A dual-targeting biomimetic liposome composite material, its preparation method and application

Abstract

This invention discloses a dual-targeting biomimetic liposome composite material, its preparation method, and its application, belonging to the field of nanomedicine technology. Using liposome nanoparticles as a carrier, evolocumab and shikonin are loaded onto the carrier to obtain drug-loaded liposomes. These drug-loaded liposomes are then fused with macrophage membranes and modified with phospholipid-modified hyaluronic acid to obtain the dual-targeting biomimetic liposome composite material. This invention utilizes evolocumab for lipid-lowering effects and shikonin for anti-inflammatory effects, employing two different mechanisms to achieve targeted therapy for atherosclerosis. The outermost biomimetic membrane leverages the inherent immune escape and recruitment characteristics of macrophages to achieve long-term blood circulation and targeted ability to lesion sites. By utilizing the interaction between hyaluronic acid and the CD44 receptor on the target cell surface, the composite material is effectively utilized by target cells, enhancing its efficacy in preventing and treating atherosclerosis.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine technology, specifically to a dual-targeting biomimetic liposome composite material, its preparation method, and its application. Background Technology

[0002] Atherosclerosis is the most common underlying cause of ischemic heart disease and stroke, accounting for a quarter of all deaths worldwide. Hyperhomocysteinemia (HHcy) is considered an independent risk factor for atherosclerosis, and it has been established that HHcy leads to endothelial cell activation and dysfunction, with mass endothelial cell apoptosis being the initiating event in atherosclerotic development. Furthermore, increased secretion of intercellular adhesion molecules recruits more macrophages, and endothelial barrier disruption and widening of the intercellular space promote the infiltration of more lipoproteins and inflammatory macrophages into the subendothelial layer. Lipoproteins are then phagocytosed by macrophages to form foam cells, thereby promoting the formation of atherosclerosis. Lipid accumulation by inflammatory macrophages to form foam cells is considered a key event in the early development of atherosclerosis. Therefore, finding ways to alleviate endothelial cell dysfunction and inhibit foam cell formation holds promise for effective treatment of atherosclerosis.

[0003] Currently, the treatment of atherosclerosis includes lowering blood lipids, antiplatelet therapy, and antihypertensive therapy. In lipid regulation, statins and proprotein convertase subtilisin 9 (PCSK9) inhibitors are the cornerstone of drug therapy for dyslipidemia, playing a crucial role in the prevention and control of atherosclerotic cardiovascular disease both domestically and internationally.

[0004] Evol is a PCSK9-specific inhibitor, targeting lipid metabolism in its early intervention phase, offering at least a step ahead of statins in targeting PCSK9. Furthermore, in the early development of atherosclerosis, the inflammatory microenvironment at plaque sites promotes PCSK9 activation, increases lipid uptake receptor (LOX-1), and decreases lipid efflux receptors A1 / G1 (ABCA1 / G1), ultimately leading to the accumulation of lipids within macrophages and the formation of foam cells. Evol effectively inhibits PCSK9, thereby suppressing foam cell formation and preventing atherosclerosis. However, current clinical studies show that after patients discontinue Evol, their blood lipid levels rise along with the increase in PCSK9, significantly increasing the risk of atherosclerosis.

[0005] Shikonin (SKN) is the main active ingredient in the traditional Chinese medicine Lithospermum erythrorhizon. As an inhibitor of key enzymes in the glycolysis pathway, it effectively inhibits the increase in glycolysis induced by homocysteine ​​(Hcy). Simultaneously, numerous studies have shown that shikonin inhibits endothelial cell apoptosis and improves endothelial cell function. Shikonin also possesses good antioxidant, anti-inflammatory, and antithrombotic effects, inhibiting the proliferation and migration of smooth muscle cells (SMCs) and regulating macrophage polarization, thereby inhibiting lipid accumulation and achieving the goal of treating atherosclerosis. However, similar to traditional hydrophobic anti-inflammatory drugs, shikonin, after oral or intravenous injection, has a short circulation time, non-specific distribution, and low drug utilization rate, severely hindering its clinical application. Summary of the Invention

[0006] This invention provides a dual-targeting biomimetic liposome composite material, its preparation method, and its application. It effectively solves the problems of short circulation time, non-specific distribution, and low drug utilization of shikonin, a hydrophobic anti-inflammatory drug, in the human body, further extending the blood circulation time of Evol and reducing treatment costs. This invention utilizes liposomes to simultaneously load the lipid-lowering drug evolocumab and the anti-inflammatory drug shikonin, while simultaneously hybridizing a macrophage membrane onto the surface of nanoparticles to obtain a dual-targeting biomimetic liposome composite material. This composite material possesses a long blood circulation half-life and good targeting ability. Co-loading evolocumab and shikonin, it utilizes evolocumab for lipid-lowering effects and shikonin for anti-inflammatory and anti-atherosclerotic effects, achieving targeted therapy for atherosclerosis.

[0007] The first objective of this invention is to provide a dual-targeting biomimetic liposome composite material, the composite material comprising drug-loaded liposomes loaded with evolocumab and shikonin, the drug-loaded liposomes being coated with macrophage membranes modified with phospholipid-modified hyaluronic acid.

[0008] A second objective of this invention is to provide a method for preparing the above-mentioned dual-targeting biomimetic liposome composite material, comprising the following steps:

[0009] Evolomab, shikonin, cholesterol, and lecithin were dissolved in a solvent, water was added, and the mixture was sonicated, evaporated under reduced pressure, hydrated, and dialyzed to obtain a liposome dispersion loaded with evolomab and shikonin.

[0010] The liposome dispersion loaded with evolocumab and shikonin was mixed with the biomimetic membrane solution, repeatedly extruded, and then phospholipid-modified hyaluronic acid solution was added. The mixture was stirred and reacted at 30℃~37℃ to obtain a dual-targeting biomimetic liposome composite material.

[0011] In a preferred embodiment, the mass ratio of the biomimetic membrane to the liposomes loaded with evolocumab and shikonin is 1 to 2:10.

[0012] In a preferred embodiment, the mass ratio of the liposome loaded with evolocumab and shikonin to phospholipidified hyaluronic acid is 1:0.1 to 0.25.

[0013] In a preferred embodiment, the mass ratio of evolocumab, lecithin, and cholesterol is 3-6:20:10; and the mass ratio of shikonin, lecithin, and cholesterol is 1-4:20:10.

[0014] In a preferred embodiment, the repeated extrusion is performed by repeatedly extruding 8 to 10 times using a micro extruder with an aperture of 220 nm.

[0015] As a preferred embodiment, the preparation method of the biomimetic membrane solution includes the following steps: ultrasonically disrupting macrophage membranes at a power of 30-50W for 3-5 minutes, repeatedly freezing and thawing at -80℃ and 37℃ 2-3 times, centrifuging at 10000-13000rpm, and then dissolving in PBS to obtain the biomimetic membrane solution.

[0016] As a preferred embodiment, the preparation method of the phospholipid-modified hyaluronic acid solution includes the following steps: dissolving hyaluronic acid and an activator in water, stirring for 30-60 minutes, adding phospholipid polyethylene glycol amino, stirring at 600-800 rpm for 12-24 hours to obtain a mixture, and dialyzing the mixture using a dialysis bag with a molecular weight cutoff of 3-3.5 kDa for 12-24 hours to obtain the phospholipid-modified hyaluronic acid solution.

[0017] It should be noted that the activator is 1(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and the mass ratio of 1(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and hyaluronic acid is 5:10:0.8 to 1.2.

[0018] A third objective of this invention is to provide the application of the above-mentioned dual-targeting biomimetic liposome composite material in the preparation of a medicament for treating atherosclerosis caused by hyperhomocysteinemia.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] To address the shortcomings of existing nanomedicines, such as rapid metabolism and lack of active targeting, this invention provides a dual-targeting biomimetic liposome composite material. Using liposome nanoparticles as a carrier, evolocumab and shikonin are loaded onto the carrier to obtain drug-loaded liposomes. These drug-loaded liposomes are then fused with macrophage membranes to obtain drug-loaded liposomes coated with a biomimetic membrane. The biomimetic membrane is further modified with phospholipid-modified hyaluronic acid to obtain the dual-targeting biomimetic liposome composite material. The liposomes simultaneously physically encapsulate evolocumab and shikonin, the biomimetic membrane is fused with the liposomes, and the phospholipid-modified hyaluronic acid is inserted into the surface of the biomimetic membrane. Utilizing the design features of the biomimetic membrane and hyaluronic acid modification, specific aggregation and penetration of the drug at the lesion site are achieved, while simultaneously improving the bioavailability of the drug to target cells. This invention utilizes evolocumab for lipid-lowering effects and shikonin for anti-inflammatory effects, employing two different mechanisms of action to achieve targeted therapy for atherosclerosis. The inflammatory environment at atherosclerotic plaque sites secretes chemokines to recruit macrophages. The outermost biomimetic membrane, through its source cells' (macrophages) inherent immune escape and recruitment characteristics, enables the composite material to achieve long blood circulation and targeted ability to the lesion site. Utilizing the interaction between hyaluronic acid and the target cell surface receptor CD44, the composite material can be effectively utilized by target cells, improving its efficacy in preventing and treating atherosclerosis.

[0021] This invention improves the blood circulation half-life of the drug and achieves targeted effects on atherosclerotic plaques and target cells, providing new theoretical support for the development of anti-atherosclerotic drugs and related clinical testing and treatment. It has significant scientific, practical and economic value.

[0022] In addition to loading drugs for treating atherosclerosis, the biomimetic liposome composite material with co-loaded drugs provided by this invention can replace different types of biomimetic membranes and drugs, and utilize the "homing effect" of biomimetic membranes and the mechanisms of action of different drugs as an alternative platform for the treatment of other diseases, such as tumors and rheumatoid arthritis. Therefore, this invention combines biomimetic membrane coatings and targeted modified nanocomposite materials, which can provide multiple functions and advantages for the treatment of a variety of diseases. Attached Figure Description

[0023] Figure 1 Transmission electron microscopy (TEM) images of liposomes loaded with evolocumab and shikonin (L@ES NPs) and dual-targeting biomimetic liposome composites (HA-ML@ES NPs) prepared in Example 1 of this invention; wherein, image A is L@ES NPs and image B is HA-ML@ES NPs.

[0024] Figure 2 The images show Coomassie Brilliant Blue staining of the biomimetic membrane in Example 1 of this invention; where A is Coomassie Brilliant Blue staining and B is Western blotting of proteins.

[0025] Figure 3 The biosafety of the dual-targeted biomimetic liposome composite material HA-ML@ES NPs prepared in Example 1 of this invention is shown in Figure A, where hemolysis rate is shown in Figure B, and red blood cell morphology is shown in Figure B.

[0026] Figure 4 The dual-targeting biomimetic liposome composite material HA-ML@ES NPs prepared in Example 1 of this invention targets damaged endothelial cells and inflammatory macrophages. Figure A is a schematic diagram of the experimental principle; Figure B is a comparison diagram of HA-ML@SENPs and L@ES targeting damaged endothelial cells and inflammatory macrophages.

[0027] Figure 5 The blood half-life of the HA-ML@ES NPs of this invention in Kunming mice is shown.

[0028] Figure 6 This invention demonstrates the targeting ability of HA-M@ES NPs to atherosclerotic plaque sites in mice.

[0029] Figure 7 The present invention describes HA-ML@ES NPs to inhibit endothelial cell apoptosis and macrophage lipid accumulation. Figure A shows the flow cytometry results of endothelial cell apoptosis, with control being the negative group and model being the positive group. Evol+SKN represents the combination of the two drugs in free form. Figure B shows Oil Red O staining of foam cells to detect the accumulation of oxLDL in macrophages.

[0030] Figure 8 This invention relates to the use of HA-M@ES NPs in mice to treat early atherosclerosis. Detailed Implementation

[0031] 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 and accompanying drawings. However, the embodiments described are not intended to limit the invention. Unless otherwise specified, the following test methods and detection methods are conventional methods; unless otherwise specified, the reagents and raw materials are commercially available.

[0032] The technical content of this invention will be analyzed and explained in detail below.

[0033] This invention provides a dual-targeting biomimetic liposome composite material, the composite material comprising drug-loaded liposomes loaded with evolocumab and shikonin, the drug-loaded liposomes being coated with macrophage membranes modified with phospholipid-modified hyaluronic acid.

[0034] In the above technical solution, liposomes are used to simultaneously load the lipid-lowering drug evolocumab and the anti-inflammatory drug shikonin, and a macrophage membrane is hybridized on the surface of nanoparticles to obtain a dual-targeting biomimetic liposome composite material. This composite material has a long blood circulation half-life and good targeting ability. It co-loads evolocumab and shikonin, and uses evolocumab to lower lipids and shikonin to reduce inflammation and achieve anti-atherosclerotic effects, thereby realizing targeted therapy for atherosclerosis.

[0035] The preparation method of the above-mentioned dual-targeting biomimetic liposome composite material includes the following steps:

[0036] Evolomab, shikonin, cholesterol, and lecithin were dissolved in a solvent, water was added, and the mixture was sonicated, evaporated under reduced pressure, hydrated, and dialyzed to obtain a liposome dispersion loaded with evolomab and shikonin.

[0037] The liposome dispersion loaded with evolocumab and shikonin was mixed with a biomimetic membrane solution and repeatedly extruded 8–10 times using a micro-extruder with a pore size of 220 nm. Then, a phospholipid-modified hyaluronic acid solution was added, and the mixture was stirred and reacted at 30°C–37°C to obtain a dual-targeting biomimetic liposome composite material. If the reaction temperature is below 30°C, the resulting composite material will have a large and uneven particle size, leading to unstable performance. If the reaction temperature is too high, above 37°C, the efficacy of evolocumab will be reduced, failing to achieve the desired effect.

[0038] In a preferred embodiment, the mass ratio of the biomimetic membrane to the liposomes loaded with evolocumab and shikonin is 1–2:10. If the mass ratio is not within the above-defined range, the efficacy will be affected to varying degrees. If the mass ratio is 0.5:10, i.e., the biomimetic membrane mass is low, immune escape will occur, blood circulation time will be prolonged, and efficacy will be greatly reduced. If the mass ratio is 3:10, i.e., the concentration of the biomimetic membrane is high, the stability of the liposomes loaded with evolocumab and shikonin will decrease.

[0039] To achieve better targeting of the composite material, the mass ratio of the liposomes loaded with evolocumab and shikonin to phospholipidized hyaluronic acid is 1:0.1–0.25. If the proportion of phospholipidized hyaluronic acid is too low, the targeting ability to damage endothelium and inflammatory macrophages will be poor; if the proportion of phospholipidized hyaluronic acid is too high, the hyaluronic acid loading will be insufficient, and the drug will not achieve optimal specific targeting.

[0040] To further achieve better lipid-lowering and anti-inflammatory effects, the mass ratio of evolocumab, lecithin, and cholesterol is 3–6:20:10; the mass ratio of shikonin, lecithin, and cholesterol is 1–4:20:10, and a more preferred mass ratio is 5:3:20:10. At the above loading levels of evolocumab and shikonin, better lipid-lowering and anti-inflammatory effects can be achieved.

[0041] It should be noted that the preparation method of the biomimetic membrane solution used in this invention includes the following steps: the macrophage membrane is ultrasonically disrupted at a power of 30-50W for 3-5 minutes, repeatedly frozen and thawed at -80℃ and 37℃ 2-3 times, centrifuged at 10000-13000rpm, and then dissolved in PBS to obtain the biomimetic membrane solution.

[0042] The preparation method of the phospholipid-modified hyaluronic acid solution used in this invention includes the following steps: dissolving hyaluronic acid and an activator in water, stirring for 30-60 min, adding phospholipid polyethylene glycol amino, stirring at 600-800 rpm for 12-24 h to obtain a mixture, and dialyzing the mixture using a dialysis bag with a molecular weight cutoff of 3-3.5 kDa for 12-24 h to obtain the phospholipid-modified hyaluronic acid solution.

[0043] In a preferred embodiment, the activator is 1(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, wherein the mass ratio of 1(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and hyaluronic acid is 5:10:0.8 to 1.2.

[0044] The effects are explained below with reference to specific embodiments.

[0045] Example 1

[0046] A method for preparing a dual-targeting biomimetic liposome composite material includes the following steps:

[0047] S1. Preparation of liposome dispersion loaded with evolocumab and shikonin: The liposome dispersion was prepared using a reverse evaporation method. The specific procedure was as follows: 20 mg lecithin and 10 mg cholesterol were dissolved in 2 mL of chloroform, and 5 mg evolocumab and 3 mg shikonin were dissolved in 2 mL of chloroform. All these were added to a 250 mL round-bottom flask, along with 6 mL anhydrous diethyl ether and 4 mL ddH2O. After thorough mixing, the mixture was sonicated in a water bath (50 W) for 5 min to form a homogeneous W / O emulsion. The emulsion was then subjected to rotary evaporation under reduced pressure until the organic solvent was completely evaporated, resulting in a homogeneous gel phase at the bottom of the flask. Finally, 10 mL of liposome dispersion was added... 2% Tween 80 PBS buffer was used to sonicate the round-bottom flask to ensure that the liposomes on the flask wall were completely detached into the buffer solution. Rotary evaporation was then continued to remove residual organic solvents and form a liposome suspension with uniform particle size. The liposome suspension was then dialyzed in a 300 kDa dialysis bag for 12 h to remove free evolocumab and shikonin, resulting in a liposome dispersion loaded with evolocumab and shikonin, denoted as L@ES NPs.

[0048] S2. Preparation of biomimetic membrane dispersion: Macrophage membrane was prepared according to the instructions of the membrane protein extraction kit. Macrophages were washed twice with PBS (pre-cooled at 4℃) and resuspended in membrane protein extraction agent A (containing 1mM PMSF (phenylmethylsulfonyl fluoride)). The macrophages were lysed on ice for 30 min and sonicated in a water bath at 4℃ for 10 min (80W). The macrophage membrane was repeatedly frozen and thawed five times at -80℃ and 37℃ for 30 min each time. The macrophage membrane precipitate was obtained by centrifugation at 12000 rpm and 4℃ for 30 min, and denoted as M.

[0049] S3. Preparation of phospholipid-modified hyaluronic acid: 8 mg EDC (1-(3-dimethylaminopropyl)3-ethylcarbodiimide hydrochloride), 16 mg NHS (N-hydroxysuccinimide), and 1 mg hyaluronic acid were dissolved in PBS and stirred at 800 rpm for 30 min at room temperature to activate the carboxyl groups on the surface of the hyaluronic acid. Subsequently, 1 mg DSPE-PEG2000-NH2 (phospholipid polyethylene glycol amino) was added to the above solution and stirred at 800 rpm for 24 h at room temperature. The above mixed solution was dialyzed in a 3.5 kDa dialysis bag for 24 h to remove free EDC, NHS, and DSPE-PEG2000-NH2. Finally, the purified material was lyophilized using a freeze-drying method. The lyophilized phospholipid-modified hyaluronic acid was stored at -20°C for later use and denoted as HA.

[0050] S4. Preparation of dual-targeting biomimetic liposome nanomedicine composite material: liposomes and biomimetic membrane dispersions loaded with evolocumab and shikonin were prepared at a mass ratio of 10:1. 1 mg of L@ES NPs and 0.1 mg of biomimetic membrane were resuspended in 1 mL of PBS and repeatedly extruded 10 times through a micro-extruder with a pore size of 220 nm to obtain a biomimetic liposome nanomedicine composite material co-loaded with evolocumab and shikonin (denoted as ML@ES). Phospholipid-modified hyaluronic acid was redissolved in PBS solution, and 250 μg of the phospholipid-modified hyaluronic acid solution was added to the ML@ES NPs solution. The mixture was stirred in a water bath at 37 °C and 800 rpm for 30 min to obtain a dual-targeting biomimetic nanomedicine, namely HA-ML@ES NPs.

[0051] Example 2

[0052] A method for preparing a dual-targeting biomimetic liposome composite material includes the following steps:

[0053] S1. Preparation of liposome dispersion loaded with evolocumab and shikonin: The liposome dispersion was prepared using a reverse evaporation method. The specific procedure was as follows: 20 mg lecithin and 10 mg cholesterol were dissolved in 2 mL of chloroform, and 3 mg evolocumab and 1 mg shikonin were dissolved in 2 mL of chloroform. All these were added to a 250 mL round-bottom flask, along with 6 mL anhydrous diethyl ether and 4 mL ddH2O. After thorough mixing, the mixture was sonicated in a water bath (50 W) for 5 min to form a homogeneous W / O emulsion. The emulsion was then subjected to rotary evaporation under reduced pressure until the organic solvent was completely evaporated, resulting in a homogeneous gel phase at the bottom of the flask. Finally, 10 mL of liposome dispersion was added... 2% Tween 80 PBS buffer was used to sonicate the round-bottom flask to ensure that the liposomes on the flask wall were completely detached into the buffer solution. Rotary evaporation was then continued to remove residual organic solvents and form a liposome suspension with uniform particle size. The liposome suspension was then dialyzed in a 300 kDa dialysis bag for 12 h to remove free evolocumab and shikonin, resulting in a liposome dispersion loaded with evolocumab and shikonin, denoted as L@ES NPs.

[0054] S2. Preparation of biomimetic membrane dispersion: Macrophage membrane was prepared according to the instructions of the membrane protein extraction kit. Macrophages were washed twice with PBS (pre-cooled at 4℃) and resuspended in membrane protein extraction agent A (containing 1mM PMSF (phenylmethylsulfonyl fluoride)). The macrophages were lysed on ice for 30 min and sonicated in a water bath at 4℃ for 10 min (80W). The macrophage membrane was repeatedly frozen and thawed five times at -80℃ and 37℃ for 30 min each time. The macrophage membrane precipitate was obtained by centrifugation at 12000 rpm and 4℃ for 30 min, and denoted as M.

[0055] S3. Preparation of phospholipid-modified hyaluronic acid: 8 mg EDC (1-(3-dimethylaminopropyl)3-ethylcarbodiimide hydrochloride), 16 mg NHS (N-hydroxysuccinimide), and 1 mg hyaluronic acid were dissolved in PBS and stirred at 800 rpm for 30 min at room temperature to activate the carboxyl groups on the surface of the hyaluronic acid. Subsequently, 1 mg DSPE-PEG2000-NH2 (phospholipid polyethylene glycol amino) was added to the above solution and stirred at 800 rpm for 24 h at room temperature. The above mixed solution was dialyzed in a 3.5 kDa dialysis bag for 24 h to remove free EDC, NHS, and DSPE-PEG2000-NH2. Finally, the purified material was lyophilized using a freeze-drying method. The lyophilized phospholipid-modified hyaluronic acid was stored at -20°C for later use and denoted as HA.

[0056] S4. Preparation of dual-targeting biomimetic liposome nanomedicine composite material: liposomes and biomimetic membrane dispersions loaded with evolocumab and shikonin were prepared at a mass ratio of 10:1. 1 mg of L@ES NPs and 0.1 mg of biomimetic membrane were resuspended in 1 mL of PBS and repeatedly extruded 10 times through a micro-extruder with a pore size of 220 nm to obtain a biomimetic liposome nanomedicine composite material co-loaded with evolocumab and shikonin (denoted as ML@ES). Phospholipid-modified hyaluronic acid was redissolved in PBS solution, and 250 μg of the phospholipid-modified hyaluronic acid solution was added to the ML@ES NPs solution. The mixture was stirred in a water bath at 37 °C and 800 rpm for 30 min to obtain a dual-targeting biomimetic nanomedicine, namely HA-ML@ES NPs.

[0057] Example 3

[0058] A method for preparing a dual-targeting biomimetic liposome composite material includes the following steps:

[0059] S1. Preparation of liposome dispersion loaded with evolocumab and shikonin: The liposome dispersion was prepared using a reverse evaporation method. The specific procedure was as follows: 20 mg lecithin and 10 mg cholesterol were dissolved in 2 mL of chloroform, and 6 mg evolocumab and 4 mg shikonin were dissolved in 2 mL of chloroform. All these were added to a 250 mL round-bottom flask, along with 6 mL anhydrous diethyl ether and 4 mL ddH2O. After thorough mixing, the mixture was sonicated in a water bath (50 W) for 5 min to form a homogeneous W / O emulsion. The emulsion was then subjected to rotary evaporation under reduced pressure until the organic solvent was completely evaporated, resulting in a homogeneous gel phase at the bottom of the flask. Finally, 10 mL of the emulsion was added... 2% Tween 80 PBS buffer was used to sonicate the round-bottom flask to ensure that the liposomes on the flask wall were completely detached into the buffer solution. Rotary evaporation was then continued to remove residual organic solvents and form a liposome suspension with uniform particle size. The liposome suspension was then dialyzed in a 300 kDa dialysis bag for 12 h to remove free evolocumab and shikonin, resulting in a liposome dispersion loaded with evolocumab and shikonin, denoted as L@ES NPs.

[0060] S2. Preparation of biomimetic membrane dispersion: Macrophage membrane was prepared according to the instructions of the membrane protein extraction kit. Macrophages were washed twice with PBS (pre-cooled at 4℃) and resuspended in membrane protein extraction agent A (containing 1mM PMSF (phenylmethylsulfonyl fluoride)). The macrophages were lysed on ice for 30 min and sonicated in a water bath at 4℃ for 10 min (80W). The macrophage membrane was repeatedly frozen and thawed five times at -80℃ and 37℃ for 30 min each time. The macrophage membrane precipitate was obtained by centrifugation at 12000 rpm and 4℃ for 30 min, and denoted as M.

[0061] S3. Preparation of phospholipid-modified hyaluronic acid: 8 mg EDC (1-(3-dimethylaminopropyl)3-ethylcarbodiimide hydrochloride), 16 mg NHS (N-hydroxysuccinimide), and 1 mg hyaluronic acid were dissolved in PBS and stirred at 800 rpm for 30 min at room temperature to activate the carboxyl groups on the surface of the hyaluronic acid. Subsequently, 1 mg DSPE-PEG2000-NH2 (phospholipid polyethylene glycol amino) was added to the above solution and stirred at 800 rpm for 24 h at room temperature. The above mixed solution was dialyzed in a 3.5 kDa dialysis bag for 24 h to remove free EDC, NHS, and DSPE-PEG2000-NH2. Finally, the purified material was lyophilized using a freeze-drying method. The lyophilized phospholipid-modified hyaluronic acid was stored at -20°C for later use and denoted as HA.

[0062] S4. Preparation of dual-targeting biomimetic liposome nanomedicine composite material: liposomes and biomimetic membrane dispersions loaded with evolocumab and shikonin were prepared at a mass ratio of 10:1. 1 mg of L@ES NPs and 0.1 mg of biomimetic membrane were resuspended in 1 mL of PBS and repeatedly extruded 8 times through a micro-extruder with a pore size of 220 nm to obtain a biomimetic liposome nanomedicine composite material co-loaded with evolocumab and shikonin (denoted as ML@ES). Phospholipid-modified hyaluronic acid was redissolved in PBS solution, and 150 μg of the phospholipid-modified hyaluronic acid solution was added to the ML@ES NPs solution. The mixture was stirred in a water bath at 37 °C and 800 rpm for 30 min to obtain a dual-targeting biomimetic nanomedicine, namely HA-ML@ES NPs.

[0063] Example 4

[0064] A method for preparing a dual-targeting biomimetic liposome composite material includes the following steps:

[0065] S1. Preparation of liposome dispersion loaded with evolocumab and shikonin: The liposome dispersion was prepared using a reverse evaporation method. The specific procedure was as follows: 20 mg lecithin and 10 mg cholesterol were dissolved in 2 mL of chloroform, and 5 mg evolocumab and 3 mg shikonin were dissolved in 2 mL of chloroform. All these were added to a 250 mL round-bottom flask, along with 6 mL anhydrous diethyl ether and 4 mL ddH2O. After thorough mixing, the mixture was sonicated in a water bath (50 W) for 5 min to form a homogeneous W / O emulsion. The emulsion was then subjected to rotary evaporation under reduced pressure until the organic solvent was completely evaporated, resulting in a homogeneous gel phase at the bottom of the flask. Finally, 10 mL of liposome dispersion was added... 2% Tween 80 PBS buffer was used to sonicate the round-bottom flask to ensure that the liposomes on the flask wall were completely detached into the buffer solution. Rotary evaporation was then continued to remove residual organic solvents and form a liposome suspension with uniform particle size. The liposome suspension was then dialyzed in a 300 kDa dialysis bag for 12 h to remove free evolocumab and shikonin, resulting in a liposome dispersion loaded with evolocumab and shikonin, denoted as L@ES NPs.

[0066] S2. Preparation of biomimetic membrane dispersion: Macrophage membrane was prepared according to the instructions of the membrane protein extraction kit. Macrophages were washed twice with PBS (pre-cooled at 4℃) and resuspended in membrane protein extraction agent A (containing 1mM PMSF (phenylmethylsulfonyl fluoride)). The macrophages were lysed on ice for 30 min and sonicated in a water bath at 4℃ for 10 min (80W). The macrophage membrane was repeatedly frozen and thawed five times at -80℃ and 37℃ for 30 min each time. The macrophage membrane precipitate was obtained by centrifugation at 12000 rpm and 4℃ for 30 min, and denoted as M.

[0067] S3. Preparation of phospholipid-modified hyaluronic acid: 8 mg EDC (1-(3-dimethylaminopropyl)3-ethylcarbodiimide hydrochloride), 16 mg NHS (N-hydroxysuccinimide), and 1 mg hyaluronic acid were dissolved in PBS and stirred at 800 rpm for 30 min at room temperature to activate the carboxyl groups on the surface of the hyaluronic acid. Subsequently, 1 mg DSPE-PEG2000-NH2 (phospholipid polyethylene glycol amino) was added to the above solution and stirred at 800 rpm for 24 h at room temperature. The above mixed solution was dialyzed in a 3.5 kDa dialysis bag for 24 h to remove free EDC, NHS, and DSPE-PEG2000-NH2. Finally, the purified material was lyophilized using a freeze-drying method. The lyophilized phospholipid-modified hyaluronic acid was stored at -20°C for later use and denoted as HA.

[0068] S4. Preparation of dual-targeting biomimetic liposome nanomedicine composite material: liposomes and biomimetic membrane dispersions loaded with evolocumab and shikonin were prepared at a mass ratio of 10:2. 1 mg of L@ES NPs and 0.2 mg of biomimetic membrane were resuspended in 1 mL of PBS and repeatedly extruded 9 times through a micro-extruder with a pore size of 220 nm to obtain a biomimetic liposome nanomedicine composite material co-loaded with evolocumab and shikonin (denoted as ML@ES). Phospholipid-modified hyaluronic acid was redissolved in PBS solution, and 100 μg of phospholipid-modified hyaluronic acid solution was added to the ML@ES NPs solution. The mixture was stirred in a water bath at 37℃ and 800 rpm for 30 min to obtain a dual-targeting biomimetic nanomedicine, namely HA-ML@ES NPs.

[0069] Example 5

[0070] A method for preparing a dual-targeting biomimetic liposome composite material includes the following steps:

[0071] S1. Preparation of liposome dispersion loaded with evolocumab and shikonin: The liposome dispersion was prepared using a reverse evaporation method. The specific procedure was as follows: 20 mg lecithin and 10 mg cholesterol were dissolved in 2 mL of chloroform, and 6 mg evolocumab and 1 mg shikonin were dissolved in 2 mL of chloroform. All these were added to a 250 mL round-bottom flask, along with 6 mL anhydrous diethyl ether and 4 mL ddH2O. After thorough mixing, the mixture was sonicated in a water bath (50 W) for 5 min to form a homogeneous W / O emulsion. The emulsion was then subjected to rotary evaporation under reduced pressure until the organic solvent was completely evaporated, resulting in a homogeneous gel phase at the bottom of the flask. Finally, 10 mL of liposome dispersion was added... 2% Tween 80 PBS buffer was used to sonicate the round-bottom flask to ensure that the liposomes on the flask wall were completely detached into the buffer solution. Rotary evaporation was then continued to remove residual organic solvents and form a liposome suspension with uniform particle size. The liposome suspension was then dialyzed in a 300 kDa dialysis bag for 12 h to remove free evolocumab and shikonin, resulting in a liposome dispersion loaded with evolocumab and shikonin, denoted as L@ES NPs.

[0072] S2. Preparation of biomimetic membrane dispersion: Macrophage membrane was prepared according to the instructions of the membrane protein extraction kit. Macrophages were washed twice with PBS (pre-cooled at 4℃) and resuspended in membrane protein extraction agent A (containing 1mM PMSF (phenylmethylsulfonyl fluoride)). The macrophages were lysed on ice for 30 min and sonicated in a water bath at 4℃ for 10 min (80W). The macrophage membrane was repeatedly frozen and thawed five times at -80℃ and 37℃ for 30 min each time. The macrophage membrane precipitate was obtained by centrifugation at 12000 rpm and 4℃ for 30 min, and denoted as M.

[0073] S3. Preparation of phospholipid-modified hyaluronic acid: 8 mg EDC (1-(3-dimethylaminopropyl)3-ethylcarbodiimide hydrochloride), 16 mg NHS (N-hydroxysuccinimide), and 1 mg hyaluronic acid were dissolved in PBS and stirred at 800 rpm for 30 min at room temperature to activate the carboxyl groups on the surface of the hyaluronic acid. Subsequently, 1 mg DSPE-PEG2000-NH2 (phospholipid polyethylene glycol amino) was added to the above solution and stirred at 800 rpm for 24 h at room temperature. The above mixed solution was dialyzed in a 3.5 kDa dialysis bag for 24 h to remove free EDC, NHS, and DSPE-PEG2000-NH2. Finally, the purified material was lyophilized using a freeze-drying method. The lyophilized phospholipid-modified hyaluronic acid was stored at -20°C for later use and denoted as HA.

[0074] S4. Preparation of dual-targeting biomimetic liposome nanomedicine composite material: liposomes and biomimetic membrane dispersions loaded with evolocumab and shikonin were prepared at a mass ratio of 10:1.5. 1 mg of L@ES NPs and 0.15 mg of biomimetic membrane were resuspended in 1 mL of PBS and repeatedly extruded 10 times through a micro-extruder with a pore size of 220 nm to obtain a biomimetic liposome nanomedicine composite material co-loaded with evolocumab and shikonin (denoted as ML@ES). Phospholipid-modified hyaluronic acid was redissolved in PBS solution, and 200 μg of the phospholipid-modified hyaluronic acid solution was added to the ML@ES NPs solution. The mixture was stirred in a water bath at 37 °C and 800 rpm for 30 min to obtain a dual-targeting biomimetic nanomedicine, namely HA-ML@ES NPs.

[0075] The performance of the dual-targeting biomimetic liposome composite material prepared above was tested, and the specific process and results are as follows.

[0076] Figure 1 Transmission electron microscopy (TEM) images and particle size potential analysis of L@ES NPs and HA-ML@ES NPs prepared in Example 1 of this invention are shown. Image A shows L@ES NPs, and image B shows HA-ML@ES NPs. Figure 1 As shown, transmission electron microscopy (TEM) images of the L@ESNPs and HA-ML@ES NPs prepared in Example 1 were analyzed. The results showed that uniformly dispersed spherical L@ES NPs nanoparticles were successfully prepared with a particle size of approximately 150 nm. The HA-modified biomimetic membrane liposomes, after being co-loaded with evolocumab and shikonin, had a uniform particle size of approximately 180 nm.

[0077] Figure 2 These are images of the biomimetic membrane of this invention, stained with Coomassie Brilliant Blue and showing Western blotting of proteins. Image A shows Coomassie Brilliant Blue staining, and image B shows Western blotting of proteins. Figure 2 As shown, the biomimetic membrane prepared in Example 1 was analyzed for membrane characteristic proteins. The results showed that the biomimetic membrane successfully disguised itself on the surface of the nanocomposite and retained the characteristic proteins of the cell membrane surface.

[0078] The biosafety, pharmacokinetics, and targeting properties of the dual-targeting biomimetic liposome composite material prepared in this invention were investigated.

[0079] Biosafety verification of dual-targeted biomimetic liposome composite materials

[0080] Figure 3 The biosafety of the dual-targeting biomimetic liposome composite material HA-ML@ES NPs prepared in Example 1 of this invention was investigated. Figure 3 As shown in Figure A, in the hemolysis experiment, compared with the H2O treatment group, the hemolysis rates of both the Evol+SKN and HA-ML@ES groups were less than 5%, and from... Figure 3 No atypical red blood cells were found in the red blood cell morphology image in Figure B, indicating that intravenous administration is safe.

[0081] Using the dual-targeting biomimetic liposome nanomedicine composite material prepared in Example 1, the cellular uptake, blood half-life, and in vivo targeting of the HA-ML@ES NPs biomimetic nanomaterial were determined by a semi-quantitative method of detecting fluorescence intensity.

[0082] Figure 4 Using transwell chambers, endothelial cells were seeded on the upper layer and macrophages on the lower layer to simulate vascular plaques. After adding Dil-labeled HA-ML@ES NPs to the upper layer and culturing for 4 hours, the cells were washed and fixed with PBS and then confocal images were taken. Compared with the normal group, it was observed that the Dil fluorescence signal in endothelial cells and macrophages was enhanced after Hcy stimulation. Figure 4 Figure A in the diagram is a schematic diagram of the experimental principle. The upper layer consists of endothelial cells, and the lower layer consists of macrophages, simulating the environment of atherosclerotic plaques in vitro. Figure B illustrates that HA-ML@SE has a stronger ability to target endothelial cells and macrophages than L@ES.

[0083] Figure 5 HA-ML was prepared using dihydroporphyrin e6 (Ce6) and Ce6-labeled HA-ML@ES NPs. ce6 Kunming mice were injected via tail vein with 200 μL of Ce6 and HA-ML at a concentration of 5 mg / kg. ce6 Subsequently, blood samples were collected at different time points (0h, 6h, 12h, 18h, 24h) for fluorescence intensity measurement.

[0084] Among them, L Ce6 It was prepared by the following method: L was prepared by reverse evaporation. Ce6 The specific procedure for NPs is as follows: Dissolve 20 mg of lecithin and 10 mg of cholesterol in 2 mL of chloroform, and dissolve 10 mg of Ce6 in 2 mL of chloroform. Add both solutions to a 250 mL round-bottom flask, add 6 mL of anhydrous diethyl ether and 4 mL of ddH2O, mix thoroughly, and sonicate in a water bath (50 W) for 5 min to form a homogeneous W / O emulsion. Rotary evaporation under reduced pressure continues until the organic solvent is completely evaporated, forming a homogeneous gel phase at the bottom of the flask. Then, add 10 mL of 2% Tween 80 PBS buffer, and perform simple sonication on the round-bottom flask to ensure that the liposomes on the flask wall are completely detached into the buffer solution. Continue rotary evaporation to remove residual organic solvent and form a liposome suspension with uniform particle size. Dialyze the liposome suspension in a 300 kDa dialysis bag for 12 h to remove free Ce6.

[0085] HA-ML Ce6The liposomes loaded with CE6 were prepared as follows: a biomimetic membrane (M) (1 mL, 1 mg / mL) subjected to ultrasonic treatment in a water bath was mixed at a mass ratio of 10:1, and repeatedly extruded through a micro-extruder with a pore size of 220 nm at least 10 times to obtain ML. Ce6 Biomimetic nano-formulation: Phospholipid-modified hyaluronic acid was redissolved in PBS solution. 5 mg of the phospholipid-modified hyaluronic acid solution was added to MLCe6 solution, and the mixture was stirred in a water bath at 37°C and 600–800 rpm for 60 min to obtain HA-ML. Ce6 .

[0086] Figure 5 This represents the blood half-life of the HA-ML@ES NPs of this invention in C57 mice. The results are as follows: Figure 5 As shown, Ce6 and HA-ML Ce6 The blood circulation half-lives were 0.52 h and 1.31 h, respectively. This indicates that the biomimetic membrane-masked nano-formulation is beneficial for prolonging blood circulation time.

[0087] ApoE - / - Mice were fed a high-methionine diet for 3 months and then injected via tail vein with 200 μL of Ce6 and HA-ML at a concentration of 5 mg / kg. Ce6 Twelve hours later, the mouse aorta was harvested for fluorescence imaging.

[0088] Figure 6 This demonstrates the targeting ability of the HA-ML@ES NPs of this invention at atherosclerotic plaque sites in mice. The results are as follows: Figure 6 As shown, with Ce6 and HA-ML Ce6 Compared to the group, HA-ML Ce6 The group showed significant aggregation at the aortic plaque site, indicating that the HA-ML@ES NPs biomimetic nano-formulation prepared in Example 1 can effectively target atherosclerotic plaque sites.

[0089] The HA-ML@ES NPs biomimetic nanoparticle formulation prepared in Example 1 was used to inhibit endothelial cell apoptosis and foam cell formation.

[0090] After incubating human umbilical vein endothelial cells (HUVECs) with 100 μM Hcy for 24 hours, we further incubated the cells with Evol+SKN and HA-ML@ES NPs for an additional 24 hours. Apoptosis of HUVECs was assessed using flow cytometry and annexin V-FITC / PI assay, following the manufacturer's instructions (BD, CA, USA).

[0091] After incubating with 100 μM Hcy for 24 hours, RAW264.7 cells were further treated with Evol+SKN and HA-ML@ES NPs at the same concentrations of Evol (1.25 nM) and SKN (250 nM). Then, ox-LDL (80 μg / mL) was added and the cells were incubated for 48 hours before staining with 3% Oil Red O (ORO).

[0092] Figure 7 HA-ML@ES NPs biomimetic nanoparticle formulations are used to inhibit endothelial cell apoptosis and foam cell formation. Figure 7 Figure A shows the flow cytometry results of endothelial cell apoptosis; control is the negative group, model is the positive group, and Evol+SKN represents the free Evol+SKN combined with the drug. Figure B shows Oil Red O staining of foam cell formation to detect the accumulation of oxLDL in macrophages. Figure 7 As shown, the HA-ML@ES NPs biomimetic nanoparticle formulation effectively inhibits endothelial cell apoptosis and foam cell formation.

[0093] The HA-ML@ES NPs biomimetic nanoformulation prepared in Example 1 was used for the early treatment of atherosclerosis.

[0094] ApoE - / - After one month of a high-methionine diet, mice were given an early preventative treatment group. The treatment groups were PBS, Evol+SKN group (2.5 mg / kg Evol + 1.2 mg / kg SKN), and HA-ML@ES NPs group (2.5 mg / kg Evol + 1.2 mg / kg SKN), administered twice a week for two months, during which time they were on a high-methionine diet. Three days after the end of treatment, the aorta of the mice was dissected and Oil Red O staining was performed to examine the therapeutic effects of different treatment groups.

[0095] Figure 8 These are bright-field images and gross staining images of the aortic arch in different treatment groups of atherosclerosis according to the present invention. Results are as follows: Figure 8 As shown, the HA-ML@ES NPs treatment group significantly reduced the formation of atherosclerotic plaques in the early treatment regimen, indicating that the biomimetic nano-formulation constructed in this invention can effectively prevent the early progression of atherosclerosis.

[0096] 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 dual-targeting biomimetic liposome composite material, characterized in that, The composite material includes drug-loaded liposomes loaded with evolocumab and shikonin, and the drug-loaded liposomes are coated with macrophage membranes modified with phospholipid-modified hyaluronic acid. The preparation method of the above-mentioned dual-targeting biomimetic liposome composite material includes the following steps: Evolomab, shikonin, cholesterol and lecithin were dissolved in a solvent, water was added, and the mixture was sonicated, evaporated under reduced pressure, hydrated and dialyzed to obtain a liposome dispersion loaded with evolomab and shikonin. The liposome dispersion loaded with evolocumab and shikonin was mixed with a biomimetic membrane solution, repeatedly extruded, and then a phospholipid-modified hyaluronic acid solution was added. The mixture was stirred and reacted at 30℃~37℃ to obtain a dual-targeting biomimetic liposome composite material. The mass ratio of the biomimetic membrane to the liposomes loaded with evolocumab and shikonin was 1~2:10; the mass ratio of the liposomes loaded with evolocumab and shikonin to the phospholipid-modified hyaluronic acid was 1:0.1~0.25; the repeated extrusion was performed by repeatedly extruding 8~10 times using a micro-extruder with a pore size of 220nm.

2. The dual-targeting biomimetic liposome composite material according to claim 1, characterized in that, The mass ratio of evolocumab, lecithin, and cholesterol is 3–6:20:10; the mass ratio of shikonin, lecithin, and cholesterol is 1–4:20:

10.

3. The dual-targeting biomimetic liposome composite material according to claim 1, characterized in that, The preparation method of the biomimetic membrane solution includes the following steps: the macrophage membrane is ultrasonically disrupted at a power of 30-50W for 3-5 minutes, repeatedly frozen and thawed at -80℃ and 37℃ 2-3 times, centrifuged at 10000-13000rpm, and then dissolved in PBS to obtain the biomimetic membrane solution.

4. The dual-targeting biomimetic liposome composite material according to claim 1, characterized in that, The preparation method of the phospholipid-modified hyaluronic acid solution includes the following steps: dissolving hyaluronic acid and an activator in water, stirring for 30-60 minutes, adding phospholipid polyethylene glycol amino, stirring at 600-800 rpm for 12-24 hours to obtain a mixture, and dialyzing the mixture for 12-24 hours using a dialysis bag with a molecular weight cutoff of 3-3.5 kDa to obtain the phospholipid-modified hyaluronic acid solution.

5. The dual-targeting biomimetic liposome composite material according to claim 4, characterized in that, The activator is 1(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, wherein the mass ratio of 1(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and hyaluronic acid is 5:10:0.8~1.

2.

6. The use of the dual-targeting biomimetic liposome composite material of claim 1 in the preparation of a medicament for treating atherosclerosis caused by hyperhomocysteinemia.

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