Bionic PLGA composite nanomaterial as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to a bionic PLGA composite nano-material and a preparation method and application thereof.The bionic PLGA composite nano-material is characterized in that after curcumin and iloumab are co-loaded and packaged through a polylactic acid-glycolic acid copolymer, a bionic membrane is disguised on the outer layer of the polylactic acid-glycolic acid copolymer; and modifying the surface of the biomimetic membrane with phosphatidylated hyaluronic acid. According to the biomimetic PLGA composite nano-material disclosed by the invention, by utilizing the design characteristics of biomimetic membrane and hyaluronic acid modification and combining two anti-atherosclerosis effects of curcumin and iloumab with different mechanisms, the specific aggregation and permeation of a drug at a diseased region are realized, and the bioavailability of a target cell to the drug is improved; the purpose of treating atherosclerosis through synergistic interaction is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a bionic PLGA composite nano material and a preparation method and application thereof. Background Art

[0002] Atherosclerosis, referred to as AS, its pathogenesis mainly involves high levels of plasma cholesterol, especially plasma low-density lipoprotein cholesterol LDL-C, and the accumulation of inflammatory cells in atherosclerotic plaques. The early lesions of atherosclerosis originate from the accumulation of cholesterol under the endothelium. With the continuous development of atherosclerosis, the expression of adhesion molecules on the endothelium increases, recruiting monocytes to the lesion plaques, and then monocytes differentiate into macrophages and ingest excessive LDL-C to form foam cells, and accelerate the recruitment of more immune cells to the inflammatory site of the plaque through a positive feedback mechanism, thereby promoting the further development of atherosclerotic plaques. Existing methods for treating atherosclerosis mainly include drug therapy, vascular intervention therapy and surgical treatment, and oral drug therapy is the main treatment. However, clinical oral medication has the disadvantages of low drug targeting, poor water solubility and rapid clearance, resulting in limited efficacy. Therefore, the development of a new drug formulation is a problem that needs to be solved urgently.

[0003] Eloumab is a proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitor, also known as a PCSK9 inhibitor, which has attracted widespread attention in the treatment of atherosclerosis due to its function of lowering LDL-C. It is well known that the low-density lipoprotein cholesterol receptor LDLR is the main pathway for the removal of cholesterol from the circulation. Eloumab can block the PCSK9 protein-mediated LDLR degradation process, thereby upregulating the cell surface LDLR level and ultimately achieving the effect of lowering plasma LDL. However, the current LDL-lowering therapy cannot fully address the residual cardiovascular risk caused by potential inflammation. Therefore, the combination of anti-inflammatory drugs is an effective way to solve this problem. Curcumin is a natural polyphenol derived from the rhizome of turmeric. It has attracted widespread attention due to its anti-inflammatory, immunosuppressive, antioxidant, antiviral, antitumor activities and its lipid-lowering function. Previous studies have shown that curcumin may play an anti-atherosclerotic role by regulating macrophage polarization and inhibiting the transmission of inflammatory signals. However, the poor water solubility and low bioavailability of this natural drug have greatly limited its clinical use. Therefore, it is considered to adjust the administration method and use the two drugs in combination for the treatment of atherosclerosis. However, how to deliver the two drugs to the lesion area at the same time and increase the accumulation of the drugs in the inflammatory area remains a major challenge. Summary of the invention

[0004] The purpose of the present invention is to provide a bionic PLGA composite nanomaterial, which can deliver two drugs, evolocumab and curcumin, to the lesion area at the same time and improve the accumulation of drugs in the inflammatory area.

[0005] The technical solution adopted by the present invention is:

[0006] The invention provides a bionic PLGA composite nanomaterial. The bionic PLGA composite nanomaterial comprises a polylactic acid-glycolic acid copolymer for co-loading and encapsulating curcumin and eloumab, a bionic membrane is camouflaged on the outer layer of the polylactic acid-glycolic acid copolymer, and then the surface of the bionic membrane is modified with phospholipidated hyaluronic acid.

[0007] Preferably, the particle size of the bionic PLGA composite nanomaterial is 100 nm to 300 nm.

[0008] The present invention provides a method for preparing the bionic PLGA composite nanomaterial, comprising the following steps:

[0009] Curcumin is encapsulated in a polylactic acid-glycolic acid copolymer to prepare a polylactic acid-glycolic acid copolymer dispersion loaded with curcumin; eloumab is encapsulated in a polylactic acid-glycolic acid copolymer to prepare a polylactic acid-glycolic acid copolymer dispersion loaded with eloumab; a biomimetic membrane dispersion and a phospholipidated hyaluronic acid are prepared; the polylactic acid-glycolic acid copolymer dispersion loaded with curcumin, the polylactic acid-glycolic acid copolymer dispersion loaded with eloumab and the biomimetic membrane dispersion are mixed and extruded to prepare a biomimetic membrane-coated composite material loaded with curcumin and eloumab; the phospholipidated hyaluronic acid is mixed with the biomimetic membrane-coated composite material loaded with curcumin and eloumab, so that the phospholipidated hyaluronic acid is inserted into the surface of the biomimetic membrane-coated composite material loaded with curcumin and eloumab to obtain the biomimetic PLGA composite nanomaterial.

[0010] Preferably, the method of encapsulating the curcumin in poly(lactic acid-co-glycolic acid) comprises any one of a nanoprecipitation method, an emulsification solvent volatilization method, a liquid / solvent evaporation method, a liquid-driven co-flow focusing method and an emulsification / solvent diffusion method.

[0011] Preferably, the method of encapsulating the evolocumab in poly(lactic acid-co-glycolic acid) comprises any one of a spray drying method, an emulsification solvent volatilization method, a liquid / solvent evaporation method and a nanoprecipitation method.

[0012] Preferably, when preparing the polylactic acid-co-glycolic acid dispersion loaded with curcumin, the mass ratio of curcumin to the polylactic acid-co-glycolic acid is 1:10-20.

[0013] Preferably, when preparing the polylactic acid-co-glycolic acid dispersion loaded with evolocumab, the mass ratio of evolocumab to the polylactic acid-co-glycolic acid is 0.025 to 0.5:1.

[0014] Preferably, when preparing the biomimetic membrane-coated composite material loaded with curcumin and eloumab, the molar ratio of the polylactic acid-co-glycolic acid dispersion loaded with curcumin to the polylactic acid-co-glycolic acid dispersion loaded with eloumab is 20 to 40:1.

[0015] The present invention provides an application of the bionic PLGA composite nano material. The bionic PLGA composite nano material is used for preparing a preparation for treating atherosclerosis.

[0016] Preferably, the atherosclerosis includes at least one of aortic atherosclerosis, coronary atherosclerosis, cerebral atherosclerosis, renal artery atherosclerosis and limb artery atherosclerosis.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention provides a biomimetic PLGA composite nanomaterial, wherein the biomimetic PLGA composite nanomaterial is a polylactic acid-glycolic acid copolymer that co-loads and encapsulates curcumin and eloumab, and then a biomimetic membrane is camouflaged on the outer layer of the polylactic acid-glycolic acid copolymer, and then phospholipidated hyaluronic acid is modified on the surface of the biomimetic membrane. The biomimetic PLGA composite nanomaterial of the present invention utilizes the design features of biomimetic membrane and hyaluronic acid modification, combines the anti-atherosclerotic effects of curcumin and eloumab of two different mechanisms, realizes the specific aggregation and penetration of drugs in the lesion site, and improves the bioavailability of drugs to target cells, so as to achieve the purpose of synergistically enhancing the treatment of atherosclerosis.

[0019] In addition to loading drugs for treating and / or preventing atherosclerosis, the present invention provides a bionic nanocomposite material that is co-loaded with drugs, which 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 immune escape ability of the bionic membrane and the mechanism of action of different drugs. Therefore, the present invention combines a bionic membrane coating with a targeted modified nanocomposite material, which can provide multiple functions and advantages to treat a variety of diseases.

[0020] The present invention compensates 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 related clinical treatments, and has important scientific significance, practical value and economic value.

[0021] The bionic PLGA composite nanomaterial of the present invention utilizes the anti-atherosclerotic effects of two different mechanisms of curcumin and eloumab to achieve targeted treatment of atherosclerosis. Compared with a single drug, the dosage is halved and the combined use of nano-sized drugs enhances efficacy and reduces toxicity. The inflammatory environment at the site of atherosclerotic plaques secretes inflammatory factors to recruit macrophages, and the outermost bionic membrane achieves the ability of the nanocomposite material to achieve long blood circulation and target the lesion site through its inherent immune escape and recruitment characteristics of the source cells. By utilizing the characteristics of the interaction between hyaluronic acid and the target cell surface receptor CD44, the target cells can effectively utilize the nanocomposite material and improve its effect in preventing and treating atherosclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Coomassie brilliant blue staining and protein immunoblotting of HA-M@P@(Evol+Cur)NPs prepared in the present invention; A is Coomassie brilliant blue staining, and B is protein immunoblotting.

[0023] Figure 2 Transmission electron microscopy images of HA-M@P@(Evol+Cur)NPs and empty carrier PLGANPs prepared in the present invention, A is PLGANPs, and B is HA-M@P@(Evol+Cur)NPs.

[0024] Figure 3 This is a fusion experiment of macrophage membrane wrapped PLGA of the present invention, A to C are respectively: PLGA and M@P@(Evol+Cur).

[0025] Figure 4 The HA-M@P@(Evol+Cur) NPs prepared in the present invention inhibit the uptake of DiI-oxLDL and the formation of foam cells. A is the experimental result of DiI-oxLDL uptake; B is the experimental result of inhibiting foam cell formation, from left to right are Control group, Model group, Cur+Evol group and HA-M@P@(Evol+Cur) group.

[0026] Figure 5 The ability of HA-M@P@(Evol+Cur)NPs prepared by the present invention to regulate macrophage polarization and reduce inflammation. A~L are the treatment conditions of different component drugs after iNOS incubation; a~l are the treatment conditions of different component drugs after Arg-1 incubation; M is the statistical result corresponding to A~L; N is the statistical result corresponding to a~l; O is the expression of different immune factors.

[0027] Figure 6 The HA-M@P@(Evol+Cur)NPs prepared by the present invention are - / -Prolonged blood half-life in mice, A is the test results of different groups at different time points; B is the statistical result corresponding to A.

[0028] Figure 7 The targeting of HA-M@P@(Evol+Cur)NPs prepared in the present invention to atherosclerotic plaques in mice. A is the result of three parallel experiments after using P@Ce6, and B is the result of three parallel experiments after using HA-M@P@Ce6.

[0029] Figure 8 These are the gross staining pictures of blood vessels in different treatment groups of the present invention for the treatment of atherosclerosis. A is the ApoE after treatment with different drugs - / - Representative images of ORO staining of the mouse aortic arch, from left to right: 3 parallel experiments without any drug injection, 3 parallel experiments with PBS injection, 3 parallel experiments with 20 mg / kg Cur injection, 3 parallel experiments with 5 mg / kg Evol injection, 3 parallel experiments with 10 mg / kg Cur + 2.5 mg / kg Evol injection, and 3 parallel experiments with the same dose of HA-M@P@(Evol+Cur); B is ApoE - / - ORO staining of frozen sections of mouse aortic root; C is the level of Hcy in serum after treatment with different drugs.

[0030] Fig. 9 These are H&E staining images of the heart, liver, spleen, lung and kidney of mice with advanced atherosclerosis treated with different treatment groups of the present invention. DETAILED DESCRIPTION

[0031] The present invention is further described below by specific examples, but the scope of the present invention is not limited thereto. The details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0032] The inventive concept of the present invention is as follows:

[0033] Existing methods for treating atherosclerosis mainly include drug therapy, vascular intervention therapy and surgical treatment, and oral drug therapy is the main method. However, clinical oral medication has disadvantages such as low drug targeting, poor water solubility and rapid clearance, which leads to limited efficacy. Therefore, the development of a new drug dosage form is a problem that needs to be solved urgently.

[0034] As a drug for the treatment of atherosclerosis, eloumab blocks the PCSK9 protein-mediated LDLR degradation process, upregulates the cell surface LDLR level and ultimately reduces plasma LDL. However, the current LDL-lowering therapy cannot fully address the residual cardiovascular risk caused by underlying inflammation, so anti-inflammatory drugs are usually used in combination to solve this problem. Curcumin, as a natural polyphenol, can be used as a potential synergistic therapeutic drug. However, the poor water solubility and low bioavailability of natural drugs have greatly limited their clinical use. Therefore, it is considered to adjust the administration method and use the two drugs in combination for the treatment of atherosclerosis. However, how to deliver the two drugs to the lesion area at the same time and increase the accumulation of drugs at the site of inflammation remains a major challenge.

[0035] Based on this, the present invention provides a biomimetic PLGA composite nanomaterial, wherein the biomimetic PLGA composite nanomaterial is a polylactic acid-glycolic acid copolymer that co-loads and encapsulates curcumin and eloumab, and then a biomimetic membrane is camouflaged on the outer layer of the polylactic acid-glycolic acid copolymer, and then phospholipidated hyaluronic acid is modified on the surface of the biomimetic membrane. The biomimetic PLGA composite nanomaterial of the present invention utilizes the design features of biomimetic membrane and hyaluronic acid modification, combines the anti-atherosclerotic effects of curcumin and eloumab of two different mechanisms, realizes the specific aggregation and penetration of drugs in the lesion site, and improves the bioavailability of drugs to target cells, so as to achieve the purpose of synergistic treatment of atherosclerosis.

[0036] 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. In the description of the present invention, unless otherwise specified, the reagents used are all commercially available, and the methods used are all conventional techniques in the art.

[0037] The abbreviations of the present invention are shown in Table 1.

[0038] Table 1 Abbreviations

[0039]

[0040]

[0041] Example 1

[0042] A method for preparing a bionic PLGA composite material comprises the following steps:

[0043] S1. Prepare PLGA dispersion loaded with curcumin by nanoprecipitation method. The specific operation process is as follows:

[0044] Dissolve 20 mg PLGA in 1 mL DMSO to prepare PLGA-DMSO solution. Take 50 μL of 20 mg / mL curcumin solution prepared with DMSO and add it to 500 μL PLGA-DMSO solution. Mix thoroughly and ultrasonicate in a 50W water bath for 5 min. Add the mixture dropwise to a 5 mL PVA aqueous solution with a mass fraction of 2% under water bath ultrasonication. Mix well and place in a 3.5 kDa dialysis bag and dialyze with deionized water for 24 hours to obtain a PLGA dispersion loaded with curcumin, recorded as P@Cur.

[0045] S2. The PLGA dispersion loaded with evolocumab was prepared by the emulsification solvent evaporation method. The specific operation process is as follows:

[0046] Dissolve 10 mg PLGA in 1 mL dichloromethane solution to prepare PLGA-dichloromethane solution. Take 12.5 μL of 10 mg / mL eloumab and add it dropwise to 500 μL PLGA-dichloromethane solution under water bath ultrasound, and mix thoroughly at 10% power ultrasound for 1 min. Add the mixture dropwise to 5 mL of 2% PVA under stirring, and ultrasound at 40% power for 1 min; stir at 300 rpm for 12 h to evaporate dichloromethane, centrifuge at 12000 rpm for 30 min, and re-dissolve the precipitate with PBS to obtain PLGA dispersion loaded with eloumab, recorded as P@Evol.

[0047] S3, preparing biomimetic membrane dispersion, the specific operation process is as follows:

[0048] Prepare macrophage membranes according to the instructions of the membrane protein extraction kit. Wash the macrophages twice with 4°C precooled PBS and resuspend them in membrane protein extractor A, which contains 1mM PMSF. Lyse on ice for 30 minutes, sonicate in a 4°C 80W water bath for 10 minutes; freeze and thaw repeatedly 5 times in -80°C and 37°C environments, each for 30 minutes; centrifuge at 12000rpm, 4°C for 30 minutes to obtain macrophage membrane precipitates, recorded as or M.

[0049] S4, preparing phospholipidated hyaluronic acid, the specific operation process is as follows:

[0050] 8 mg EDC, 16 mg NHS and 1 mg hyaluronic acid were dissolved in PBS and stirred at 800 rpm for 30 min at room temperature to activate the carboxyl groups on the surface of hyaluronic acid; then, 1 mg DSPE-PEG 2000 -NH2 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-PEG 2000-NH2. Finally, the purified material was freeze-dried by freeze drying, and the freeze-dried phospholipid hyaluronic acid was placed in a -80°C refrigerator for later use, and was recorded as HA.

[0051] S5: Preparation of biomimetic PLGA composite materials, the specific operation process is as follows:

[0052] P@Cur and P@Evol were mixed at a molar ratio of 20:1. 1 mL of 3 mg / mL mixed P@Cur and P@Evol was mixed with 0.187 mL of biomimetic membrane dispersion, and extruded repeatedly for at least 10 times through a micro extruder with a pore size of 200 nm to obtain a biomimetic membrane-coated composite material loaded with curcumin and eloumab, recorded as: M@P@(Evol+Cur); phospholipidated hyaluronic acid was redissolved in PBS solution, 750 μg of phospholipidated hyaluronic acid solution was added to M@P@(Evol+Cur), and stirred in a water bath at 37°C and 800 rpm for 60 min to obtain a biomimetic PLGA composite material co-loaded with curcumin and eloumab, namely HA-M@P@(Evol+Cur)NPs.

[0053] Figure 1 Coomassie blue staining and protein immunoblotting of HA-M@P@(Evol+Cur)NPs prepared in the present invention. Figure 1 In the figure, A is Coomassie Brilliant Blue staining, and B is protein immunoblotting. Figure 1 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.

[0054] Figure 2 Transmission electron microscopy images of HA-M@P@(Evol+Cur)NPs and empty carrier PLGANPs prepared in the present invention. Figure 2 In the figure, A is PLGANPs and B is HA-M@P@(Evol+Cur)NPs. Figure 2 As shown, the HA-M@P@(Evol+Cur)NPs and empty carrier PLGANPs prepared in Example 1 were subjected to transmission electron microscopy image analysis, and the results showed that uniformly dispersed spherical PLGA nanoparticles were successfully prepared with a particle size of about 100nm to 200nm; the HA-modified biomimetic membrane was wrapped in the outer layer of PLGA co-loaded with curcumin and eloumab, showing an obvious "core-shell" structure with a particle size of about 200nm.

[0055] Figure 3 This is a fusion experiment of macrophage membrane encapsulating PLGA of the present invention, indicating that the macrophage membrane successfully encapsulates PLGA nanoparticles.

[0056] Example 2

[0057] Application of a biomimetic PLGA composite material in the prevention and treatment of homocysteine-induced atherosclerosis

[0058] The HA-M@P@(Evol+Cur)NPs prepared in Example 1 were used for subsequent experiments.

[0059] (1) Methods for determination of DiI-oxLDL uptake assay and foam cell formation assay.

[0060] The DiI-oxLDL uptake experimental procedure was as follows: 1.5 × 10 5 RAW264.7 cells were inoculated into 24-well plates, and 500 μL 100 μM Hcy was added and incubated for 24 hours; different components of drugs were added for treatment for 24 hours, and then 40 μg / mL DiI-oxLDL was added for treatment for 12 hours; the cells were removed and washed with PBS three times, fixed with 4% paraformaldehyde for 10 minutes, and stained with DAPI for 30 minutes, and photographed under a confocal microscope.

[0061] The different component drugs are as follows: Control group: no treatment; Model group: Hcy was added; Cur+Evol group: 3μL 10μM Cur+2.7μL 1.25nM Evol was added after Hcy was added; HA-M@P@(Evol+Cur) group: 18.8μL 60μg / mL HA-M@P@(Evol+Cur) NPs was added after Hcy was added.

[0062] The steps of the foam cell formation experiment are as follows: 1.5×10 5 RAW264.7 cells were inoculated into 24-well plates, and 100 μM Hcy was added and incubated for 24 hours; different components of drugs diluted in serum-free medium were used for 2 hours; 80 μg / mL ox-LDL was added for 48 hours; the medium was removed, PBS was washed 3 times, and 3% ORO staining solution was added for 30 minutes; the microscope was used for photography. Different components of drugs were the same as above.

[0063] The DiI-oxLDL uptake experiment and foam cell formation experiment were used to determine the ability of HA-M@P@(Evol+Cur)NPs prepared in Example 1 to inhibit foam cell formation. Figure 4 The HA-M@P@(Evol+Cur)NPs of the present invention inhibit the uptake of DiI-oxLDL and inhibit the formation of foam cells. Figure 4 In the figure, A is the uptake of DiI-oxLDL, and B is the inhibition of foam cells. Figure 4 It was shown that HA-M@P@(Evol+Cur)NPs biomimetic nanoformulation could effectively inhibit the formation of foam cells.

[0064] (2) Experimental determination method of HA-M@P@(Evol+Cur)NPs regulating macrophage polarization to reduce inflammation.

[0065] The experimental steps of HA-M@P@(Evol+Cur) regulating macrophage polarization are as follows:

[0066] RAW264.7 cells were seeded in 24-well plates containing cell slides at a density of 1 × 10 cells per well. 4 cells. After incubation with 100 μM Hcy overnight, M1 / M2 macrophages were polarized in vitro. After polarization, the cells were fixed with 4% paraformaldehyde and then incubated with 0.3% TritonX-100 for 30 minutes. After washing with PBS, the cells were blocked with a blocking solution containing 10% NGS and 0.3% TritonX-100 at room temperature for 2 hours. Then, the cells were incubated with the primary antibodies iNOS and Arg-1, respectively, at 4°C overnight. Subsequently, the fluorescent secondary antibody was incubated 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 confocal laser scanning microscope. The experimental results are shown in Figure 5 It was shown that HA-M@P@(Evol+Cur)NPs could regulate the M1-M2 transition of macrophages.

[0067] The experimental steps of HA-M@P@(Evol+Cur)NPs inhibiting macrophage inflammatory response are as follows:

[0068] RAW264.7 cells were inoculated into 24-well plates and cultured for 24 hours, and then different components of drugs were added and incubated for 48 hours; the culture medium was collected and the expression of inflammatory factors was detected by ELISA kit. Figure 5 It was shown that HA-M@P@(Evol+Cur)NPs could inhibit the expression of inflammatory factors.

[0069] (3) The blood half-life and in vivo targeting of HA-M@P@(Evol+Cur)NPs biomimetic nanoformulation were determined by semi-quantitative means of detecting fluorescence intensity.

[0070] Experiment 1: PLGA and HA-M@P@(Evol+Cur)NPs were labeled with dihydrochlorin e6, i.e. Ce6. 200 μL of 5 mg / kg P@Ce6 and HA-M@P@Ce6 were injected into the tail vein of C57BL / 6 mice, and blood samples were collected at different time points, 0h, 1h, 2h, 4h, 4h, 8h, 12h and 24h for fluorescence intensity measurement.

[0071] Among them, P@Ce6 was prepared by the following method: 10 mg PLGA was dissolved in 1 mL dichloromethane solution to obtain PLGA solution, 100 μL 20 mg / mL Ce6 was added dropwise to the PLGA solution, and after sufficient mixing, 60W water bath ultrasound was applied for 5 min. The above mixture was added dropwise to 10 mL PVA with a mass fraction of 1%, and ultrasound was applied at 10% power for 10 min. Stir at 100 rpm for 12 h to evaporate dichloromethane. Centrifuge at 10000 rpm for 10 min, and the precipitate was re-dissolved with PBS to obtain a PLGA dispersion loaded with Ce6, which was recorded as P@Ce6.

[0072] HA-M@P@Ce6 was prepared by the following method: the PLGA dispersion loaded with Ce6 was mixed with 1 mL, 1 mg / mL biomimetic membrane M after water bath ultrasonic treatment in a mass ratio of 1:2, and repeatedly extruded at least 10 times through a micro-extruder with a pore size of 200 nm to obtain the M@P@Ce6 biomimetic nanopreparation; the phospholipidated hyaluronic acid was redissolved in PBS solution, 250 ug of the phospholipidated hyaluronic acid solution was added to the M@P@Ce6 solution, and stirred in a water bath at 37°C and 800 rpm for 60 minutes to obtain HA-M@P@Ce6.

[0073] Figure 6 is the blood half-life of HA-M@P@Ce6 in C57 mice. Figure 6 As shown in the figure, the blood circulation half-lives of P@Ce6 and HA-M@P@Ce6 are 1.313h and 2.457h, respectively, indicating that the biomimetic membrane-camouflaged nanoformulation is beneficial to prolong the blood circulation time.

[0074] Experiment 2: ApoE - / - After mice were fed a high-fat and high-protein diet for 3 months, 200 μL of P@Ce6 and HA-M@P@Ce6 at a concentration of 5 mg / kg were injected into the tail vein, respectively. As in experiment 1, the mouse aorta was taken out for fluorescence imaging 12 hours later.

[0075] Figure 7 The results show that the HA-M@P@Ce6 of the present invention is targeted to the atherosclerotic plaques in mice. Figure 7 As shown, compared with the Ce6 group, the HA-M@P@Ce6 group significantly aggregated at the aortic plaque, indicating that the bionic nanoformulation prepared in Example 1 can effectively target the atherosclerotic plaque site.

[0076] (4) Use HA-M@P@(Evol+Cur)NPs bionic nanoformulation for the treatment of Hcy-induced atherosclerosis.

[0077] ApoE - / -After mice were fed a high-fat, high-protein HMD+HFD diet for 3 months, the treatment groups were divided into Model group, Cur group, Evol group, Cur+Evol group and HA-M@P@(Evol+Cur) group. The specific groups are shown in Table 2. The drugs were administered twice a week for 3 consecutive months, during which a high-fat, high-protein diet was maintained. All drugs were administered via the tail vein. Three days after the end of the treatment, the aorta of the mice was stripped for sectioning and Oil Red O staining to examine the therapeutic effects of the different treatment groups.

[0078] Table 2 Treatment groups

[0079] Treatment Group Injectable drugs and dosage Model Group Equal volume of PBS Cur Group 20mg / kgCur Evol Group 5mg / kgEvol Cur+Evol group 10mg / kgCur+2.5mg / kgEvol HA-M@P@(Evol+Cur) group 10mg / kgCur+2.5mg / kgEvol

[0080] Figure 8 The following are the gross staining pictures of blood vessels of different treatment groups of the present invention for the treatment of Hcy-induced atherosclerosis. Figure 8 As shown in the figure, in the HA-M@P@(Evol+Cur) treatment group, gross examination by surface Oil Red O staining showed that the arteries including the aortic arch, thoracic artery, abdominal artery and common iliac artery contained extensive atherosclerotic plaques throughout the arterial trunk after 12 weeks of HMD+HFD feeding. Compared with the Model group, lipid staining of the entire aorta was significantly increased, as shown in the figure. Figure 8 As shown, the ORO-stained aortic lipids observed in mice treated with HA-M@P@(Evol+Cur) were significantly reduced. Consistent with these results, ORO-stained sections of the aortic root in each group also showed that HA-M@P@(Evol+Cur) treatment had a stronger anti-atherosclerotic ability and significantly reduced homocysteine ​​levels. This indicates that the biomimetic nanoparticles constructed by the present invention can effectively treat atherosclerosis induced by high homocysteine.

[0081] Figure 8 In the figure, A represents ApoE after treatment with different drugs - / - Representative images of ORO staining of mouse aortic arch; B is ApoE - / - ORO staining of frozen sections of mouse aortic root; C is the level of Hcy in serum after treatment with different drugs.

[0082] (5) Continue to use HA-M@P@(Evol+Cur)NPs biomimetic nanoformulation for in vivo biosafety testing. - / - After the treatment of mice, the main organs of the mice in the control group with normal diet, the Model group with high methionine diet, the 20 mg / kg Cur group, the 5 mg / kg Evol group, the 10 mg / kg Cur + 2.5 mg / kg Evol group, and the HA-M@P@(Evol + Cur)NPs group, including the heart, liver, spleen, lung, and kidney, were collected for H&E staining to investigate the biosafety of different treatment groups.

[0083] 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 bionic nanoparticles constructed by the present invention have good biosafety.

[0084] 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.

[0085] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A biomimetic PLGA composite nanomaterial, characterized in that: The bionic PLGA composite nanomaterial is a polylactic acid-glycolic acid copolymer that co-loads and encapsulates curcumin and eloumab, and then a bionic membrane is camouflaged on the outer layer of the polylactic acid-glycolic acid copolymer, and then the surface of the bionic membrane is modified with phospholipidated hyaluronic acid.

2. The biomimetic PLGA composite nanomaterial according to claim 1, characterized in that: The particle size of the bionic PLGA composite nano material is 100nm-300nm.

3. The method for preparing the biomimetic PLGA composite nanomaterial according to claim 1, characterized in that: The following steps are involved: encapsulating curcumin in poly(lactic acid-glycolic acid) copolymer to prepare a poly(lactic acid-glycolic acid) copolymer dispersion loaded with curcumin; Encapsulating evolocumab in poly(lactic acid-co-glycolic acid) to prepare a poly(lactic acid-co-glycolic acid) dispersion loaded with evolocumab; preparing a biomimetic membrane dispersion and phospholipidated hyaluronic acid; The polylactic acid-co-glycolic acid dispersion loaded with curcumin, the polylactic acid-co-glycolic acid dispersion loaded with eloumab and the biomimetic membrane dispersion are mixed and extruded to prepare a biomimetic membrane-coated composite material loaded with curcumin and eloumab; The phospholipidated hyaluronic acid is mixed with the composite material of curcumin and eloumab coated by a biomimetic membrane, so that the phospholipidated hyaluronic acid is inserted into the surface of the composite material of curcumin and eloumab coated by a biomimetic membrane to obtain the biomimetic PLGA composite nanomaterial.

4. The preparation method according to claim 3, characterized in that: The method of encapsulating the curcumin in the polylactic acid-co-glycolic acid copolymer comprises any one of a nanoprecipitation method, an emulsification solvent volatilization method, a liquid / solvent evaporation method, a liquid-driven co-flow focusing method and an emulsification / solvent diffusion method.

5. The preparation method according to claim 3, characterized in that: The method of encapsulating the evolocumab in the polylactic acid-glycolic acid copolymer includes any one of a spray drying method, an emulsification solvent volatilization method, a liquid / solvent evaporation method and a nanoprecipitation method.

6. The preparation method according to claim 3, characterized in that: When preparing the polylactic acid-glycolic acid copolymer dispersion loaded with curcumin, the mass ratio of curcumin to the polylactic acid-glycolic acid copolymer is 1:10-20.

7. The preparation method according to claim 3, characterized in that: When preparing the polylactic acid-glycolic acid copolymer dispersion loaded with eloumab, the mass ratio of eloumab to the polylactic acid-glycolic acid copolymer is 0.025-0.5:

1.

8. The preparation method according to claim 3, characterized in that: When preparing the biomimetic membrane-coated composite material loaded with curcumin and eloumab, the molar ratio of the polylactic acid-glycolic acid copolymer dispersion loaded with curcumin to the polylactic acid-glycolic acid copolymer dispersion loaded with eloumab is 20-40:

1.

9. The use of the biomimetic PLGA composite nanomaterial as claimed in claim 1, characterized in that: The bionic PLGA composite nano material is used for preparing a preparation for treating atherosclerosis.

10. The use according to claim 9, characterized in that The atherosclerosis includes at least one of aortic atherosclerosis, coronary atherosclerosis, cerebral atherosclerosis, renal artery atherosclerosis and limb artery atherosclerosis.

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