Bionic liposome co-drug-loaded nano-particles as well as preparation method and application thereof

Abstract

The invention discloses bionic lipidosome co-drug-loaded nanoparticles as well as a preparation method and application thereof, and relates to the technical field of new dosage form pharmaceutical preparations. The preparation method comprises the following steps: loading hydroxysafflor yellow A and statins into double layers or a core of a liposome, adding a biomimetic membrane on the outer layer of the liposome and modifying with phosphatidylated hyaluronic acid to obtain the biomimetic liposome co-drug-loaded nano-particles with the particle size of 60-100 nm; wherein the liposome is prepared from lecithin and cholesterol; the biomimetic membrane is prepared from macrophages; the phosphatidylated hyaluronic acid is prepared from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, hyaluronic acid and phospholipid polyethylene glycol amino groups, and the phosphatidylated hyaluronic acid is prepared from the phosphatidylated hyaluronic acid, the N-hydroxysuccinimide, the hyaluronic acid and the phospholipid polyethylene glycol amino groups. According to the invention, two medicines can be loaded at the same time and can be sent to a lesion area at the same time.

Description

Technical Field

[0001] The present invention relates to the technical field of new dosage form pharmaceutical preparations, and more specifically to a bionic liposome co-drug-loaded nanoparticle and a preparation method and application thereof. Background Art

[0002] Atherosclerosis, abbreviated as AS in English, is a complex, progressive and multifaceted disease characterized by lipid deposition, inflammatory cell aggregation and atherosclerotic plaque formation in the arterial wall. It poses a major challenge to human health and generates a huge economic burden. Although the cause is still unclear, more and more people believe that systemic and chronic inflammation are closely related to the occurrence and development of atherosclerosis, and the pathological basis of atherosclerosis mainly involves lipid metabolism disorders and chronic inflammatory responses of the vascular wall. Existing methods for the treatment of atherosclerosis mainly include drug therapy, vascular interventional therapy and surgical treatment, with drug therapy being the main method.

[0003] Natural products, including monomer components or extracts of Chinese herbal medicines, have the advantages of good efficacy and low toxicity when used in the treatment of atherosclerosis. Hydroxysafflor yellow A is a yellow powder extracted from the dried flowers of the safflower plant of the Asteraceae family, the Latin name of which is Carthamus tinctorius L. It is a compound with a single chalcone glycoside structure and has multiple biological activities, including anti-platelet aggregation, anti-inflammatory, antioxidant, blood circulation and blood stasis. It has a wide range of applications in the medical field, such as for the preparation of drugs to treat coronary heart disease, cerebral thrombosis, hyperlipidemia and other diseases. Due to the high water solubility and low permeability of hydroxysafflor yellow A, it often faces absorption barriers when administered orally, resulting in low bioavailability. This limits the application effect of hydroxysafflor yellow A in the treatment of various diseases, such as cardiovascular and cerebrovascular diseases, anti-inflammatory, antioxidant, etc. Clinically, statins can effectively reduce low-density lipoprotein cholesterol to the level recommended by the guidelines, but because a single drug cannot eliminate inflammation, adverse cardiovascular events still occur frequently. Therefore, the combined use of Chinese and Western medicines with a halved dosage is expected to achieve effective treatment of atherosclerosis and its complications. However, existing technologies mostly use combined treatment in the form of oral administration of two drugs separately, and there is a lack of products that can carry two drugs at the same time and deliver the two drugs to the lesion area at the same time. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a bionic liposome co-drug-loaded nanoparticle and a preparation method and application thereof, which are used to load two drugs at the same time and deliver the two drugs to the lesion area at the same time.

[0005] The present invention is achieved through the following technical solutions:

[0006] Provided are bionic liposome co-drug-loaded nanoparticles, wherein hydroxysafflor yellow A and statins are loaded into the double layer or core of the liposome, and the outer layer of the liposome is modified with a bionic membrane and phospholipidated hyaluronic acid to obtain bionic liposome co-drug-loaded nanoparticles with a particle size of 60nm to 100nm.

[0007] Among them, liposomes are prepared from lecithin and cholesterol.

[0008] The biomimetic membrane is made from macrophages.

[0009] The phospholipidated hyaluronic acid is prepared from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, hyaluronic acid and phospholipid polyethylene glycol amino.

[0010] Preferably, the method for preparing the biomimetic liposome-drug-loaded nanoparticles specifically comprises the following steps:

[0011] Lecithin and cholesterol are dissolved in an organic solvent, and the organic solvent is removed after the reaction to obtain liposomes.

[0012] The liposomes are mixed with hydroxysafflor yellow A, statins and PBS solution for hydration reaction, so that the hydroxysafflor yellow A and statins are loaded into the bilayer or core of the liposomes, thereby obtaining liposome nanoparticles co-loaded with hydroxysafflor yellow A and statins.

[0013] Preparation of biomimetic membrane dispersions using macrophages.

[0014] Phospholipidated hyaluronic acid is prepared by using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, hyaluronic acid and phospholipid polyethylene glycol amino groups, and the phospholipidated hyaluronic acid is dissolved in a PBS solution to obtain a phospholipidated hyaluronic acid solution; in a PBS environment, a biomimetic membrane dispersion liquid and a phospholipidated hyaluronic acid solution are sequentially added to liposome nanoparticles co-loaded with hydroxysafflor yellow A and statins, so that the outer layer of the liposome is modified with a biomimetic membrane and the phospholipidated hyaluronic acid, and biomimetic liposome co-loaded with drugs nanoparticles are obtained.

[0015] Preferably, the organic solvent in the method for preparing the biomimetic liposome co-drug-loaded nanoparticles is chloroform.

[0016] Preferably, in the method for preparing the bionic liposome co-drug-loaded nanoparticles, the ratio of lecithin, cholesterol and organic solvent is 2 mg to 3 mg: 1 mg: 1 mL.

[0017] Preferably, in the method for preparing the bionic liposome co-drug-loaded nanoparticles, the mass ratio of the liposome, hydroxysafflor yellow A and statin is 10-11:1:1.

[0018] Preferably, in the method for preparing the biomimetic liposome co-drug-loaded nanoparticles, the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, hyaluronic acid, and phospholipid polyethylene glycol amino is 25:50:5:24-25.

[0019] Preferably, in the method for preparing the biomimetic liposome co-drug-loaded nanoparticles, the volume ratio of the biomimetic membrane dispersion to the phospholipidated hyaluronic acid solution is 0.05-0.06:0.2-0.24.

[0020] Preferably, the statin used in the bionic liposome co-drug-loaded nanoparticles is any one of rosuvastatin, pitavastatin and simvastatin.

[0021] Furthermore, the bionic liposome co-drug-loaded nanoparticles are used in the preparation of drugs for treating atherosclerosis.

[0022] Furthermore, a drug for treating atherosclerosis is provided, wherein the active ingredient of the drug includes the bionic liposome co-drug-loaded nanoparticles.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The biomimetic liposome co-drug-loaded nanoparticles provided by the present invention load hydroxysafflor yellow A and statins into the bilayer or core of the liposome, and the outer layer of the liposome is modified with a biomimetic membrane and phospholipidated hyaluronic acid to obtain biomimetic liposome co-drug-loaded nanoparticles. The biomimetic liposome co-drug-loaded nanoparticles load hydroxysafflor yellow A and statins into the bilayer or core of the liposome, and are modified with a macrophage membrane and phospholipidated hyaluronic acid at the same time. Hydroxysafflor yellow A can reduce the dosage of statins while being anti-inflammatory. The outermost macrophage membrane and hyaluronic acid modification play a homing effect and actively target atherosclerotic plaque lesions. The introduction of the macrophage membrane prolongs the blood circulation time of the nanocomposite preparation, and realizes that the two drugs are simultaneously delivered to the lesion area to improve the specific accumulation and penetration of the drugs in the inflammatory site and improve the bioavailability of the drugs to the target cells, so as to achieve the purpose of synergistic treatment of atherosclerosis.

[0025] (2) The biomimetic liposome co-drug-loaded nanoparticles prepared by the present invention utilize the anti-atherosclerotic effects of hydroxysafflor yellow A and statins of two different mechanisms to achieve targeted treatment of atherosclerosis. Compared with a single drug, the dosage is halved and the combined use of nanosized drugs enhances efficacy and reduces toxicity. It makes up for the common liver function abnormalities, muscle effects, blood sugar abnormalities, gastrointestinal reactions and other possible side effects of long-term use of statins, while improving the half-life of the drug in the blood and the plaque targeting effect, providing a new option for carrying out relevant clinical treatments, and has important scientific significance, practical value and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 The Coomassie blue staining and protein immunoblotting images of HA-ML@(H+R)NPs of the present invention are shown in FIG. For macrophage membrane;

[0028] Figure 1 In the figure, A is the Coomassie blue staining of the biomimetic membrane; B is the protein immunoblotting;

[0029] Figure 2 Transmission electron microscopy images of Lip@(H+R)NPs and HA-ML@(H+R)NPs of the present invention;

[0030] Figure 2 In the figure, A is the transmission electron microscopy image of Lip@(H+R)NPs; B is the transmission electron microscopy image of HA-ML@(H+R)NPs;

[0031] Figure 3 This is a diagram of the fusion experiment of macrophage membrane and liposome of the present invention;

[0032] Figure 3 In the figure, A is liposome, which is labeled with Dil red fluorescence; B is macrophage membrane Labeled with green fluorescence; C is the fusion of red fluorescence label and green fluorescence label into yellow fluorescence;

[0033] Figure 4 This is a diagram showing that HA-ML@(H+R)NPs of the present invention inhibit the uptake of DiI-oxLDL and inhibit the formation of foam cells;

[0034] Figure 4In the figure, A is the image of HA-ML@(H+R)NPs inhibiting the uptake of DiI-oxLDL, Bright field refers to the indicated field, Dil-oxLDL refers to the image of oxidized low-density lipoprotein labeled with red fluorescence, DAPI refers to the image of DAPI-stained cell nuclei with blue fluorescence, Overlay refers to the image of the cells in the indicated field, red fluorescence, and blue fluorescence overlapping; B is the experimental image of HA-ML@(H+R)NPs inhibiting the formation of foam cells, ORO refers to the staining with Oil Red O staining solution;

[0035] Figure 5 The HA-ML@(H+R)NPs of the present invention promote macrophage polarization and inhibit inflammation;

[0036] Figure 5 In the figure, A is the image of HA-ML@(H+R)NPs promoting macrophage polarization; B is the image of HA-ML@(H+R)NPs inhibiting inflammation;

[0037] Figure 6 Prolonging the blood half-life of HA-ML@(H+R)NPs of the present invention in C57 mice;

[0038] Figure 6 In the figure, A is the fluorescence graph of the blood circulation half-life at different time points after the tail vein injection of Ce6 and HA-ML@Ce6 NPs in mice; B is the in vivo pharmacokinetic curve of Ce6 and HA-ML@Ce6NPs;

[0039] Figure 7 This is a diagram showing the targeting of HA-ML@(H+R)NPs of the present invention to atherosclerotic plaques in mice;

[0040] Figure 8 This is a diagram showing the effect of HA-ML@(H+R)NPs of the present invention on treating Hcy-induced atherosclerosis;

[0041] Figure 8 In the figure, A represents ApoE after treatment with different drugs - / - Representative images of ORO staining of mouse aortic arch. Nine parallel samples were set up for each treatment in each group; B is ApoE - / - ORO staining of frozen sections of mouse aortic root; C is a graph of Hcy levels in serum after treatment with different drugs;

[0042] Fig. 9 These are H&E staining images of the heart, liver, spleen, lung and kidney of mice with atherosclerosis treated with different treatment groups of the present invention. DETAILED DESCRIPTION

[0043] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are provided. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0045] Table 1 Chinese-English comparison table

[0046]

[0047] The structure of traditional nanomaterials loaded with drugs is usually a single combination of drug molecules and the surface of nanomaterials. The drug molecules are connected to the surface of polymer nanoparticles through covalent bonds or the drug molecules are adsorbed on the surface of nanomaterials through physical forces such as van der Waals forces or electrostatic forces. However, these structures lack targeting and cannot deliver drugs to the lesion area, thus reducing the treatment efficiency.

[0048] The present invention proposes a novel bionic liposome co-drug-loaded nanoparticle, the structure of which is to load hydroxysafflor yellow A and statins into the double layer or core of the liposome, and to modify the outer layer of the liposome with a bionic membrane and phospholipidated hyaluronic acid to obtain the bionic liposome co-drug-loaded nanoparticle, thereby solving the problem of insufficient targeting, inability to deliver drugs to the lesion area, and reduced treatment efficiency.

[0049] Based on the same inventive concept, the present invention also provides a method for preparing biomimetic liposome co-drug-loaded nanoparticles, which specifically comprises the following steps:

[0050] Lecithin and cholesterol are dissolved in an organic solvent, and the organic solvent is removed after the reaction to obtain liposomes.

[0051] The liposomes are mixed with hydroxysafflor yellow A, statins and PBS solution for hydration reaction, so that the hydroxysafflor yellow A and statins are loaded into the bilayer or core of the liposomes, thereby obtaining liposome nanoparticles co-loaded with hydroxysafflor yellow A and statins.

[0052] Preparation of biomimetic membrane dispersions using macrophages.

[0053] Phospholipidated hyaluronic acid is prepared by using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, hyaluronic acid and phospholipid polyethylene glycol amino groups, and the phospholipidated hyaluronic acid is dissolved in a PBS solution to obtain a phospholipidated hyaluronic acid solution; in a PBS environment, a biomimetic membrane dispersion liquid and a phospholipidated hyaluronic acid are sequentially added to liposome nanoparticles co-loaded with hydroxysafflor yellow A and statins, so that the outer layer of the liposome is modified with the biomimetic membrane and the phospholipidated hyaluronic acid, and biomimetic liposome co-loaded with drugs nanoparticles are obtained.

[0054] Liposomes are closed vesicles formed by lipid bilayers. They are composed of phospholipids and other lipid-like materials and have a bilayer structure. The hydrophilic head faces the water phase, and the hydrophobic tails gather together on the inside to form a stable closed vesicle. Different types of active drugs can be encapsulated in the water phase and the bilayer inside the vesicles, thereby achieving drug protection and targeted delivery. Liposomes are based on the characteristics of nanostructures, so they can improve drug permeability and solubility, increase the oral bioavailability of poorly soluble drugs, and improve the in vivo and in vitro stability of preparations by encapsulating active drugs, improve efficacy, reduce toxic side effects, and improve patient compliance.

[0055] Hydroxysafflor yellow A and statins are physically encapsulated by liposomes to obtain liposome nanoparticles co-loaded with hydroxysafflor yellow A and statins. The macrophage biomimetic membrane is camouflaged on the outermost layer of the liposome nanoparticles co-loaded with hydroxysafflor yellow A and statins. Phospholipidated hyaluronic acid is inserted into the surface of the macrophage biomimetic membrane. By utilizing the design characteristics of the biomimetic membrane and hyaluronic acid modification, the inflammatory environment at the atherosclerotic plaque site secretes inflammatory factors to recruit macrophages. The outermost macrophage biomimetic membrane realizes the co-loaded hydroxysafflor yellow A and statins liposome nanoparticles to deliver the two drugs to the lesion area at the same time through its inherent immune escape and recruitment characteristics of the source cells, and improves the accumulation of the drugs at the inflammatory site. It has a long blood circulation half-life and good targeting ability, and realizes the halving of the dose and synergistic targeted treatment of atherosclerosis. By utilizing the characteristics of the interaction between hyaluronic acid and the target cell surface receptor CD44, the target cells can effectively utilize the nanoparticles and improve their effect in preventing and treating atherosclerosis.

[0056] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0057] Example 1

[0058] A method for preparing biomimetic liposome co-drug-loaded nanoparticles comprises the following steps:

[0059] Dissolve 10 mg of lecithin and 5 mg of cholesterol in 5 mL of chloroform solution, mix thoroughly, and evaporate under reduced pressure at 37°C until a uniform transparent film is formed on the inner wall of the bottle to obtain liposomes.

[0060] Liposomes, HSYA, RVS and 5 mL PBS solution were mixed for hydration reaction at 37°C for 60 min, so that HSYA and RVS were loaded into the bilayer or core of the liposomes to obtain liposome nanoparticles co-loaded with HSYA and RVS, denoted as: Lip@(H+R)NPs. The mass ratio of liposomes, HSYA and RVS was 10:1:1.

[0061] According to the instructions of the membrane protein extraction preparation kit, macrophage membranes were prepared. 20 million macrophages were washed twice with 2 mL PBS and resuspended in 1 mL membrane protein extractant A containing 1 mM PMSF, with membrane protein extractant A: PMSF = 100: 1. Lysed on ice for 30 min, ultrasonicated in a water bath at 80 W and 4 ° C for 10 min; frozen in a -80 ° C environment, thawed in a 37 ° C environment, repeated 5 times, each time for 30 min; centrifuged at 12000 rpm and 4 ° C for 30 min to obtain macrophage membranes, 1 mL PBS was added to the macrophage membranes for ultrasonication, and the ultrasonic conditions were ultrasonicated at 40 W for 4 s each time and paused for 4 s. The number of ultrasonications was 30 times to prepare a biomimetic membrane dispersion, recorded as: M.

[0062] 125mg EDC, 250mg NHS and 25mg hyaluronic acid were dissolved in 5mL PBS and stirred at 800rpm for 30min at room temperature to activate the carboxyl groups on the surface of hyaluronic acid; then, 120mg DSPE-PEG2000-NH2 was added to the above solution and stirred at 800rpm for 24h at room temperature; the above mixed solution was placed in a 2.5kD dialysis bag and dialyzed for 24h to remove free EDC, NHS and DSPE-PEG2000-NH2. Finally, the purified material was freeze-dried by freeze drying to obtain phospholipidated hyaluronic acid, recorded as: HA. 5mg HA was dissolved in 1mL PBS solution to obtain HA solution for standby use.

[0063] 10 mg of Lip@(H+R)NPs was dissolved in 1 mL of PBS to obtain a Lip@(H+R)NPs solution with a concentration of 10 mg / mL. 1 mL of Lip@(H+R)NPs solution was mixed with 0.05 mL of M with a concentration of 10 mg / mL, and the mixture was repeatedly extruded 10 times through a micro-extruder with a pore size of 200 nm to obtain bionic nanoparticles co-loaded with HSYA and RVS, recorded as: ML@(H+R)NPs; 0.2 mL of HA solution was added to ML@(H+R)NPs, and stirred in a water bath at 37°C and 800 rpm for 60 min, so that the outer layer of the liposome was modified with a bionic membrane and phospholipidated hyaluronic acid to obtain bionic liposome co-drug nanoparticles, named HA-ML@(H+R)NPs.

[0064] Example 2

[0065] A method for preparing biomimetic liposome co-drug-loaded nanoparticles comprises the following steps:

[0066] Dissolve 12 mg of lecithin and 5 mg of cholesterol in 5 mL of chloroform solution, mix thoroughly, and evaporate under reduced pressure at 37°C until a uniform transparent film is formed on the inner wall of the bottle to obtain liposomes.

[0067] Liposomes, HSYA, RVS and 5 mL PBS solution were mixed for hydration reaction at 37°C for 60 min, so that HSYA and RVS were loaded into the bilayer or core of the liposomes to obtain liposome nanoparticles co-loaded with HSYA and RVS, denoted as: Lip@(H+R)NPs. The mass ratio of liposomes, HSYA and RVS was 11:1:1.

[0068] According to the instructions of the membrane protein extraction preparation kit, macrophage membranes were prepared. 20 million macrophages were washed twice with 2 mL PBS and resuspended in 1 mL membrane protein extractant A containing 1 mM PMSF, with membrane protein extractant A: PMSF = 100: 1. Lysed on ice for 30 min, ultrasonicated in a water bath at 80 W and 4 ° C for 10 min; frozen in a -80 ° C environment, thawed in a 37 ° C environment, repeated 5 times, each time for 30 min; centrifuged at 12000 rpm and 4 ° C for 30 min to obtain macrophage membranes, 1 mL PBS was added to the macrophage membranes for ultrasonication, and the ultrasonic conditions were ultrasonicated at 40 W for 4 s each time and paused for 4 s. The number of ultrasonications was 30 times to prepare a biomimetic membrane dispersion, recorded as: M.

[0069] 125mg EDC, 250mg NHS and 25mg hyaluronic acid were dissolved in 5mL PBS and stirred at 800rpm for 30min at room temperature to activate the carboxyl groups on the surface of hyaluronic acid; then, 125mg DSPE-PEG2000-NH2 was added to the above solution and stirred at 800rpm for 24h at room temperature; the above mixed solution was placed in a 2.5kD dialysis bag and dialyzed for 24h to remove free EDC, NHS and DSPE-PEG2000-NH2. Finally, the purified material was freeze-dried by freeze drying to obtain phospholipidated hyaluronic acid, which was recorded as: HA. 5mg HA was redissolved in 1mL PBS solution to obtain HA solution for standby use.

[0070] 10 mg of Lip@(H+R)NPs was dissolved in 1 mL of PBS to obtain a Lip@(H+R)NPs solution with a concentration of 10 mg / mL. 1 mL of Lip@(H+R)NPs solution was mixed with 0.06 mL of M with a concentration of 10 mg / mL, and the mixture was repeatedly extruded 10 times through a micro-extruder with a pore size of 200 nm to obtain bionic nanoparticles co-loaded with HSYA and RVS, recorded as: ML@(H+R)NPs; 0.24 mL of HA solution was added to ML@(H+R)NPs, and stirred in a water bath at 37°C and 800 rpm for 60 min, so that the outer layer of the liposome was modified with a bionic membrane and phospholipidated hyaluronic acid, to obtain bionic liposome co-drug-loaded nanoparticles, named HA-ML@(H+R)NPs.

[0071] It should be noted that, in addition to rosuvastatin, pitavastatin or simvastatin can also be used to prepare biomimetic liposome co-drug-loaded nanoparticles.

[0072] Characteristic protein analysis was performed on the HA-ML@(H+R)NPs prepared in Example 1 and Example 2. The results are as follows: Figure 1 As shown, the results showed that the biomimetic membrane dispersion M successfully camouflaged onto the surface of liposome nanoparticles Lip@(H+R)NPs co-loaded with HSYA and RVS, and retained the characteristic proteins on the cell membrane surface.

[0073] This embodiment is subjected to electron microscopy transmission, as shown in FIG. Figure 2 As shown in A, uniformly dispersed spherical liposome nanoparticles were observed, indicating that the liposome nanoparticles Lip@(H+R)NPs co-loaded with HSYA and RVS were successfully prepared with a particle size of 50nm to 80nm; Figure 2 As shown in B, it was observed that the HA-modified biomimetic membrane M was wrapped in the core of the liposome nanoparticles Lip@(H+R)NPs co-loaded with HSYA and RVS, with a particle size of 60nm to 100nm.

[0074] Figure 3 This is an experiment of the fusion of macrophage membrane and liposome of the present invention, which shows that the hybridization of macrophage membrane and liposome is successful.

[0075] Example 3

[0076] The HA-ML@(H+R)NPs prepared in Example 1 and Example 2 were tested using the DiI-oxLDL uptake experiment and the foam cell formation experiment.

[0077] The experimental steps of DiI-oxLDL uptake were as follows: THP-1 cells were inoculated into 24-well plates and divided into the Control group without Hcy, the Model group with 100μM Hcy and the drug treatment group. The drug treatment groups were divided into HSYA=50μM group, RVS=10μM group, HSYA+RVS=25μM+5μM group and HA-ML@(H+R)NPs=25μM HSYA+5μM RVS group, which were incubated together for 24h, and then treated with 40μg / mL DiI-oxLDL for 12h; the cells were removed and washed with PBS three times, fixed with 4% paraformaldehyde for 10min and stained with DAPI for 30min, and photographed with a confocal microscope.

[0078] The steps of the foam cell formation experiment are as follows: THP-1 cells were inoculated in 24-well plates and divided into the Control group without Hcy, the Model group with 100μM Hcy and the drug treatment group. The drug treatment groups were divided into HSYA=50μM group, RVS=10μM group, HSYA+RVS=25μM+5μM group and HA-ML@(H+R)NPs=25μM+5μM group and incubated together for 24h; 80μg / ml ox-LDL was added respectively for 48h; the culture medium was removed, PBS was washed 3 times, and 3% ORO staining solution was added for staining for 30min; the microscope was used for photography. The ORO staining solution in the cells was extracted with 60% isopropanol for quantification.

[0079] The results are as follows Figure 4 As shown, Figure 4 A in the figure shows that HA-ML@(H+R)NPs inhibit the uptake of DiI-oxLDL, Brightfield indicates the field, Dil-oxLDL refers to oxidized low-density lipoprotein labeled with red fluorescence, DAPI stains the cell nucleus with blue fluorescence, and Overlay indicates the overlapping images of the field cell, red fluorescence, and blue fluorescence; Figure 4 Figure B shows that HA-ML@(H+R)NPs can effectively inhibit foam cell formation.

[0080] Example 4

[0081] The ability of HA-ML@(H+R)NPs prepared in Example 1 and Example 2 to promote macrophage polarization and inhibit inflammation was determined.

[0082] The experimental steps of HA-ML@(H+R)NPs promoting macrophage polarization are as follows: THP-1 cells were seeded in a 24-well plate containing a cell slide at a density of 1×10 ^4cells. They were divided into the Control group without Hcy, the Model group with 100μMHcy, and the drug-treated group. The drug-treated groups were divided into the HSYA=50μM group, the RVS=10μM group, the HSYA+RVS=25μM+5μM group, and the HA-ML@(H+R)NPs=25μM+5μM group, and were incubated together for 24 hours; the cells were fixed with 4% paraformaldehyde, and then incubated with 0.3% TritonX-100 for 30 minutes. Washed with PBS and blocked with 5% goat serum at room temperature for 2 hours. Then, the cells were incubated with the primary antibody CD80 or CD206 at 4°C overnight. Subsequently, the fluorescent secondary antibody was incubated at room temperature for 2 hours, and the cell nuclei were counterstained with DAPI staining solution. Finally, the slides were sealed with an anti-fluorescence quencher, and the cells were observed and photographed under a laser scanning confocal microscope. The experimental results are shown in the figure. Figure 5 A shows that HA-ML@(H+R)NPs can promote the M1-M2 transition of macrophages.

[0083] The experimental steps of HA-ML@(H+R)NPs inhibiting macrophage inflammatory response are as follows: RAW264.7 cells were inoculated into 24-well plates and divided into the Control group without Hcy, the Model group with 100μM Hcy, and the drug treatment group. The drug treatment groups were divided into HSYA=50μM group, RVS=10μM group, HSYA+RVS=25μM+5μM group and HA-ML@(H+R)NPs=25μM+5μM group and incubated together for 24h; the culture medium was collected and the expression of inflammatory factors was detected by ELISA kit. The experimental results are shown in Figure 5 B shows that HA-ML@(H+R)NPs can inhibit the expression of inflammatory factors.

[0084] Example 5

[0085] Using the HA-ML@(H+R)NPs prepared in Example 1 and Example 2, the blood half-life and in vivo targeting of HA-ML@(H+R)NPs were determined by semi-quantitative means of detecting fluorescence intensity.

[0086] Experiment 1: Ce6 was used to label Lip and HA-ML@(H+R)NPs to prepare L@Ce6 NPs and HA-ML@Ce6 NPs. After 200 μL of 5 mg / kg Ce6 and HA-ML@Ce6 NPs were injected into the tail vein of C57BL / 6 mice, blood samples were collected at 0.5 h, 1 h, 2 h, 4 h, 8 h, 10 h, 12 h, and 24 h for fluorescence intensity measurement.

[0087] Among them, HA-ML@Ce6 NPs were prepared by the following method: 20 mg Ce6 was dissolved in 1 ml DMSO solution to obtain a Ce6 mother solution with a concentration of 20 mg / mL, 6.6 mg lecithin and 3.3 mg cholesterol, 66 μL Ce6 mother solution were dissolved in 5 mL chloroform solution, fully mixed, vacuum decompression evaporated to remove the organic solvent, liposomes were obtained, 1 mL PBS was added for hydration for 1 hour, and water bath ultrasound was performed at 60W for 5 min. The Ce6-loaded liposome dispersion was prepared, 0.33 mg macrophage membrane was added according to the mass ratio of lecithin to macrophage membrane of 1:20, stirred at 37°C for 30 min, and then 0.66 mg phospholipidated hyaluronic acid was added and stirred at 37°C for 60 min, centrifuged at 13600 rpm for 10 min, and the precipitate was re-dissolved with PBS to obtain HA-ML@Ce6 NPs.

[0088] Figure 6 is the blood half-life of HA-ML@(H+R)NPs of the present invention in C57 mice. Figure 6 As shown in the figure, the blood circulation half-lives of Ce6 and HA-ML@Ce6 NPs are 0.51h and 1.69h, respectively, indicating that HA-ML@(H+R)NPs is beneficial to prolong the blood circulation time.

[0089] Experiment 2: ApoE - / - After mice were fed a high-fat and high-protein diet for 3 months, 200 μL of 5 mg / kg Ce6 and HA-ML@Ce6 NPs obtained in Experiment 1 were injected into the tail vein. Twelve hours later, the mouse aorta was taken for fluorescence imaging.

[0090] The results are as follows Figure 7 As shown, compared with the Ce6 and HA-ML@Ce6 NPs groups, the HA-ML@Ce6 NPs group significantly aggregated in the aortic plaque, indicating that the HA-ML@(H+R)NPs prepared in Example 1 can effectively target the atherosclerotic plaque site.

[0091] Example 6

[0092] The HA-ML@(H+R)NPs prepared in Example 1 and Example 2 are used for the treatment of advanced stage of Hcy-induced atherosclerosis.

[0093] ApoE - / -After 3 months of high-methionine diet, mice were divided into a control group with normal diet, a model group with high-methionine diet, a 10 mg / kg HSYA group, a 5 mg / kg RVS group, a 5 mg / kg HSYA+2.5 mg / kg RVS group, and a HA-ML@(H+R)NPs group, including 5 mg / kg HSYA+2.5 mg / kg RVS. The drug was administered by tail vein injection once every 3 days for 8 consecutive weeks, during which the high-methionine diet was maintained. Three days after the end of the treatment, the mouse aorta was stripped for sectioning and ORO staining to examine the therapeutic effects of the different treatment groups.

[0094] The results are as follows Figure 8 As shown in Figure 2, in the HA-ML@(H+R)NPs treated group, gross examination by surface ORO staining showed that arteries including the aortic arch, thoracic artery, abdominal artery, and common iliac artery contained extensive atherosclerotic plaques throughout the arterial trunk after 16 weeks of high-lipid and high-methionine feeding. Compared with the control group, lipid staining was significantly increased throughout the aorta, as shown in Figure 2. Figure 8 As shown in A, a significant reduction in ORO-stained aortic lipids was observed in mice treated with HA-ML@(H+R)NPs. Consistent with these results, ORO-stained sections of the aortic root in each group also showed that HA-ML@(H+R)NPs treatment had a stronger anti-atherosclerotic ability, with a significant reduction in Hcy levels, as shown in Figure 2. Figure 8 As shown in B. This indicates that the HA-ML@(H+R)NPs constructed in the present invention can effectively treat high Hcy-induced atherosclerosis.

[0095] Example 7

[0096] The HA-ML@(H+R)NPs prepared in Example 1 and Example 2 were used for in vivo biosafety testing. - / - After the treatment of mice, the control group with normal diet, the model group with high methionine diet, the 10mg / kg HSYA group, the 5mg / kg RVS group, the 5mg / kg HSYA+2.5mg / kg RVS group, and the HA-ML@(H+R)NPs group (including 5mg / kg HSYA+2.5mg / kg RVS) were collected for H&E staining of the main organs of the mice, including the heart, liver, spleen, lung, and kidney, to investigate the biosafety of different treatment groups.

[0097] The results are as follows Fig. 9 As shown, no obvious organic lesions were found in the organs of mice in different treatment groups, indicating that the HA-ML@(H+R)NPs constructed in the present invention have good biosafety.

[0098] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0099] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A biomimetic liposome co-drug-loaded nanoparticle, It is characterized in that Hydroxysafflor yellow A and statins are loaded into the bilayer or core of liposomes, and the outer layer of the liposomes is modified with a biomimetic membrane and phospholipidated hyaluronic acid to obtain biomimetic liposome co-drug-loaded nanoparticles with a particle size of 60nm to 100nm; Among them, liposomes are prepared from lecithin and cholesterol; The biomimetic membrane is prepared from macrophages; The phospholipidated hyaluronic acid is prepared from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, hyaluronic acid and phospholipid polyethylene glycol amino.

2. The method for preparing biomimetic liposome-co-drug-loaded nanoparticles according to claim 1, It is characterized in that The specific steps include: Dissolving lecithin and cholesterol in an organic solvent, removing the organic solvent after the reaction, and obtaining liposomes; The liposomes are mixed with hydroxysafflor yellow A, a statin and a PBS solution for hydration reaction, so that the hydroxysafflor yellow A and the statin are loaded into the bilayer or core of the liposomes, thereby obtaining liposome nanoparticles co-loaded with hydroxysafflor yellow A and the statin; Preparation of biomimetic membrane dispersions using macrophages; Phospholipidated hyaluronic acid is prepared by using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, hyaluronic acid and phospholipid polyethylene glycol amino groups, and the phospholipidated hyaluronic acid is dissolved in a PBS solution to obtain a phospholipidated hyaluronic acid solution; in a PBS environment, a biomimetic membrane dispersion liquid and a phospholipidated hyaluronic acid solution are sequentially added to liposome nanoparticles co-loaded with hydroxysafflor yellow A and statins, so that the outer layer of the liposome is modified with a biomimetic membrane and the phospholipidated hyaluronic acid, and biomimetic liposome co-loaded with drugs nanoparticles are obtained.

3. The method for preparing the biomimetic liposome-drug-loaded nanoparticles as claimed in claim 2, It is characterized in that The organic solvent is chloroform.

4. The method for preparing the biomimetic liposome-drug-loaded nanoparticles according to claim 2, It is characterized in that The ratio of the lecithin, cholesterol and organic solvent is 10mg-12mg:5mg:5mL.

5. The method for preparing the biomimetic liposome-drug-loaded nanoparticles as claimed in claim 2, It is characterized in that The mass ratio of the liposome, hydroxysafflor yellow A and statins is 10-11:1:

1.

6. The method for preparing the biomimetic liposome-drug-loaded nanoparticles as claimed in claim 2, It is characterized in that The mass ratio of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, hyaluronic acid and phospholipid polyethylene glycol amino group is 25:50:5:24-25.

7. The method for preparing the biomimetic liposome-drug-loaded nanoparticles as claimed in claim 2, It is characterized in that The volume ratio of the bionic membrane dispersion liquid to the phospholipidated hyaluronic acid solution is 0.05-0.06:0.2-0.

24.

8. The biomimetic liposome-co-drug-loaded nanoparticles as claimed in claim 1, It is characterized in that The statin is any one of rosuvastatin, pitavastatin and simvastatin.

9. Use of the biomimetic liposome co-drug-loaded nanoparticles as claimed in claim 1 in the preparation of drugs for treating atherosclerosis.

10. A drug for treating atherosclerosis, It is characterized in that The active ingredient of the drug includes the biomimetic liposome co-drug-loaded nanoparticles according to claim 1.

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