material, characterized in that, The ROS-responsive liposome composite material is obtained by loading shikonin and rosuvastatin onto liposomes modified with the ROS-sensitive linker DSPE-TK-PEG2000, and then further encapsulating the outer layer with a biomimetic membrane.

2. The method for preparing the ROS-responsive liposome composite material as described in claim 1, characterized in that, Includes the following steps: Shikonin, DSPE-TK-PEG2000, lecithin and cholesterol were dissolved together in an organic solvent, and the organic solvent was removed by vacuum distillation to obtain a liposome membrane loaded with shikonin. The liposome membrane loaded with rosuvastatin and shikonin was hydrated to obtain a liposome dispersion co-loaded with shikonin and rosuvastatin. Preparation of biomimetic membrane dispersion; The biomimetic membrane dispersion was mixed with a liposome dispersion co-loaded with shikonin and rosuvastatin, and the mixture was reacted to obtain the ROS-responsive liposome composite material.

3. The preparation method according to claim 2, characterized in that, The mass ratio of shikonin, DSPE-TK-PEG2000, lecithin and cholesterol is 0.069~0.08:0.25:5:2.

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4. The preparation method according to claim 2, characterized in that, The pressure during vacuum distillation is controlled at 0.07 MPa to 0.1 MPa.

5. The preparation method according to claim 2, characterized in that, The hydration conditions are: hydration at 35℃~40℃ for 1 hour.

6. The preparation method according to claim 2, characterized in that, The mass ratio of the biomimetic membrane dispersion to the liposome dispersion co-loaded with shikonin and rosuvastatin is 2.5~5:0.25~0.

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7. The preparation method according to claim 2, characterized in that, The conditions for the reaction are: Ultrasonication for 10 minutes, followed by stirring at 37°C for 60 minutes.

8. The application of the ROS-responsive liposome composite material as described in claim 1, characterized in that, The ROS-responsive liposome composite material is used to prepare drugs for treating atherosclerosis.

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