PLGA targeted drug composite material as well as preparation method and application thereof

Abstract

The invention discloses a PLGA (poly (lactic-co-glycolic acid)) targeted drug composite material as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The preparation method of the PLGA targeted drug composite material comprises the following steps: dissolving astaxanthin, dihydroartemisinin and a polylactic acid-glycolic acid copolymer in an organic solvent to obtain a mixed solution, adding the mixed solution into a surfactant solution, loading the astaxanthin and dihydroartemisinin on the polylactic acid-glycolic acid copolymer to form drug-loaded PLGA, and preparing a dispersion liquid; and adding an activated hyaluronic acid solution into the dispersion liquid, and stirring to wrap the hyaluronic acid on the drug-loaded PLGA to obtain the PLGA targeted drug composite material. The dihydroartemisinin and the astaxanthin are loaded on the polylactic acid-glycolic acid copolymer and coat the hyaluronic acid, and the active targeting function of the hyaluronic acid targets inflammatory macrophages at a focus part, so that the nano composite material is effectively utilized by target cells, and the atherosclerosis prevention and treatment effect of the nano composite material is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and more specifically to a PLGA targeted drug composite material and a preparation method and application thereof. Background Art

[0002] As the disease with the highest morbidity and mortality in the world, the initial pathological mechanism of atherosclerosis (As) is that oxidized low-density lipoprotein (ox-LDL) damages vascular endothelial cells, leading to local inflammatory response. Cytokines are secreted at the site of injury to recruit monocytes in the circulating system and differentiate into macrophages to phagocytize ox-LDL, eventually forming As plaques. As the disease progresses, a large amount of lipids accumulate in late-stage As plaques. A large number of basic and clinical studies have found that there is infiltration of various immune cells in atherosclerotic plaques. After a large amount of ox-LDL is deposited in macrophages, antigen presenting proteins are expressed on the surface of macrophages, activating T cells, which in turn causes the above cells to participate in immune inflammatory lesions at atherosclerotic plaques.

[0003] Although dihydroartemisinin can inhibit antigen presentation on the surface of macrophages and regulate the plaque microenvironment, maintaining the clearance function of macrophages also requires timely promotion of the decomposition of deposited lipids in macrophages and cholesterol efflux. Studies have shown that astaxanthin promotes macrophage lipoprotein degradation and cholesterol efflux by upregulating lipophage function.

[0004] However, when used alone, dihydroartemisinin and astaxanthin have the defects of low drug targeting, poor water solubility and rapid clearance, resulting in limited efficacy. Summary of the invention

[0005] In view of the above problems, the present invention provides a PLGA targeted drug composite material and its preparation method and application. In the present invention, dihydroartemisinin and astaxanthin are used together, loaded on a polylactic acid-glycolic acid copolymer, and the outer layer is coated with hyaluronic acid, and the active targeting function of hyaluronic acid targets the inflammatory macrophages in the lesion site. The target cells can effectively utilize the nanocomposite material and improve its effect in preventing and treating atherosclerosis.

[0006] The first object of the present invention is to provide a method for preparing a PLGA targeted drug composite material, comprising the following steps:

[0007] Dissolving astaxanthin, dihydroartemisinin and polylactic acid-co-glycolic acid in an organic solvent to obtain a mixed solution, adding the mixed solution to a surfactant solution, and loading astaxanthin and dihydroartemisinin on the polylactic acid-co-glycolic acid to form drug-loaded PLGA by a nanoprecipitation method to prepare a dispersion;

[0008] Adding activated hyaluronic acid solution into the dispersion, stirring the reaction to wrap the hyaluronic acid on the drug-loaded PLGA, and obtaining the PLGA targeted drug composite material.

[0009] In a preferred embodiment of the present invention, the mass ratio of astaxanthin to polylactic acid-co-glycolic acid is 0.5 to 2:10. For example, the mass ratio of astaxanthin to polylactic acid-co-glycolic acid is 0.5:10, 1:10, 1.5:10, 2:10, etc., but is not limited to the listed values, and other values ​​not listed within the above numerical range are also applicable.

[0010] In a preferred embodiment of the present invention, the mass ratio of dihydroartemisinin and polylactic acid-glycolic acid copolymer is 2 to 5:10. For example, the mass ratio of dihydroartemisinin and polylactic acid-glycolic acid copolymer is 2:10, 2.5:10, 3:10, 3.5:10, 4:10, 4.5:10, 5:10, etc., but is not limited to the listed values. Other values ​​not listed within the above numerical range are also applicable.

[0011] In a preferred embodiment of the present invention, the nanoprecipitation method is reacted at room temperature for 12h to 14h, for example, the reaction time is 12h, 12.5h, 13h, 13.5h, 14h, etc., but is not limited to the listed values, and other unlisted values ​​within the above numerical range are also applicable.

[0012] In a preferred embodiment of the present invention, the mass ratio of the polylactic acid-glycolic acid copolymer and the surfactant is 1:5-7. For example, the mass ratio of the polylactic acid-glycolic acid copolymer and the surfactant is 1:5, 1:5.5, 1:6, 1:6.5, 1:7, etc., but is not limited to the listed values. Other values ​​not listed within the above numerical range are also applicable.

[0013] In a preferred embodiment of the present invention, the activated hyaluronic acid is obtained by activating hyaluronic acid using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide as activators.

[0014] In a preferred embodiment of the present invention, the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and hyaluronic acid is 5 to 10:16:1. For example, the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and hyaluronic acid is 5:16:1, 5.5:16:1, 6:16:1, 6.5:16:1, 7:16:1, 7.5:16:1, 8:16:1, 8.5:16:1, 9:16:1, 9.5:16:1, 10:16:1, etc., but is not limited to the listed values, and other values ​​not listed within the above numerical range are equally applicable.

[0015] In a preferred embodiment of the present invention, the mass ratio of PLGA to activated hyaluronic acid is 8 to 12:1. For example, the mass ratio of PLGA to activated hyaluronic acid is 8:1, 8.5:1, 9:1, 9.5:1, 10:1, 10.5:1, 11:1, 11.5:1, 12:1, etc., but is not limited to the listed values. Other values ​​not listed within the above numerical range are also applicable.

[0016] The second object of the present invention is to provide a PLGA targeted drug composite material prepared by the above preparation method.

[0017] The third object of the present invention is to provide the use of the above PLGA targeted drug composite material in the preparation of a targeted therapeutic agent for atherosclerosis.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) The present invention designs a highly safe and biodegradable PLGA nanocomposite material. The composite nanomaterial is physically encapsulated by PLGA for astaxanthin and dihydroartemisinin, thereby improving the water solubility of astaxanthin and dihydroartemisinin. Hyaluronic acid is coated on the outermost layer of the nanocomposite material to achieve specific aggregation and penetration of the drug at the lesion site and improve the bioavailability of the drug to the target cell. In addition, the present invention combines astaxanthin and dihydroartemisinin, which can promote macrophage lipoprotein degradation and cholesterol efflux by upregulating the lipophage function, inhibit macrophage surface antigen presentation to regulate the plaque microenvironment, and combine the anti-atherosclerotic effects of two different mechanisms, ultimately effectively treating atherosclerosis. By utilizing the characteristics of the interaction between hyaluronic acid and the target cell surface receptor CD44, the active targeting function of the outermost layer of hyaluronic acid targets the inflammatory macrophages at the lesion site. The target cells can effectively utilize the nanocomposite material and improve its effect in preventing and treating atherosclerosis.

[0020] (2) The present invention can improve drug solubility, improve the blood half-life of the drug, and achieve targeted effects on plaques in atherosclerotic areas. It provides 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Transmission electron microscopy images of different materials, where A is PLGA NPs and B is HA@PLGA@(AST+DHA)NPs.

[0022] Figure 2 are infrared spectra of different materials, where A is a comparison of infrared spectra of AST and HA@PLGA@(AST+DHA)NPs, and B is a comparison of infrared spectra of DHA and HA@PLGA@(AST+DHA)NPs.

[0023] Figure 3 Result graph (A) and bar graph (B) showing the ROS scavenging performance of HA@PLGA@(AST+DHA) NPs.

[0024] Figure 4 These are the results of HA@PLGA@(AST+DHA)NPs inhibiting the foam cell formation of DiI-oxLDL, where Figure A is a laser scanning confocal microscopy image, Figure B is a bar graph based on the laser scanning confocal microscopy image, Figure C is an Oil Red O staining result, and Figure D is a bar graph based on the Oil Red O staining result.

[0025] Figure 5 The blood half-life of different materials in C57 mice; (A) is the fluorescence image of serum of Ce6, PLGA@Ce6 and HA@PLGA@Ce6 at different time points, and (B) is the fitting curve of serum fluorescence values ​​of Ce6, PLGA@Ce6NP and HA@PLGA@Ce6 NPs at different time points.

[0026] Figure 6 The results of the targeting of PLGA@Ce6 and HA@PLGA@Ce6 to atherosclerotic plaques in mice are shown in figure (A) and bar graph (B).

[0027] Figure 7 These are the stained images of frozen sections of blood vessels in different treatment groups for atherosclerosis, from left to right: Control group, Model group, AT group, AST+DHA group and HPAD group.

[0028] Figure 8These are the results of Western blotting analysis of the HA@PLGA@(AST+DHA) NPs prepared in Example 1 for the treatment of atherosclerosis, wherein A is a protein band graph and B is a bar graph. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] When used alone, dihydroartemisinin and astaxanthin have the defects of low drug targeting, poor water solubility and fast clearance, resulting in limited efficacy. Based on this, the present invention creatively combines astaxanthin and dihydroartemisinin to promote macrophage lipophage function and regulate plaque microenvironment, respectively, so as to achieve the synergistic maintenance of macrophage clearance of lipid deposition, and ultimately effectively treat atherosclerosis. However, how to implement the treatment strategy of combining astaxanthin and dihydroartemisinin to achieve drug regulation of target cells at the plaque site. Simple nanomedicines have the disadvantages of being easily metabolized and cleared quickly and lacking active targeting.

[0031] Based on this, the present invention further seeks a therapeutic agent with a long blood circulation period, good lesion site targeting, and can alleviate atherosclerosis. Macrophages, as the main component cells of plaques, highly express CD44 molecules on the surface. Hyaluronic acid molecules are natural ligands of CD44, which can specifically target the CD44 receptors of macrophages, thereby improving cell targeting. At the same time, hyaluronic acid, as a substance naturally present in the human body, has very low immunogenicity. In addition, hyaluronic acid has extremely strong hydrophilicity and can bind a large amount of water. This characteristic contributes to its stable presence in the blood and reduces rapid clearance in the blood.

[0032] The present invention provides a method for preparing a PLGA targeted drug composite material, comprising the following steps:

[0033] Dissolving astaxanthin, dihydroartemisinin and polylactic acid-co-glycolic acid in an organic solvent to obtain a mixed solution, adding the mixed solution to a surfactant solution, and loading astaxanthin and dihydroartemisinin on the polylactic acid-co-glycolic acid to form drug-loaded PLGA by a nanoprecipitation method to obtain a dispersion;

[0034] Add the activated hyaluronic acid solution into the dispersion, stir to wrap the hyaluronic acid on the drug-loaded PLGA, and obtain the PLGA targeted drug composite material.

[0035] During the preparation of the present invention, astaxanthin and dihydroartemisinin are first loaded on polylactic acid-glycolic acid copolymer by nanoprecipitation method to form drug-loaded PLGA, and then hyaluronic acid is coated on the drug-loaded PLGA to obtain a PLGA targeted drug composite material. During the preparation process, the addition of raw materials and the change of preparation conditions will affect the efficiency of encapsulation and loading of astaxanthin and dihydroartemisinin, that is, affect the content of delivered drugs, and may also affect the particle size of the PLGA targeted drug composite material.

[0036] The PLGA targeted drug composite material prepared by the present invention is composed of astaxanthin, dihydroartemisinin, PLGA and hyaluronic acid targeting modification layer. Astaxanthin and dihydroartemisinin are physically encapsulated in PLGA, and the hyaluronic acid modification layer is coated on the outermost layer of the nanocomposite material. The final nanocomposite material has a particle size of 100nm to 150nm. The inner core PLGA is used as a carrier to load the two drugs, astaxanthin and dihydroartemisinin. The outer shell: the hyaluronic acid molecule is a natural ligand of CD44, which can specifically target the CD44 receptor of inflammatory macrophages, thereby improving cell targeting. At the same time, hyaluronic acid, as a substance naturally present in the human body, has very low immunogenicity. In addition, hyaluronic acid has extremely strong hydrophilicity and can bind a large amount of water. This property helps it to exist stably in the blood and reduce rapid clearance in the blood; the lesion cells in the atherosclerotic plaque site are inflammatory macrophages, and the two drugs, astaxanthin and dihydroartemisinin, can be targeted and delivered to the macrophages in the plaque site.

[0037] The polylactic acid-glycolic acid copolymer used in the present invention was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. with the trade name of poly(D,L-lactide-co-glycolide), the product number of 719900, and the molecular weight of 38000-54000. Polyvinyl alcohol was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. with the product number of 767385 and the degree of glycolysis of 98.0-99.0 mol.

[0038] Unless otherwise specified, in the present invention, polylactic acid-co-glycolic acid is denoted as PLGA, dimethyl sulfoxide is denoted as DMSO, polyvinyl alcohol is denoted as PVA, deionized water is denoted as ddH2O, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is denoted as EDC, N-hydroxysuccinimide is denoted as NHS, dihydroartemisinin is denoted as DHA, and astaxanthin is denoted as ASH.

[0039] Example 1

[0040] Step 1, preparing a PLGA dispersion co-loaded with astaxanthin and dihydroartemisinin: preparing PLGA@(AST+DHA)NPs by solvent evaporation method, the specific operation process is as follows:

[0041] Preparation of astaxanthin solution: Add astaxanthin to DMSO to prepare an astaxanthin solution with a concentration of 1 mg / mL;

[0042] Preparation of dihydroartemisinin solution: Add dihydroartemisinin into DMSO to prepare a dihydroartemisinin solution with a concentration of 5 mg / mL;

[0043] Preparation of PLGA solution: PLGA was added to DMSO to prepare a PLGA solution with a concentration of 10 mg / mL;

[0044] Preparation of PVA solution: PVA was added into ddH2O to prepare a PVA solution with a mass concentration of 1%;

[0045] 300 μL of 1 mg / mL astaxanthin solution and 198 μL of 5 mg / mL dihydroartemisinin were added to 300 μL of 10 mg / mL PLGA solution, mixed thoroughly, and ultrasonicated at 30 W for 1 min. The above mixture was added dropwise to 5 mL of 1% PVA solution, ultrasonicated at 30 W for 5 min, stirred at 25 ° C and 1000 rpm for 12 h, and a mixed solution was obtained after stirring. The above mixture was placed in a 3.5 kDa dialysis bag and dialyzed with deionized water for 24 h. After dialysis, a PLGA dispersion with a concentration of 1 mg / ml co-loaded astaxanthin and dihydroartemisinin was obtained, which was recorded as PLGA@(AST+DHA)NPs dispersion.

[0046] Step 2: Preparation of activated hyaluronic acid

[0047] 8 mg EDC, 16 mg NHS and 1 mg hyaluronic acid were dissolved in 1 ml PBS buffer solution with a concentration of 0.01 mol / L and a pH value of 7.2-7.4, and stirred at 800 rpm for 30 min at room temperature to activate the groups on the surface of hyaluronic acid to obtain a mixed solution. The mixed solution was placed in a 3.5 kDa dialysis bag and dialyzed for 24 h to remove free EDC and NHS, and then the activated hyaluronic acid was freeze-dried by freeze drying to obtain activated hyaluronic acid, recorded as HA, and the freeze-dried hyaluronic acid was placed in a -20°C refrigerator for use.

[0048] Step 3, preparing a hyaluronic acid-modified PLGA dispersion co-loaded with astaxanthin and dihydroartemisinin:

[0049] 1 mg of HA was dissolved in a PBS buffer solution with a concentration of 0.01 mol / L and a pH value of 7.2-7.4 to obtain a hyaluronic acid solution. 100 μL of the hyaluronic acid solution was added to 1 ml of a 1 mg / ml PLGA@(AST+DHA)NPs dispersion, and the mixture was stirred in a water bath at 25°C and 800 rpm for 30 min to obtain a mixed solution. The mixed solution was placed in a 3.5 kDa dialysis bag and dialyzed with deionized water for 24 h to obtain a PLGA targeted composite material co-loaded with astaxanthin and dihydroartemisinin, namely, HA@PLGA@(AST+DHA)NPs, abbreviated as HPAD.

[0050] Example 2

[0051] Step 1, preparing a PLGA dispersion co-loaded with astaxanthin and dihydroartemisinin: preparing PLGA@(AST+DHA)NPs by solvent evaporation method, the specific operation process is as follows:

[0052] Preparation of astaxanthin solution: Add astaxanthin to DMSO to prepare an astaxanthin solution with a concentration of 1 mg / mL;

[0053] Preparation of dihydroartemisinin solution: Add dihydroartemisinin into DMSO to prepare a dihydroartemisinin solution with a concentration of 5 mg / mL;

[0054] Preparation of PLGA solution: PLGA was added to DMSO to prepare a PLGA solution with a concentration of 10 mg / mL;

[0055] Preparation of PVA solution: PVA was added into ddH2O to prepare a PVA solution with a mass concentration of 1%;

[0056] 150 μL of 1 mg / mL astaxanthin solution and 120 μL of 5 mg / mL dihydroartemisinin were added to 300 μL of 10 mg / mL PLGA solution, mixed thoroughly, and ultrasonicated at 30 W for 1 min. The above mixture was added dropwise to 7 mL of 1% PVA solution, ultrasonicated at 30 W for 5 min, stirred at 25 ° C and 700 rpm for 24 h, and a mixed solution was obtained after stirring. The above mixture was placed in a 3.5 kDa dialysis bag and dialyzed with deionized water for 24 h. After the dialysis was completed, a PLGA dispersion with a concentration of 1 mg / ml co-loaded astaxanthin and dihydroartemisinin was obtained, which was recorded as PLGA@(AST+DHA)NPs dispersion.

[0057] Step 2: Preparation of activated hyaluronic acid

[0058] 5 mg EDC, 16 mg NHS and 1 mg hyaluronic acid were dissolved in 1 ml PBS buffer solution with a concentration of 0.01 mol / L and a pH value of 7.2-7.4, and stirred at 800 rpm for 30 min at room temperature to activate the groups on the surface of hyaluronic acid to obtain a mixed solution. The mixed solution was placed in a 3.5 kDa dialysis bag and dialyzed for 24 h to remove free EDC and NHS. The activated hyaluronic acid was then freeze-dried by freeze-drying to obtain activated hyaluronic acid, recorded as HA, and the freeze-dried hyaluronic acid was placed in a -20°C refrigerator for use.

[0059] Step 3, preparing a hyaluronic acid-modified PLGA dispersion co-loaded with astaxanthin and dihydroartemisinin:

[0060] 1 mg of HA was dissolved in 1 ml of PBS buffer solution with a concentration of 0.01 mol / L and a pH value of 7.2-7.4 to obtain a hyaluronic acid solution; 120 μL of the hyaluronic acid solution was added to 1 ml of PLGA@(AST+DHA) NPs dispersion with a concentration of 1 mg / ml, and the mixture was stirred in a water bath at 25°C and 800 rpm for 30 min to obtain a mixed solution; the mixed solution was placed in a 3.5 kDa dialysis bag and dialyzed with deionized water for 24 h to obtain a PLGA targeted composite material co-loaded with astaxanthin and dihydroartemisinin.

[0061] Example 3

[0062] Step 1, preparing a PLGA dispersion co-loaded with astaxanthin and dihydroartemisinin: preparing PLGA@(AST+DHA)NPs by solvent evaporation method, the specific operation process is as follows:

[0063] Preparation of astaxanthin solution: Add astaxanthin to DMSO to prepare an astaxanthin solution with a concentration of 1 mg / mL;

[0064] Preparation of dihydroartemisinin solution: Add dihydroartemisinin into DMSO to prepare a dihydroartemisinin solution with a concentration of 5 mg / mL;

[0065] Preparation of PLGA solution: PLGA was added to DMSO to prepare a PLGA solution with a concentration of 10 mg / mL;

[0066] Preparation of PVA solution: PVA was added into ddH2O to prepare a PVA solution with a mass concentration of 1%;

[0067] 300 μL of 1 mg / mL astaxanthin solution and 300 μL of 5 mg / mL dihydroartemisinin were added to 300 μL of 10 mg / mL PLGA solution, mixed thoroughly, and ultrasonicated at 30 W for 1 min. The above mixture was added dropwise to 6 mL of 1% PVA solution, ultrasonicated at 30 W for 5 min, stirred at 25 ° C and 700 rpm for 24 h, and a mixed solution was obtained after stirring. The mixed solution was placed in a 3.5 kDa dialysis bag and dialyzed with deionized water for 24 h. After dialysis, a PLGA dispersion with a concentration of 1 mg / ml co-loaded astaxanthin and dihydroartemisinin was obtained, which was recorded as PLGA@(AST+DHA)NPs dispersion.

[0068] Step 2: Preparation of activated hyaluronic acid

[0069] 10 mg EDC, 16 mg NHS and 1 mg hyaluronic acid were dissolved in 1 ml of PBS buffer solution with a concentration of 0.01 mol / L and a pH value of 7.2-7.4, and stirred at 800 rpm for 30 min at room temperature to activate the groups on the surface of hyaluronic acid to obtain a mixed solution. The mixed solution was placed in a 3.5 kDa dialysis bag and dialyzed for 24 h to remove free EDC and NHS, and then the activated hyaluronic acid was freeze-dried by freeze drying to obtain activated hyaluronic acid, recorded as HA, and the freeze-dried hyaluronic acid was placed in a -20°C refrigerator for use.

[0070] Step 3, preparing a hyaluronic acid-modified PLGA dispersion co-loaded with astaxanthin and dihydroartemisinin:

[0071] 1 mg of HA was dissolved in 1 mL of PBS buffer solution with a concentration of 0.01 mol / L and a pH of 7.2-7.4 to obtain a hyaluronic acid solution; 80 μL of the hyaluronic acid solution was added to 1 ml of PLGA@(AST+DHA) NPs dispersion with a concentration of 1 mg / ml, and the mixture was stirred in a water bath at 25°C and 800 rpm for 30 min to obtain a mixed solution; the mixed solution was placed in a 3.5 kDa dialysis bag and dialyzed with deionized water for 24 h to obtain a PLGA targeted composite material co-loaded with astaxanthin and dihydroartemisinin.

[0072] Comparative Example 1

[0073] 10 mg PLGA was dissolved in 1 ml dichloromethane. 100 μL of the above solution was added dropwise into 1 ml PVA solution with a mass concentration of 1%. Ultrasonication was performed at 30 W for 5 min. The mixture was stirred at 25 ° C and 700 rpm for 24 h. After the stirring was completed, a mixed solution was obtained. The mixed solution was placed in a 3.5 kDa dialysis bag and dialyzed with deionized water for 24 h. After the dialysis was completed, the dried PLGA nanoparticles were recorded as PLGANPs.

[0074] The PLGA targeting composite material co-loaded with astaxanthin and dihydroartemisinin prepared in Example 1 was characterized below.

[0075] Figure 1 Figure 1 is a transmission electron micrograph of the PLGANPs of the present invention and the HA@PLGA@(AST+DHA)NPs prepared in Example 1. Figure 1 As shown, the results show that PLGANPs are spherical and uniformly dispersed, with a particle size of about 106nm; in the HA@PLGA@(AST+DHA)NPs prepared in Example 1, hyaluronic acid is modified in the outer layer of PLGA@(AST+DHA)NPs, presenting an obvious core-shell structure, indicating that PLGA encapsulates astaxanthin and dihydroartemisinin, and the hyaluronic acid molecules in the outer layer have a targeting effect. The particle size of the HA@PLGA@(AST+DHA)NPs prepared in Example 1 is about 122nm.

[0076] Figure 2 The infrared spectrum of HA@PLGA@(AST+DHA)NPs prepared in Example 1 of the present invention is compared with the infrared spectrum of AST and DHA. Infrared spectrum detection shows that the infrared characteristic peak of AST (1700-750cm -1 ) and DHA infrared characteristic peaks (1275-1060cm -1 ), indicating that PLGA was successfully loaded with AST and DHA.

[0077] Example 4

[0078] A PLGA targeted drug composite material is used in the prevention and treatment of atherosclerosis, wherein the HA@PLGA@(AST+DHA) NPs prepared in Example 1 are used in the prevention and treatment of atherosclerosis.

[0079] Figure 3 In the control, ApoE - / - Mice are fed a normal diet; Model is ApoE - / - Mice were fed a high-fat diet: 40% fat, 40% carbohydrates, 20% protein, and 1.25% cholesterol.

[0080] Fluorescence imaging was used to determine the ability of HA@PLGA@(AST+DHA)NPs prepared in Example 1 to scavenge oxidative stress in macrophages. Figure 3 As shown. ROS is a manifestation of cellular oxidative stress. The 2,7-dichlorofluorescein diacetate (DCFH-DA) probe emits green light in the presence of ROS. Figure 3From the intensity of green fluorescence, it can be seen that compared with the Model, the Control exhibits a weak green light; compared with the Model, the green fluorescence of the AST+DHA group is weakened, and the HA@PLGA@(AST+DHA)NPs is further weakened, indicating that HA@PLGA@(AST+DHA)NPs can effectively clear the ROS level in macrophages.

[0081] The ability of HA@PLGA@(AST+DHA)NPs prepared in Example 1 to delay foam cell formation was determined by DiI-labeled oxidized low-density lipoprotein (DiI-oxLDL) uptake experiment and foam cell formation experiment. Figure 4 It was shown that during the lipid uptake inhibition phase, Figure 4 As shown in Figures A and B, compared with the Control, Model and AST+DHA combination groups, HA@PLGA@(AST+DHA)NPs significantly inhibited the uptake of DiI-oxLDL; in the stage of reducing lipid deposition, Figure 4 As can be seen from Figures C and D, compared with the Control, Model and AST+DHA combination groups, the Oil Red O staining results showed that HA@PLGA@(AST+DHA)NPs significantly inhibited the deposition of oxLDL in macrophages. The two experiments showed that HA@PLGA@(AST+DHA)NPs can effectively delay the formation of foam cells.

[0082] The blood half-life and in vivo targeting of HA@PLGA@(AST+DHA) NPs prepared in Example 1 were determined by semi-quantitative means of detecting fluorescence intensity.

[0083] PLGA NPs and HA@PLGA were labeled with dihydrochlorin e6(ce6) to prepare PLGA@ce6 and HA@PLGA@ce6, respectively.

[0084] Among them, PLGA@ce6 was prepared by the following method: 10 mg PLGA was dissolved in 1 mL dichloromethane solution to obtain a PLGA solution;

[0085] Dissolve ce6 in DMSO to obtain 100 μL of ce6 solution with a concentration of 20 mg / ml. Add the ce6 solution dropwise to the PLGA solution, mix thoroughly, and ultrasonicate in a water bath at 60 W for 5 min to obtain a mixed solution. Add the above mixed solution dropwise to 10 mL of a 1% PVA solution, and ultrasonicate for 5 min at an ultrasonic power of 30 W. Stir at 1000 rpm for 12 h to evaporate dichloromethane. Centrifuge at 10000 rpm for 10 min, and re-dissolve the precipitate with PBS to obtain a PLGA dispersion loaded with ce6, recorded as PLGA@ce6.

[0086] HA@PLGA@ce6 was prepared by the following method:

[0087] 1 mg HA was redissolved in 1 ml of PBS buffer solution with a concentration of 0.01 mol / L and a pH value of 7.2-7.4 to obtain a hyaluronic acid solution. 100 μL of the hyaluronic acid solution was added to 1 ml of PLGA@ce6 NPs solution with a concentration of 1 mg / ml. The mixture was stirred in a water bath at 25°C and 800 rpm for 60 min to obtain a ce6-loaded HA and PLGA dispersion, which was recorded as HA@PLGA@ce6.

[0088] Experiment 1: Determination of blood half-life

[0089] After C57BL / 6 mice were injected with 200 μL of 5 mg / kg ce6, PLGA@ce6 and HA@PLGA@ce6 through the tail vein, blood samples were collected at different time points of 0h, 0.5h, 2h, 4h, 8h, 12h and 24h for fluorescence intensity measurement.

[0090] Figure 5 is the blood half-life of HA@PLGA@ce6 in C57 mice. Figure 5 As shown in the figure, the blood circulation half-lives of ce6, PLGA@ce6 and HA@PLGA@ce6 are 0.42h, 0.73h and 1.07h respectively. This shows that hyaluronic acid modification is beneficial to prolonging blood circulation time. This is because hyaluronic acid has extremely strong hydrophilicity and can bind a large amount of water. This property helps it to exist stably in the blood and reduce rapid clearance; as a substance naturally present in the human body, hyaluronic acid has very low immunogenicity and is not easily recognized and cleared by the immune system. Hyaluronic acid can be modified to increase its affinity for specific cells or tissues, such as by modifying it to enable it to specifically bind to the CD44 receptor on the surface of tumor cells, thereby prolonging its time in the blood circulation and improving the targeting of drugs.

[0091] Experiment 2: In vivo targeting assay

[0092] ApoE- / - After mice were fed a high-fat diet for one month, 200 μL of ce6, PLGA@ce6, and HA@PLGA@ce6 at a concentration of 5 mg / kg were injected through the tail vein. Twelve hours later, the mouse aorta was taken for fluorescence imaging.

[0093] Figure 6 The results show that the HA@PLGA@ce6 of the present invention is targeted to atherosclerotic plaques in mice. Figure 6 As shown in the figure, compared with the ce6 and PLGA@ce6 groups, the HA@PLGA@ce6 group significantly aggregated in the aortic plaque, indicating that HA@PLGA@ce6 can effectively target the site of atherosclerotic plaques. The aortic plaques are prone to form in the aortic arch, the aorta and the renal aorta, and the aorta and the femoral artery. Plaque formation is closely related to the deposition of lipids in the lower layer of blood vessels after macrophages phagocytize them. CD44 is highly expressed on the surface of inflammatory macrophages. HA hyaluronic acid, as a ligand for CD44, actively targets macrophages in plaques.

[0094] ApoE - / - After mice were fed a high-fat diet for one month, HA@PLGA@(AST+DHA)NPs were used for targeted treatment of atherosclerosis.

[0095] The drug-dosing groups were divided into PBS group, Model group, AT group (atorvastatin, 2 mg / kg), AST+DHA group (0.63 mg / kg AST+5.0 mg / kg DHA) and HA@PLGA@(AST+DHA)NPs group (0.63 mg / kg AST+5.0 mg / kg DHA), which were administered twice a week for 2 consecutive months, and a high-fat diet was maintained during the period. All drugs were administered by tail vein injection. Three days after the end of the treatment, the mouse aorta was stripped for macroscopic staining of the blood vessels to examine the therapeutic effects of different treatment groups.

[0096] Figure 7 The following are the gross staining pictures of blood vessels in different treatment groups of the present invention for the treatment of atherosclerosis. Figure 7 As shown, Figure 7 The white part in the blood vessels in the middle circle represents plaques, and the rectangular part below is stained with Oil Red O in the aorta, and the red part is plaque. Compared with the model group, the plaques in the AST+DHA group and HA@PLGA@(AST+DHA)NPs group were significantly reduced, and the efficacy of HA@PLGA@(AST+DHA)NPs was equivalent to that of the AT group. The reduction in plaque formation is closely related to AST promoting macrophage lipid decomposition and DHA regulating the plaque microenvironment. The HA@PLGA@(AST+DHA)NPs treatment group significantly reduced the formation of atherosclerotic plaques, indicating that the biomimetic nanoformulation constructed by the present invention can effectively treat atherosclerosis.

[0097] Figure 8 The results of Western blot analysis of HA@PLGA@(AST+DHA)NPs prepared in Example 1 for treating atherosclerosis are shown in Table 1. Figure 8 A and B are the protein band diagrams and bar graphs of the expression of human microtubule-associated protein light chain 3B (LC3B), ubiquitin binding protein (p62), adenosine triphosphate binding cassette transporter A1 (ABCA1), ATP binding cassette transporter G1 (ABCG1), and sialic acid binding Ig-like lectin 1 (Siglecl-1) protein after treatment with HA@PLGA@(AST+DHA)NPs prepared in Example 1, respectively. In the protein blotting experiment at the cellular level, ABCA1 and ABCG1 indicate the cholesterol efflux level. LC3B and P62 indicate the autophagy level. The changes in the protein levels of ABCA1, ABCG1, LC3B, and p62 indicate the upregulation of the function of macrophages in decomposing lipids. Figure 8 In the experiment, the expression of ABCA1, ABCG1 and LC3B proteins increased, indicating that the function of macrophages in decomposing lipids was upregulated, while p62 decreased. This is because p62 not only acts as an adaptor protein between autophagosomes and substrates, but also regulates the process of autophagy. When autophagic activity weakens, p62 protein accumulates in cells, and the accumulation of p62 can negatively regulate the autophagic activity of cells and inhibit the autophagic activity of cells by activating the mTORC1 signaling pathway.

[0098] The functions of Siglec-1 include the ability to internalize antigens and deliver them to lymphocytes via macrophages as antigen-presenting cells, which makes Siglec-1 play a key role in a variety of human disease states, including atherosclerosis, coronary artery disease, autoimmune diseases, etc. Figure 8 It can be seen that the HA@PLGA@(AST+DHA) NPs prepared in Example 1 of the present invention effectively reduced Siglec-1. The reduction of Siglec-1 indicates that the antigenicity of lipids is reduced and the immune microenvironment of the plaque site is improved.

[0099] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0100] 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 method for preparing a PLGA targeted drug composite material, characterized in that: The following steps are involved: Dissolving astaxanthin, dihydroartemisinin and polylactic acid-co-glycolic acid copolymer in an organic solvent to obtain a mixed solution, adding the mixed solution to a surfactant solution, and loading astaxanthin and dihydroartemisinin on the polylactic acid-co-glycolic acid copolymer by a nanoprecipitation method to form drug-loaded PLGA to prepare a dispersion; The activated hyaluronic acid solution is added to the dispersion, and the hyaluronic acid is wrapped on the drug-loaded PLGA by stirring to obtain a PLGA targeted drug composite material.

2. The method for preparing a PLGA targeted drug composite material according to claim 1, characterized in that: The mass ratio of astaxanthin to polylactic acid-glycolic acid copolymer is 0.5 to 2:

10.

3. The method for preparing a PLGA targeted drug composite material according to claim 1, characterized in that: The mass ratio of dihydroartemisinin to polylactic acid-glycolic acid copolymer is 2 to 5:

10.

4. The method for preparing a PLGA targeted drug composite material according to claim 1, characterized in that: The nanoprecipitation method is to react at room temperature for 12h to 14h.

5. The method for preparing a PLGA targeted drug composite material according to claim 1, characterized in that: The mass ratio of the polylactic acid-glycolic acid copolymer to the surfactant is 1:5-7.

6. The method for preparing a PLGA targeted drug composite material according to claim 1, characterized in that: The activated hyaluronic acid is obtained by activating hyaluronic acid using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide as activators.

7. The method for preparing a PLGA targeted drug composite material according to claim 1, characterized in that: The mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and hyaluronic acid is 5-10:16:

1.

8. The method for preparing a PLGA targeted drug composite material according to claim 1, characterized in that: The mass ratio of PLGA to activated hyaluronic acid is 8 to 12:

1.

9. A PLGA targeted drug composite material prepared according to any one of claims 1 to 8.

10. Use of the PLGA targeted drug composite material according to claim 9 in preparing a drug for targeted treatment of atherosclerosis.

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