Double-targeting bionic liposome composite material as well as preparation method and application thereof

Abstract

The invention discloses a dual-targeting bionic liposome composite material and a preparation method and application thereof, and belongs to the technical field of nano-medices.The dual-targeting bionic liposome composite material is prepared by taking liposome nano-particles as a carrier, loading iloumab and alkannin on the carrier to obtain drug-loaded liposome, and fusing the drug-loaded liposome with a macrophage membrane to obtain the dual-targeting bionic liposome composite material. And modifying with phospholipidic hyaluronic acid to obtain the double-targeting bionic liposome composite material. According to the invention, two anti-atherosclerosis effects with different mechanisms, i.e., lipid lowering by Elolukumab and anti-inflammation by alkannin, are utilized to realize targeted therapy of atherosclerosis, and the bionic membrane on the outermost layer realizes long blood circulation and lesion site targeting ability of the composite material through inherent immune escape and recruitment characteristics of macrophages. The characteristic that hyaluronic acid interacts with a target cell surface receptor CD44 is utilized, effective utilization of the target cell on the composite material is achieved, and the atherosclerosis prevention and treatment effect of the composite material is improved.

Description

Technical Field

[0001] The invention relates to the technical field of nanomedicine, and in particular to a dual-targeting bionic liposome composite material and a preparation method and application thereof. Background Art

[0002] Atherosclerosis is the most common underlying cause of ischemic heart disease and stroke, accounting for a quarter of deaths worldwide. Hyperhomocysteinemia (HHcy) is considered an independent risk factor for atherosclerosis. It has been determined that HHcy can cause endothelial cell activation and dysfunction, and a large number of endothelial cell apoptosis is the initiating event for the development of atherosclerosis. In addition, increased secretion of intercellular adhesion molecules will recruit more macrophages, and endothelial barrier destruction and gap expansion will promote more lipoproteins and inflammatory macrophages to infiltrate the subendothelial layer. Lipoproteins are then phagocytosed by macrophages to form foam cells, thereby promoting the formation of atherosclerosis. The accumulation of lipids in inflammatory macrophages to form foam cells is considered to be a key event in the development of early atherosclerosis. Therefore, finding ways to alleviate endothelial cell dysfunction and inhibit foam cell formation is expected to be an effective treatment for atherosclerosis.

[0003] At present, the treatment of atherosclerosis includes lowering blood lipids, antiplatelet therapy and antihypertensive therapy. In lipid regulation, statins and proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitors are the cornerstones of drug treatment for dyslipidemia and play an important role in preventing and controlling atherosclerotic cardiovascular diseases at home and abroad.

[0004] Evol is a PCSK9-specific inhibitor that targets PCSK9 for early intervention in lipid metabolism, at least one step ahead of statins. In addition, in the development of early atherosclerosis, the inflammatory microenvironment at the plaque site causes PCSK9 to be activated, lipid uptake receptors (LOX-1) to increase, and lipid efflux receptors A1 / G1 (ABCA1 / G1) to decrease, ultimately leading to the continuous accumulation of lipids in macrophages to form foam cells. Evol can effectively inhibit PCSK9 and thus inhibit the formation of foam cells, thereby avoiding the formation of atherosclerosis. However, current clinical studies have shown that after patients stop taking Evol, their blood lipid levels will increase as PCSK9 increases, greatly increasing the risk of AS.

[0005] Shikonin (SKN) is the main active ingredient in the Chinese herbal medicine Lithospermum officinale. As an inhibitor of key enzymes in the glycolysis pathway, it can effectively inhibit the increase in glycolysis caused by homocysteine ​​(Hcy). At the same time, many evidences show that shikonin can inhibit endothelial cell apoptosis and improve endothelial cell function. At the same time, shikonin has good antioxidant, anti-inflammatory and anti-thrombotic effects, can inhibit the proliferation and migration of smooth muscle cells (SMC), regulate the polarization of macrophages, thereby inhibiting lipid accumulation to achieve the purpose of treating atherosclerosis. However, similar to traditional hydrophobic anti-inflammatory drugs, shikonin has a short circulation time, non-specific distribution and low drug utilization after oral or intravenous injection into the human circulation, which seriously hinders its clinical application. Summary of the invention

[0006] The present invention provides a dual-targeted bionic liposome composite material and a preparation method and application thereof, which effectively solves the problem that shikonin, as a hydrophobic anti-inflammatory drug, has a short circulation time in the human body, non-specific distribution and low drug utilization, further prolongs the blood circulation time of Evol and reduces the cost of treatment. The present invention uses liposomes to simultaneously load the lipid-lowering drug eloumab and the anti-inflammatory drug shikonin, and hybridizes a layer of macrophage membrane on the surface of nanoparticles to obtain a dual-targeted bionic liposome composite material, which has a long blood circulation half-life and good targeting ability, and co-loads eloumab and shikonin, and uses eloumab to reduce lipids and shikonin to carry out anti-inflammatory and anti-atherosclerotic effects, thereby achieving targeted treatment of atherosclerosis.

[0007] The first object of the present invention is to provide a dual-targeting bionic liposome composite material, which includes drug-loaded liposomes, wherein the drug-loaded liposomes are loaded with evolocumab and shikonin, and the drug-loaded liposomes are coated with macrophage membranes, and the macrophage membranes are modified with phospholipidated hyaluronic acid.

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

[0009] Elotumab, shikonin, cholesterol and lecithin are dissolved in a solvent, water is added, and the mixture is subjected to ultrasound, reduced pressure evaporation, hydration and dialysis to obtain a liposome dispersion loaded with Elotumab and shikonin.

[0010] The liposome dispersion loaded with evolocumab and shikonin and the biomimetic membrane solution are mixed, extruded repeatedly, and then the phospholipidated hyaluronic acid solution is added, stirred and reacted at 30° C. to 37° C. to obtain a dual-targeting biomimetic liposome composite material.

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

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

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

[0014] As a preferred embodiment, the repeated extrusion is performed 8 to 10 times using a micro extruder with a pore size of 220 nm.

[0015] As a preferred embodiment, the preparation method of the biomimetic membrane solution includes the following steps: ultrasonically disrupting the macrophage membrane at a power of 30 to 50 W for 3 to 5 minutes, repeatedly freezing and thawing at -80°C and 37°C for 2 to 3 times, centrifuging at 10,000 to 13,000 rpm, and then dissolving with PBS to obtain a biomimetic membrane solution.

[0016] As a preferred embodiment, the preparation method of the phospholipidated hyaluronic acid solution comprises the following steps: dissolving hyaluronic acid and an activator in water, stirring for 30 to 60 minutes, adding phospholipid polyethylene glycol amino, stirring at 600 to 800 rpm for 12 to 24 hours to obtain a mixed solution, and dialyzing the mixed solution for 12 to 24 hours using a dialysis bag with a molecular weight cutoff of 3 to 3.5 kDa to obtain a phospholipidated 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 5:10:0.8-1.2.

[0018] The third object of the present invention is to provide an application of the above dual-targeting bionic liposome composite material in the preparation of a drug for treating atherosclerosis caused by hyperhomocysteinemia.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] In view of the shortcomings of existing nano drugs that are easily metabolized and cleared quickly and lack active targeting, a dual-targeted biomimetic liposome composite material provided by the present invention is provided, with liposome nanoparticles as carriers, eloumab and shikonin are loaded on the carrier to obtain drug-loaded liposomes, the drug-loaded liposomes are fused with macrophage membranes to obtain drug-loaded liposomes coated with biomimetic membranes, and then the biomimetic membranes are modified by phospholipidated hyaluronic acid to obtain dual-targeted biomimetic liposome composite materials. Eloumab and shikonin are physically encapsulated by liposomes at the same time, the biomimetic membranes are fused with liposomes, and phospholipidated hyaluronic acid is inserted into the surface of the biomimetic membranes. The design features of biomimetic membranes and hyaluronic acid modification are used to achieve specific aggregation and penetration of drugs at lesions, and the bioavailability of drugs to target cells is improved at the same time. The present invention utilizes eloumab to reduce lipids and shikonin to carry out anti-inflammatory two different mechanisms of anti-atherosclerotic effects, and achieves targeted treatment of atherosclerosis. The inflammatory environment at the site of atherosclerotic plaques secretes chemokines to recruit macrophages, and the outermost bionic membrane achieves the ability of the composite material to have long blood circulation and target the lesion site through the inherent immune escape and recruitment characteristics of its source cells (macrophages). By utilizing the interaction characteristics of hyaluronic acid and the target cell surface receptor CD44, the target cells can effectively utilize the composite material and improve its effect in preventing and treating atherosclerosis.

[0021] The present invention improves the blood circulation half-life of the drug, while achieving a targeted effect on atherosclerotic plaque sites and target cells, providing new theoretical support for the development of anti-atherosclerotic drugs and related clinical detection and treatment, and has important scientific significance, practical value and economic value.

[0022] In addition to loading drugs for treating atherosclerosis, the co-drug-loaded bionic liposome composite material provided by the present invention can be used as an alternative platform for treating other diseases, such as tumors, rheumatoid arthritis, etc., by replacing different bionic membrane types and drugs and utilizing the "homing effect" of the bionic membrane and the action mechanism of different drugs. Therefore, the present invention combines the bionic membrane coating and the targeted modified nanocomposite material, which can provide multiple functions and advantages to treat multiple diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Transmission electron micrographs of liposomes loaded with evolocumab and shikonin (L@ES NPs) and dual-targeted biomimetic liposome composites (HA-ML@ES NPs) prepared in Example 1 of the present invention; wherein, Figure A is L@ES NPs, and Figure B is HA-ML@ES NPs.

[0024] Figure 2 The Coomassie Brilliant Blue staining diagram of the biomimetic membrane of Example 1 of the present invention; wherein, Figure A is Coomassie Brilliant Blue staining, and Figure B is protein immunoblotting.

[0025] Figure 3 The biological safety of the dual-targeted bionic liposome composite material HA-ML@ES NPs prepared in Example 1 of the present invention, wherein Figure A is the hemolysis rate and Figure B is the red blood cell morphology image.

[0026] Figure 4 The dual-targeted bionic liposome composite material HA-ML@ES NPs prepared in Example 1 of the present invention targets damaged endothelial cells and inflammatory macrophages, wherein 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 is the blood half-life of the HA-ML@ES NPs of the present invention in Kunming mice.

[0028] Figure 6 The HA-M@ES NPs of the present invention are targeted to atherosclerotic plaques in mice.

[0029] Figure 7 The HA-ML@ES NPs of the present invention inhibit endothelial cell apoptosis and inhibit macrophage lipid accumulation, wherein Figure A is the flow cytometry result of endothelial cell apoptosis, control is the negative group, model is the positive group, and Evol+SKN is the free combination of the two drugs; Figure B is the Oil Red O staining of foam cell formation, detecting the accumulation of oxLDL in macrophages.

[0030] Figure 8 The HA-M@ES NPs of the present invention can treat early atherosclerosis formation in mice. DETAILED DESCRIPTION

[0031] In order to enable those skilled in the art to better understand the technical solution of the present invention and implement it, the present invention is further described below in conjunction with specific embodiments and drawings, but the embodiments are not intended to limit the present invention. The following test methods and detection methods, unless otherwise specified, are conventional methods; the reagents and raw materials, unless otherwise specified, are commercially available.

[0032] The technical contents of the present invention are analyzed and explained in detail below.

[0033] The present invention provides a dual-targeting bionic liposome composite material, which comprises a drug-loaded liposome loaded with evolocumab and shikonin, and coated with a macrophage membrane, wherein the macrophage membrane is modified with phospholipidated hyaluronic acid.

[0034] In the above technical scheme, liposomes are used to simultaneously load the lipid-lowering drug eloumab and the anti-inflammatory drug shikonin, and a layer of macrophage membrane is hybridized on the surface of the nanoparticles to obtain a dual-targeted bionic liposome composite material. The composite material has a long blood circulation half-life and good targeting ability, and co-loads eloumab and shikonin, using eloumab for lipid-lowering effects and shikonin for anti-inflammatory and anti-atherosclerotic effects, thereby achieving targeted treatment of atherosclerosis.

[0035] The preparation method of the dual-targeting bionic liposome composite material comprises the following steps:

[0036] Elotumab, shikonin, cholesterol and lecithin are dissolved in a solvent, water is added, and the mixture is subjected to ultrasound, reduced pressure evaporation, hydration and dialysis to obtain a liposome dispersion loaded with Elotumab and shikonin.

[0037] The liposome dispersion loaded with evolocumab and shikonin and the biomimetic membrane solution are mixed, and the mixture is repeatedly extruded 8 to 10 times using a micro extruder with a pore size of 220 nm, and then the phospholipidated hyaluronic acid solution is added, and the mixture is stirred and reacted at 30°C to 37°C to obtain a dual-targeting biomimetic liposome composite material. If the reaction temperature is lower than 30°C, the particle size of the composite material formed is large and uneven, and the performance of the dual-targeting biomimetic liposome composite material is unstable; if the reaction temperature is too high, higher than 37°C, the drug efficacy of evolocumab will be reduced, and the expected effect cannot be achieved.

[0038] As a preferred embodiment, the mass ratio of the bionic membrane to the liposome loaded with eloumab and shikonin is 1 to 2:10. If it is not within the mass ratio defined above, the efficacy of the drug will be affected to varying degrees. If the mass ratio is 0.5:10, that is, the mass of the bionic membrane is low, an immune escape effect will occur, the blood circulation time will be prolonged, and the efficacy will be greatly reduced; if the mass ratio is 3:10, that is, the concentration of the bionic membrane is high, the stability of the liposome loaded with eloumab and shikonin will be reduced.

[0039] In order to achieve better targeting of the composite material, the mass ratio of the liposome loaded with evolocumab and shikonin to phospholipidated hyaluronic acid is 1:0.1-0.25. If the proportion of phospholipidated hyaluronic acid is too low, the targeting ability to damage the endothelium and inflammatory macrophages will be worse; if the proportion of phospholipidated hyaluronic acid is too high, the hyaluronic acid cannot be loaded, and the drug cannot achieve better specific targeting.

[0040] In order to further achieve better lipid-lowering and anti-inflammatory effects, the mass ratio of eloumab, lecithin and cholesterol is 3-6:20:10; the mass ratio of shikonin, lecithin and cholesterol is 1-4:20:10, and the optimal mass ratio of eloumab, shikonin, lecithin and cholesterol is 5:3:20:10. Under the above loading amounts of eloumab 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 the present invention includes the following steps: ultrasonically crushing the macrophage membrane at a power of 30 to 50 W for 3 to 5 minutes, repeatedly freezing and thawing at -80°C and 37°C for 2 to 3 times, centrifuging at 10,000 to 13,000 rpm, and then dissolving with PBS to obtain a biomimetic membrane solution.

[0042] The preparation method of the phospholipidized hyaluronic acid solution adopted by the present invention comprises the following steps: dissolving hyaluronic acid and an activator in water, stirring for 30 to 60 minutes, adding phospholipid polyethylene glycol amino, stirring at 600 to 800 rpm for 12 to 24 hours to obtain a mixed solution, and dialyzing the mixed solution for 12 to 24 hours using a dialysis bag with a molecular weight cutoff of 3 to 3.5 kDa to obtain a phospholipidized hyaluronic acid solution.

[0043] As a preferred embodiment, 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 5:10:0.8-1.2.

[0044] The effects are described below in conjunction with specific embodiments.

[0045] Example 1

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

[0047] S1. Preparation of liposome dispersion loaded with eloumab and shikonin: The liposome dispersion was prepared by reverse evaporation method. The specific operation process was as follows: 20 mg of lecithin and 10 mg of cholesterol were dissolved in 2 mL of chloroform, 5 mg of eloumab and 3 mg of shikonin were dissolved in 2 mL of chloroform, and added to a 250 mL round-bottom flask, 6 mL of anhydrous ether and 4 mL of ddH2O were added, and after thorough mixing, water bath ultrasound (50 W) was applied for 5 min to form a uniform W / O emulsion, and reduced pressure rotary evaporation was performed until the organic solvent was completely evaporated and a uniform gel phase was formed at the bottom of the flask, and then 10 mL 2% Tween 80 PBS buffer, the round-bottom flask was subjected to simple ultrasonic treatment to ensure that the liposomes on the flask wall were completely detached into the buffer solution, and rotary evaporation was continued to remove the residual organic solvent and form a liposome suspension with uniform particle size. The above liposome suspension was placed in a 300KDa dialysis bag for dialyzation for 12h to remove free eloumab and shikonin, and a liposome dispersion loaded with eloumab and shikonin was obtained, which was recorded as L@ES NPs.

[0048] S2. Preparation of biomimetic membrane dispersion: prepare macrophage membrane according to the instructions of the membrane protein extraction preparation box, wash the macrophages twice with PBS (pre-cooled at 4°C), resuspend in membrane protein extractant A (containing 1 mM PMSF (phenylmethylsulfonyl fluoride)), lyse on ice for 30 min, and sonicate in a 4°C water bath for 10 min (80W); freeze-thaw repeatedly 5 times at -80°C and 37°C, each time for 30 min; centrifuge at 12000 rpm, 4°C for 30 min to obtain the macrophage membrane precipitate, recorded as M.

[0049] S3. Preparation of phospholipidated 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 at room temperature for 30 min to activate the carboxyl groups on the surface of the hyaluronic acid; then, 1 mg DSPE-PEG2000-NH2 (phospholipid polyethylene glycol amino) was added to the above solution, and stirred at 800 rpm at room temperature for 24 h; the above mixed solution was placed in a 3.5 kDa dialysis bag and dialyzed for 24 h to remove free EDC, NHS and DSPE-PEG2000-NH2. Finally, the purified material was freeze-dried by freeze-drying, and the freeze-dried phospholipidated hyaluronic acid was placed in a -20°C refrigerator for use, and recorded as HA.

[0050] S4. Preparation of dual-targeted bionic liposome nano-drug composite materials: the liposomes loaded with eloumab and shikonin and the bionic membrane dispersion are prepared in a mass ratio of 10:1, 1 mg of L@ES NPs and 0.1 mg of bionic membrane are resuspended in 1 mL of PBS, and the mixture is repeatedly extruded 10 times through a micro-extruder with a pore size of 220 nm to obtain a bionic liposome nano-composite material co-loaded with eloumab and shikonin (denoted as ML@ES); the phospholipidated hyaluronic acid is redissolved in a PBS solution, 250 μg of the phospholipidated hyaluronic acid solution is added to the ML@ES NPs solution, and the mixture is stirred in a water bath at 37°C and 800 rpm for 30 min to obtain a dual-targeted bionic nano-drug, i.e., HA-ML@ES NPs.

[0051] Example 2

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

[0053] S1. Preparation of liposome dispersion loaded with eloumab and shikonin: The liposome dispersion was prepared by reverse evaporation method. The specific operation process was as follows: 20 mg of lecithin and 10 mg of cholesterol were dissolved in 2 mL of chloroform, 3 mg of eloumab and 1 mg of shikonin were dissolved in 2 mL of chloroform, and added to a 250 mL round-bottom flask, 6 mL of anhydrous ether and 4 mL of ddH2O were added, and after thorough mixing, water bath ultrasound (50 W) was applied for 5 min to form a uniform W / O emulsion, and reduced pressure rotary evaporation was performed until the organic solvent was completely evaporated and a uniform gel phase was formed at the bottom of the flask, and then 10 mL 2% Tween 80 PBS buffer, the round-bottom flask was subjected to simple ultrasonic treatment to ensure that the liposomes on the flask wall were completely detached into the buffer solution, and rotary evaporation was continued to remove the residual organic solvent and form a liposome suspension with uniform particle size. The above liposome suspension was placed in a 300KDa dialysis bag for dialyzation for 12h to remove free eloumab and shikonin, and a liposome dispersion loaded with eloumab and shikonin was obtained, which was recorded as L@ES NPs.

[0054] S2. Preparation of biomimetic membrane dispersion: prepare macrophage membrane according to the instructions of the membrane protein extraction preparation box, wash the macrophages twice with PBS (pre-cooled at 4°C), resuspend in membrane protein extractant A (containing 1 mM PMSF (phenylmethylsulfonyl fluoride)), lyse on ice for 30 min, and sonicate in a 4°C water bath for 10 min (80W); freeze-thaw repeatedly 5 times at -80°C and 37°C, each time for 30 min; centrifuge at 12000 rpm, 4°C for 30 min to obtain the macrophage membrane precipitate, recorded as M.

[0055] S3. Preparation of phospholipidated 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 at room temperature for 30 min to activate the carboxyl groups on the surface of the hyaluronic acid; then, 1 mg DSPE-PEG2000-NH2 (phospholipid polyethylene glycol amino) was added to the above solution, and stirred at 800 rpm at room temperature for 24 h; the above mixed solution was placed in a 3.5 kDa dialysis bag and dialyzed for 24 h to remove free EDC, NHS and DSPE-PEG2000-NH2. Finally, the purified material was freeze-dried by freeze-drying, and the freeze-dried phospholipidated hyaluronic acid was placed in a -20°C refrigerator for use, and recorded as HA.

[0056] S4. Preparation of dual-targeted bionic liposome nano-drug composite materials: the liposomes loaded with eloumab and shikonin and the bionic membrane dispersion are prepared in a mass ratio of 10:1, 1 mg of L@ES NPs and 0.1 mg of bionic membrane are resuspended in 1 mL of PBS, and the mixture is repeatedly extruded 10 times through a micro-extruder with a pore size of 220 nm to obtain a bionic liposome nano-composite material co-loaded with eloumab and shikonin (denoted as ML@ES); the phospholipidated hyaluronic acid is redissolved in a PBS solution, 250 μg of the phospholipidated hyaluronic acid solution is added to the ML@ES NPs solution, and the mixture is stirred in a water bath at 37°C and 800 rpm for 30 min to obtain a dual-targeted bionic nano-drug, i.e., HA-ML@ES NPs.

[0057] Example 3

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

[0059] S1. Preparation of liposome dispersion loaded with eloumab and shikonin: The liposome dispersion was prepared by reverse evaporation method. The specific operation process was as follows: 20 mg of lecithin and 10 mg of cholesterol were dissolved in 2 mL of chloroform, 6 mg of eloumab and 4 mg of shikonin were dissolved in 2 mL of chloroform, and added to a 250 mL round-bottom flask, 6 mL of anhydrous ether and 4 mL of ddH2O were added, and after thorough mixing, water bath ultrasound (50 W) was applied for 5 min to form a uniform W / O emulsion, and reduced pressure rotary evaporation was performed until the organic solvent was completely evaporated and a uniform gel phase was formed at the bottom of the flask, and then 10 mL 2% Tween 80 PBS buffer, the round-bottom flask was subjected to simple ultrasonic treatment to ensure that the liposomes on the flask wall were completely detached into the buffer solution, and rotary evaporation was continued to remove the residual organic solvent and form a liposome suspension with uniform particle size. The above liposome suspension was placed in a 300KDa dialysis bag for dialyzation for 12h to remove free eloumab and shikonin, and a liposome dispersion loaded with eloumab and shikonin was obtained, which was recorded as L@ES NPs.

[0060] S2. Preparation of biomimetic membrane dispersion: prepare macrophage membrane according to the instructions of the membrane protein extraction preparation box, wash the macrophages twice with PBS (pre-cooled at 4°C), resuspend in membrane protein extractant A (containing 1 mM PMSF (phenylmethylsulfonyl fluoride)), lyse on ice for 30 min, and sonicate in a 4°C water bath for 10 min (80W); freeze-thaw repeatedly 5 times at -80°C and 37°C, each time for 30 min; centrifuge at 12000 rpm, 4°C for 30 min to obtain the macrophage membrane precipitate, recorded as M.

[0061] S3. Preparation of phospholipidated 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 at room temperature for 30 min to activate the carboxyl groups on the surface of the hyaluronic acid; then, 1 mg DSPE-PEG2000-NH2 (phospholipid polyethylene glycol amino) was added to the above solution, and stirred at 800 rpm at room temperature for 24 h; the above mixed solution was placed in a 3.5 kDa dialysis bag and dialyzed for 24 h to remove free EDC, NHS and DSPE-PEG2000-NH2. Finally, the purified material was freeze-dried by freeze-drying, and the freeze-dried phospholipidated hyaluronic acid was placed in a -20°C refrigerator for use, and recorded as HA.

[0062] S4. Preparation of dual-targeted bionic liposome nano-drug composite materials: the liposomes loaded with eloumab and shikonin and the bionic membrane dispersion are prepared in a mass ratio of 10:1, 1 mg of L@ES NPs and 0.1 mg of bionic membrane are 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 bionic liposome nano-composite material co-loaded with eloumab and shikonin (denoted as ML@ES); the phospholipidated hyaluronic acid is redissolved in a PBS solution, 150 μg of the phospholipidated hyaluronic acid solution is added to the ML@ES NPs solution, and the mixture is stirred in a water bath at 37°C and 800 rpm for 30 min to obtain a dual-targeted bionic nano-drug, i.e., HA-ML@ES NPs.

[0063] Example 4

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

[0065] S1. Preparation of liposome dispersion loaded with eloumab and shikonin: The liposome dispersion was prepared by reverse evaporation method. The specific operation process was as follows: 20 mg of lecithin and 10 mg of cholesterol were dissolved in 2 mL of chloroform, 5 mg of eloumab and 3 mg of shikonin were dissolved in 2 mL of chloroform, and added to a 250 mL round-bottom flask, 6 mL of anhydrous ether and 4 mL of ddH2O were added, and after thorough mixing, water bath ultrasound (50 W) was applied for 5 min to form a uniform W / O emulsion, and reduced pressure rotary evaporation was performed until the organic solvent was completely evaporated and a uniform gel phase was formed at the bottom of the flask, and then 10 mL 2% Tween 80 PBS buffer, the round-bottom flask was subjected to simple ultrasonic treatment to ensure that the liposomes on the flask wall were completely detached into the buffer solution, and rotary evaporation was continued to remove the residual organic solvent and form a liposome suspension with uniform particle size. The above liposome suspension was placed in a 300KDa dialysis bag for dialyzation for 12h to remove free eloumab and shikonin, and a liposome dispersion loaded with eloumab and shikonin was obtained, which was recorded as L@ES NPs.

[0066] S2. Preparation of biomimetic membrane dispersion: prepare macrophage membrane according to the instructions of the membrane protein extraction preparation box, wash the macrophages twice with PBS (pre-cooled at 4°C), resuspend in membrane protein extractant A (containing 1 mM PMSF (phenylmethylsulfonyl fluoride)), lyse on ice for 30 min, and sonicate in a 4°C water bath for 10 min (80W); freeze-thaw repeatedly 5 times at -80°C and 37°C, each time for 30 min; centrifuge at 12000 rpm, 4°C for 30 min to obtain the macrophage membrane precipitate, recorded as M.

[0067] S3. Preparation of phospholipidated 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 at room temperature for 30 min to activate the carboxyl groups on the surface of the hyaluronic acid; then, 1 mg DSPE-PEG2000-NH2 (phospholipid polyethylene glycol amino) was added to the above solution, and stirred at 800 rpm at room temperature for 24 h; the above mixed solution was placed in a 3.5 kDa dialysis bag and dialyzed for 24 h to remove free EDC, NHS and DSPE-PEG2000-NH2. Finally, the purified material was freeze-dried by freeze-drying, and the freeze-dried phospholipidated hyaluronic acid was placed in a -20°C refrigerator for use, and recorded as HA.

[0068] S4. Preparation of dual-targeted bionic liposome nano-drug composite materials: the liposomes loaded with eloumab and shikonin and the bionic membrane dispersion are prepared in a mass ratio of 10:2, 1 mg of L@ES NPs and 0.2 mg of bionic membrane are 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 bionic liposome nano-composite material co-loaded with eloumab and shikonin (denoted as ML@ES); the phospholipidated hyaluronic acid is redissolved in a PBS solution, 100 μg of the phospholipidated hyaluronic acid solution is added to the ML@ES NPs solution, and stirred in a water bath at 37°C and 800 rpm for 30 min to obtain a dual-targeted bionic nano-drug, i.e., HA-ML@ES NPs.

[0069] Example 5

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

[0071] S1. Preparation of liposome dispersion loaded with eloumab and shikonin: The liposome dispersion was prepared by reverse evaporation method. The specific operation process was as follows: 20 mg of lecithin and 10 mg of cholesterol were dissolved in 2 mL of chloroform, 6 mg of eloumab and 1 mg of shikonin were dissolved in 2 mL of chloroform, and added to a 250 mL round-bottom flask, 6 mL of anhydrous ether and 4 mL of ddH2O were added, and after thorough mixing, water bath ultrasound (50 W) was applied for 5 min to form a uniform W / O emulsion, and reduced pressure rotary evaporation was performed until the organic solvent was completely evaporated and a uniform gel phase was formed at the bottom of the flask, and then 10 mL 2% Tween 80 PBS buffer, the round-bottom flask was subjected to simple ultrasonic treatment to ensure that the liposomes on the flask wall were completely detached into the buffer solution, and rotary evaporation was continued to remove the residual organic solvent and form a liposome suspension with uniform particle size. The above liposome suspension was placed in a 300KDa dialysis bag for dialyzation for 12h to remove free eloumab and shikonin, and a liposome dispersion loaded with eloumab and shikonin was obtained, which was recorded as L@ES NPs.

[0072] S2. Preparation of biomimetic membrane dispersion: prepare macrophage membrane according to the instructions of the membrane protein extraction preparation box, wash the macrophages twice with PBS (pre-cooled at 4°C), resuspend in membrane protein extractant A (containing 1 mM PMSF (phenylmethylsulfonyl fluoride)), lyse on ice for 30 min, and sonicate in a 4°C water bath for 10 min (80W); freeze-thaw repeatedly 5 times at -80°C and 37°C, each time for 30 min; centrifuge at 12000 rpm, 4°C for 30 min to obtain the macrophage membrane precipitate, recorded as M.

[0073] S3. Preparation of phospholipidated 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 at room temperature for 30 min to activate the carboxyl groups on the surface of the hyaluronic acid; then, 1 mg DSPE-PEG2000-NH2 (phospholipid polyethylene glycol amino) was added to the above solution, and stirred at 800 rpm at room temperature for 24 h; the above mixed solution was placed in a 3.5 kDa dialysis bag and dialyzed for 24 h to remove free EDC, NHS and DSPE-PEG2000-NH2. Finally, the purified material was freeze-dried by freeze-drying, and the freeze-dried phospholipidated hyaluronic acid was placed in a -20°C refrigerator for use, and recorded as HA.

[0074] S4. Preparation of dual-targeted bionic liposome nano-drug composite materials: the liposomes loaded with eloumab and shikonin and the bionic membrane dispersion are prepared in a mass ratio of 10:1.5, 1 mg of L@ES NPs and 0.15 mg of bionic membrane are resuspended in 1 mL of PBS, and repeatedly extruded 10 times by a micro-extruder with a pore size of 220 nm to obtain a bionic liposome nano-composite material co-loaded with eloumab and shikonin (denoted as ML@ES); the phospholipidated hyaluronic acid is redissolved in a PBS solution, 200 μg of the phospholipidated hyaluronic acid solution is added to the ML@ES NPs solution, and the mixture is stirred in a water bath at 37°C and 800 rpm for 30 min to obtain a dual-targeted bionic nano-drug, i.e., HA-ML@ES NPs.

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

[0076] Figure 1 Transmission electron microscopy images and particle size potential analysis of L@ES NPs and HA-ML@ES NPs prepared in Example 1 of the present invention, wherein Figure A is L@ES NPs and Figure B is HA-ML@ES NPs. Figure 1 As shown, the L@ESNPs and HA-ML@ES NPs prepared in this Example 1 were subjected to transmission electron microscopy image analysis, and the results showed that uniformly dispersed spherical L@ES NPs nanoparticles were successfully prepared, with a particle size of about 150 nm; the HA-modified biomimetic membrane-melting liposomes had a uniform particle size after co-loading eloumab and shikonin, with a particle size of about 180 nm.

[0077] Figure 2 The Coomassie brilliant blue staining and protein immunoblotting of the biomimetic membrane of the present invention are shown in Figure A, where Figure A is Coomassie brilliant blue staining and Figure B is protein immunoblotting. Figure 2 As shown, the biomimetic membrane prepared in Example 1 was subjected to membrane characteristic protein analysis, and the results showed that the biomimetic membrane was successfully disguised on the surface of the nanocomposite and retained the characteristic proteins on the cell membrane surface.

[0078] The biosafety, pharmacokinetics and targeting of the dual-targeting bionic liposome composite material prepared by the present invention are investigated.

[0079] Biosafety verification of dual-targeted biomimetic liposome composites

[0080] Figure 3 The biological safety of the dual-targeted biomimetic liposome composite material HA-ML@ES NPs prepared in Example 1 of the present invention. Figure 3 As shown in Figure A, in the hemolysis experiment, compared with the H2O treatment group, the hemolysis rates of the Evol+SKN and HA-ML@ES groups were lower than 5%, and Figure 3 No abnormally shaped red blood cells were found in the red blood cell morphology image in Figure B, indicating that intravenous administration is safe.

[0081] The dual-targeted bionic liposome nano-drug composite material prepared in Example 1 was used to measure the cellular uptake, blood half-life and in vivo targeting of HA-ML@ES NPs bionic nanomaterials by semi-quantitative means of detecting fluorescence intensity.

[0082] Figure 4 Using a transwell chamber, endothelial cells were laid on the upper layer and macrophages were laid on the lower layer to simulate vascular plaques. Dil-labeled HA-ML@ES NPs were added to the upper layer and cultured 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 is a schematic diagram of the experimental principle. The upper layer is endothelial cells and the lower layer is macrophages, simulating the atherosclerotic plaque environment in vitro; Figure B shows that HA-ML@SE has a stronger ability to target endothelial cells and macrophages than L@ES.

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

[0084] Among them, L Ce6 Prepared by the following method: Using reverse evaporation method, prepare L Ce6 NPs, the specific operation process is: 20mg lecithin, 10mg cholesterol dissolved in 2mL chloroform, 10mg Ce6 dissolved in 2mL chloroform are added to a 250mL round-bottom flask, 6mL anhydrous ether and 4mL ddH2O are added, and after sufficient mixing, water bath ultrasound (50W) is performed for 5min to form a uniform W / O emulsion. Reduce pressure and rotary evaporation until the organic solvent is completely evaporated and a uniform gel phase is formed at the bottom of the bottle. Then add 10mL 2% Tween 80 PBS buffer, perform a simple ultrasonic treatment on the round-bottom flask to ensure that the liposomes on the bottle wall are completely detached into the buffer solution, continue rotary evaporation, remove the residual organic solvent and form a liposome suspension with uniform particle size, and place the above liposome suspension in a 300KDa dialysis bag for 12h to remove free Ce6.

[0085] HA-ML Ce6The ce6-loaded liposome dispersion was mixed with a biomimetic membrane (M) (1 mL, 1 mg / mL) after water bath ultrasound treatment at a mass ratio of 10:1, and the mixture was repeatedly extruded at least 10 times by a micro extruder with a pore size of 220 nm to obtain ML. Ce6 Bionic nanoparticle preparation: Redissolve the phospholipidated hyaluronic acid in PBS solution, add 5 mg of the phospholipidated hyaluronic acid solution to the MLCe6 solution, and stir in a water bath at 37°C and 600-800 rpm for 60 min to obtain HA-ML Ce6 .

[0086] Figure 5 is the blood half-life of HA-ML@ES NPs of the present invention in C57 mice. Figure 5 As shown, Ce6 and HA-ML Ce6 The blood circulation half-life of the two drugs was 0.52h and 1.31h respectively, which indicated that the nanoparticles disguised by bionic membrane were beneficial to prolong the blood circulation time.

[0087] ApoE - / - After the mice were fed a high-methionine diet for 3 months, 200 μL of 5 mg / kg Ce6 and HA-ML were injected into the tail vein. Ce6 12 hours later, the mouse aorta was taken for fluorescence imaging.

[0088] Figure 6 The results show that the HA-ML@ES NPs of the present invention are targeted to atherosclerotic plaques in mice. Figure 6 As shown, Ce6 and HA-ML Ce6 Compared with the HA-ML group Ce6 The HA-ML@ES NPs group significantly aggregated in the aortic plaque, indicating that the HA-ML@ES NPs biomimetic nanoformulation prepared in Example 1 can effectively target the atherosclerotic plaque site.

[0089] The HA-ML@ES NPs biomimetic nanoformulation 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 h, we further incubated the cells with Evol+SKN and HA-ML@ES NPs for another 24 h. The apoptosis of HUVECs was assessed using flow cytometry and Annexin V-FITC / PI assays according to the manufacturer's instructions (BD, CA, USA).

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

[0092] Figure 7 HA-ML@ES NPs biomimetic nanoformulation is used to inhibit endothelial cell apoptosis and foam cell formation. Figure 7 Figure A is the flow cytometry result of endothelial cell apoptosis, control is the negative group, model is the positive group, Evol+SKN is the free combination of the two drugs; Figure B is the Oil Red O staining of foam cell formation, detecting the accumulation of oxLDL in macrophages. Figure 7 As shown, HA-ML@ES NPs biomimetic nanoformulation effectively inhibited endothelial cell apoptosis and foam cell formation.

[0093] The HA-ML@ES NPs bionic nanoformulation prepared in accordance with Example 1 is used for the early treatment of atherosclerosis.

[0094] ApoE - / - After the mice were fed a high-methionine diet for 1 month, early preventive treatment was carried out. The drug-treated 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 / kgSKN). The drugs were administered twice a week for 2 consecutive months, during which time the mice were fed a high-methionine diet. Three days after the end of the treatment, the aorta of the mice was stripped and stained with Oil Red O to examine the therapeutic effects of the different treatment groups.

[0095] Figure 8 The bright field images and gross staining images of the aortic arch of blood vessels treated with atherosclerosis in different treatment groups of the present invention are shown in the figure below. 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 bionic nanoformulation constructed by the present invention can effectively prevent the progression of early atherosclerosis.

[0096] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A dual-targeting bionic liposome composite material, characterized in that: The composite material comprises drug-loaded liposomes, wherein the drug-loaded liposomes are loaded with evolocumab and shikonin, and the drug-loaded liposomes are coated with macrophage membranes, wherein the macrophage membranes are modified with phospholipidated hyaluronic acid.

2. A method for preparing the dual-targeting bionic liposome composite material according to claim 1, characterized in that: The following steps are involved: Dissolving evolocumab, shikonin, cholesterol and lecithin in a solvent, adding water, subjecting to ultrasound, evaporation under reduced pressure, hydration and dialysis to obtain a liposome dispersion loaded with evolocumab and shikonin; The liposome dispersion loaded with evolocumab and shikonin and the biomimetic membrane solution are mixed, extruded repeatedly, and then the phospholipidated hyaluronic acid solution is added, stirred and reacted at 30° C. to 37° C. to obtain a dual-targeting biomimetic liposome composite material.

3. The preparation method according to claim 2, characterized in that: The mass ratio of the bionic membrane to the liposome loaded with evolocumab and shikonin is 1 to 2:

10.

4. The preparation method according to claim 2, characterized in that: The mass ratio of the liposome loaded with evolocumab and shikonin to the phospholipidated hyaluronic acid is 1:0.1-0.

25.

5. The preparation method according to claim 2, characterized in that: The mass ratio of evolocumab, lecithin and cholesterol is 3 to 6:20:10; the mass ratio of shikonin, lecithin and cholesterol is 1 to 4:20:

10.

6. The preparation method according to claim 2, characterized in that: The repeated extrusion is performed by using a micro extruder with a pore size of 220 nm to repeatedly extrude 8 to 10 times.

7. The preparation method according to claim 2, characterized in that: The preparation method of the biomimetic membrane solution comprises the following steps: ultrasonically crushing the macrophage membrane at a power of 30 to 50 W for 3 to 5 minutes, repeatedly freezing and thawing at -80°C and 37°C for 2 to 3 times, centrifuging at 10,000 to 13,000 rpm, and then dissolving with PBS to obtain a biomimetic membrane solution.

8. The preparation method according to claim 2, characterized in that: The preparation method of the phospholipidated hyaluronic acid solution comprises the following steps: dissolving hyaluronic acid and an activator in water, stirring for 30 to 60 minutes, adding phospholipid polyethylene glycol amino, stirring at 600 to 800 rpm for 12 to 24 hours to obtain a mixed solution, and dialyzing the mixed solution for 12 to 24 hours using a dialysis bag with a molecular weight cutoff of 3 to 3.5 kDa to obtain a phospholipidated hyaluronic acid solution.

9. The preparation method according to claim 8, characterized in 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 5:10:0.8-1.

2.

10. Use of the dual-targeting bionic liposome composite material according to claim 1 in preparing a drug for treating atherosclerosis caused by hyperhomocysteinemia.

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