Bionic plga composite nanomaterial and preparation method and application thereof

Abstract

The application belongs to the technical field of biomedicine, and particularly relates to a kind of bionic PLGA composite nanomaterial and its preparation method and application, the bionic PLGA composite nanomaterial is loaded and encapsulated by polylactic acid-glycolic acid copolymer to curcumin and elotuzumab, then the outer layer of polylactic acid-glycolic acid copolymer is disguised and bionic film, and the surface of the bionic film is modified by phospholipidized hyaluronic acid.The bionic PLGA composite nanomaterial of the application utilizes the design characteristics of bionic film and hyaluronic acid modification, combines the anti-atherosclerosis effects of curcumin and elotuzumab with two different mechanisms, realizes the specific aggregation, penetration of drugs in the lesion site and improves the bioavailability of target cells to drugs, and achieves the purpose of synergistic treatment of atherosclerosis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical technology, and particularly relates to a kind of bionic PLGA composite nano material and its preparation method and application. BACKGROUND

[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 aggregation of inflammatory cells in atherosclerotic plaques. The early lesions of atherosclerosis originate from the accumulation of cholesterol under the endothelium, and as atherosclerosis develops, the expression of adhesion molecules on the endothelium increases, recruiting monocytes to the lesion plaque, then the monocytes differentiate into macrophages and ingest excess LDL-C to form foam cells, and through a positive feedback mechanism, more immune cells are recruited to the plaque inflammatory site, thereby promoting the further development of atherosclerotic plaques. The 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 drug therapy has the disadvantages of low drug targeting, poor water solubility and rapid clearance, etc., which limits the therapeutic effect. Therefore, developing a new drug dosage form is a difficult problem to be solved at present.

[0003] Evolocumab is a proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitor, also known as 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. Evolocumab can up-regulate the level of cell surface LDLR by blocking the PCSK9 protein-mediated LDLR degradation process, ultimately achieving the effect of lowering plasma LDL. However, current LDL-lowering therapies do not fully address residual cardiovascular risk caused by underlying inflammation, so the combination of anti-inflammatory drugs is an effective way to solve this problem. Curcumin is a natural polyphenol derived from the rhizomes of Curcuma longa, which has attracted widespread attention due to its anti-inflammatory, immunosuppressive, antioxidant, antiviral, antitumor activity and lipid-lowering function. Previous studies have shown that curcumin may play a role in anti-atherosclerosis by regulating macrophage polarization and inhibiting the transmission of inflammatory signals, but the poor water solubility and low bioavailability of this natural drug greatly limit its clinical use. Therefore, it is considered to adjust the administration method and use the two drugs together for the treatment of atherosclerosis. However, how to deliver the two drugs to the lesion area at the same time and improve the accumulation of drugs in the inflammatory site is still a major challenge. SUMMARY

[0004] The application aims to provide a bionic PLGA composite nanomaterial, which realizes the delivery of two drugs, i.e., elotuzumab and curcumin, to a lesion area and improves the accumulation of the drugs at an inflammation site.

[0005] The technical scheme adopted by the application is as follows:

[0006] The application provides a bionic PLGA composite nanomaterial, which is obtained by co-loading and encapsulating curcumin and elotuzumab in polylactic acid-glycolic acid copolymer, coating a bionic film on the outer layer of the polylactic acid-glycolic acid copolymer, and modifying the surface of the bionic film with phospholipidized hyaluronic acid.

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

[0008] The application provides a preparation method of the bionic PLGA composite nanomaterial, which comprises the following steps:

[0009] The curcumin is encapsulated in the polylactic acid-glycolic acid copolymer to prepare a polylactic acid-glycolic acid copolymer dispersion liquid loaded with curcumin; the elotuzumab is encapsulated in the polylactic acid-glycolic acid copolymer to prepare a polylactic acid-glycolic acid copolymer dispersion liquid loaded with elotuzumab; a bionic film dispersion liquid and phospholipidized hyaluronic acid are prepared; the polylactic acid-glycolic acid copolymer dispersion liquid loaded with curcumin, the polylactic acid-glycolic acid copolymer dispersion liquid loaded with elotuzumab, and the bionic film dispersion liquid are mixed and extruded to prepare a bionic film-coated composite material loaded with curcumin and elotuzumab; the phospholipidized hyaluronic acid is uniformly mixed with the bionic film-coated composite material loaded with curcumin and elotuzumab, so that the phospholipidized hyaluronic acid is inserted into the surface of the bionic film-coated composite material loaded with curcumin and elotuzumab, to obtain the bionic PLGA composite nanomaterial.

[0010] Preferably, the method for encapsulating the curcumin in the polylactic acid-glycolic acid copolymer comprises any one of a nanoprecipitation method, an emulsion solvent evaporation method, a liquid / solvent evaporation method, a liquid-driven co-flow focusing method, and an emulsion / solvent diffusion method.

[0011] Preferably, the method for encapsulating the elotuzumab in the polylactic acid-glycolic acid copolymer comprises any one of a spray drying method, an emulsion solvent evaporation method, a liquid / solvent evaporation method, and a nanoprecipitation method.

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

[0013] Preferably, when the curcumin-loaded polylactic acid-glycolic acid copolymer dispersion liquid is prepared, the mass ratio of curcumin and polylactic acid-glycolic acid copolymer is 0.025-0.5:1.

[0014] Preferably, when the biomimetic membrane-coated curcumin and elotuzumab-loaded composite material is prepared, the molar ratio of the curcumin-loaded polylactic acid-glycolic acid copolymer dispersion liquid and the elotuzumab-loaded polylactic acid-glycolic acid copolymer dispersion liquid is 20-40:1.

[0015] The application provides an application of the biomimetic PLGA composite nanomaterial.

[0016] Preferably, the atherosclerosis comprises at least one of aortic atherosclerosis, coronary atherosclerosis, cerebral atherosclerosis, renal atherosclerosis and peripheral atherosclerosis.

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

[0018] The application provides a biomimetic PLGA composite nanomaterial, which is obtained by co-loading and encapsulating curcumin and elotuzumab by polylactic acid-glycolic acid copolymer, coating a biomimetic membrane on the outer layer of the polylactic acid-glycolic acid copolymer, and modifying the surface of the biomimetic membrane by phospholipidized hyaluronic acid.

[0019] In addition to loading drugs for treating and / or preventing atherosclerosis, the biomimetic nanocomposite material co-loading the drugs can be used as a platform for treating other diseases, such as tumors and rheumatoid arthritis, by replacing different biomimetic membrane types and drugs, utilizing the immune escape ability of the biomimetic membrane and the action mechanism of different drugs.

[0020] The application can solve the problems of abnormal liver function, muscle influence, abnormal blood sugar, gastrointestinal reactions and other possible side effects caused by long-term use of statins, improve the half-life of the drug in the blood and the plaque targeting effect, provide a new choice for related clinical treatment, and has important scientific significance, practical value and economic value.

[0021] The bionic PLGA composite nanomaterials realize the targeted treatment of atherosclerosis by utilizing the anti-atherosclerosis effects of curcumin and Evol in two different mechanisms, and the synergistic effect and reduced toxicity of the halved dose of the nanomaterials combined with a single drug. The outermost bionic membrane realizes the long blood circulation and the targeting ability of the nanocomposite at the lesion site by the inherent immune escape and recruitment characteristics of the source cells of the membrane. The effective use of the nanocomposite by the target cells is realized by utilizing the characteristics of the interaction between hyaluronic acid and the target cell surface receptor CD44, and the effect of preventing and treating atherosclerosis is improved. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The figure is the Coomassie brilliant blue staining and Western blotting of HA-M@P(Evol+Cur)NPs prepared in the application; A is Coomassie brilliant blue staining, and B is Western blotting.

[0023] Figure 2 The figure is the transmission electron microscopy of HA-M@P(Evol+Cur)NPs and empty carrier PLGANPs prepared in the application, A is PLGANPs, and B is HA-M@P(Evol+Cur)NPs.

[0024] Figure 3 The figure is the fusion experiment of macrophage membrane wrapped PLGA in the application, A-C are, in sequence, PLGA and M@P(Evol+Cur).

[0025] Figure 4 The figure is the inhibition of DiI-oxLDL uptake and the inhibition of foam cell formation by HA-M@P(Evol+Cur)NPs prepared in the application, A is the DiI-oxLDL uptake experiment result; B is the foam cell formation inhibition experiment result, from left to right, in sequence, Control group, Model group, Cur+Evol group and HA-M@P(Evol+Cur) group.

[0026] Figure 5 The figure is the ability of HA-M@P(Evol+Cur)NPs prepared in the application to regulate macrophage polarization and reduce inflammation, A-L are the treatment of different components of drugs after iNOS incubation; a-l are the treatment of different components of 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 figure is the effect of HA-M@P(Evol+Cur)NPs prepared in the application on ApoE - / -Prolonged blood half-life in mice, A is the detection result of different groups at different time points; B is the statistical result corresponding to A.

[0028] Figure 7 Targeting of HA-M@P@(Evol+Cur) NPs prepared in the application to atherosclerotic plaque sites in mice, A is the result of 3 parallel experiments after using P@Ce6, B is the result of 3 parallel experiments after using HA-M@P@Ce6.

[0029] Figure 8 Pictures of gross staining of blood vessels treated with different treatment groups of the application for atherosclerosis. A is ApoE - / - Representative pictures of gross ORO staining of mouse aortic arch, from left to right: no injection of any drug for 3 parallel experiments, injection of PBS for 3 parallel experiments, injection of 20 mg / kg Cur for 3 parallel experiments, injection of 5 mg / kg Evol for 3 parallel experiments, injection of 10 mg / kg Cur+2.5 mg / kg Evol for 3 parallel experiments and injection of equal dose of HA-M@P@(Evol+Cur) for 3 parallel experiments; B is ApoE - / - Freeze section ORO staining of mouse aortic root; C is the Hcy level in serum after treatment with different drugs.

[0030] Figure 9 H&E staining of heart, liver, spleen, lung and kidney of mice treated with different treatment groups of the application for advanced atherosclerosis. DETAILED DESCRIPTION

[0031] The application will be further described below through specific examples, but the scope of the application is not limited. The details and forms of the technical solutions of the application can be modified or replaced without deviating from the spirit and scope of the application, and these modifications or replacements all fall within the protection scope of the application.

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

[0033] The existing methods for treating atherosclerosis mainly include drug therapy, vascular intervention therapy and surgical treatment, and oral drug therapy is mainly used. However, clinical oral drug therapy has the disadvantages of low drug targeting, poor water solubility and rapid clearance, etc., which limits the therapeutic effect. Therefore, developing a new drug dosage form is a difficult problem to be solved at present.

[0034] Evolocumab as a drug for treating atherosclerosis by blocking PCSK9 protein-mediated LDLR degradation process, up-regulating the level of cell surface LDLR and ultimately reducing plasma LDL. However, the current LDL-lowering therapy cannot fully solve the residual cardiovascular risk caused by underlying inflammation, so anti-inflammatory drugs are usually used 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 greatly limit their clinical use. Therefore, the combination of the two drugs for the treatment of atherosclerosis by adjusting the administration method is considered. However, how to deliver the two drugs to the lesion area at the same time and improve the accumulation of drugs in the inflammatory site is still a major challenge.

[0035] Based on this, the present application provides a kind of bionic PLGA composite nano material, the bionic PLGA composite nano material is loaded by polylactic acid-glycolic acid copolymer to curcumin and evolocumab and is encapsulated, then in the outer layer camouflage bionic film of polylactic acid-glycolic acid copolymer, again in the surface of the bionic film is phospholipidized hyaluronic acid modification.The bionic PLGA composite nano material of the application utilizes the design features of bionic film and hyaluronic acid modification, combines the anti-atherosclerotic effects of curcumin and evolocumab with two different mechanisms, realizes the specific accumulation, penetration of drugs in the lesion site and improves the bioavailability of target cells to drugs, and achieves the purpose of synergistically treating atherosclerosis.

[0036] In order to enable those skilled in the art to better understand the technical solutions of the present application and to implement them, the present application will be further described below in conjunction with specific embodiments. In the description of the present application, if not specifically stated, the reagents used are commercially available, and the methods used are conventional techniques in the art.

[0037] Abbreviations used in the present application are shown in Table 1.

[0038] Table 1 Abbreviation table

[0039]

[0040]

[0041] Example 1

[0042] A preparation method of a bionic PLGA composite material includes the following steps:

[0043] S1, a PLGA dispersion liquid loaded with curcumin is prepared by a nano-precipitation method, and the specific operation process is as follows:

[0044] PLGA-DMSO solution was prepared by dissolving 20 mg of PLGA in 1 mL of DMSO. 50 μL of curcumin solution prepared with DMSO at a concentration of 20 mg / mL was added to 500 μL of the PLGA-DMSO solution, which was then mixed thoroughly and subjected to ultrasonic treatment at 50 W in a water bath for 5 min. The mixture was added dropwise to 5 mL of a 2% PVA aqueous solution under ultrasonic treatment in a water bath, and then mixed thoroughly and placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa for dialysis against deionized water for 24 h to obtain a curcumin-loaded PLGA dispersion, which was denoted as P@Cur.

[0045] S2, a PLGA dispersion loaded with elotuzumab was prepared by an emulsification solvent evaporation method, and the specific operation process was as follows:

[0046] PLGA-dichloromethane solution was prepared by dissolving 10 mg of PLGA in 1 mL of dichloromethane. 12.5 μL of elotuzumab at a concentration of 10 mg / mL was added dropwise to 500 μL of the PLGA-dichloromethane solution under ultrasonic treatment in a water bath, and then mixed thoroughly under ultrasonic treatment at a power of 10% for 1 min. The mixture was added dropwise to 5 mL of a 2% PVA aqueous solution under stirring, and then mixed thoroughly under ultrasonic treatment at a power of 40% for 1 min; stirring was performed at 300 rpm for 12 h to volatilize the dichloromethane, and then centrifugation was performed at 12000 rpm for 30 min to obtain a PLGA dispersion loaded with elotuzumab, which was denoted as P@Evol.

[0047] S3, a biomimetic membrane dispersion was prepared, and the specific operation process was as follows:

[0048] According to the instructions of the membrane protein extraction kit, macrophage membranes were prepared, and the macrophages were washed twice with PBS pre-cooled at 4°C, resuspended in membrane protein extraction agent A containing 1 mM PMSF, and lysed on ice for 30 min. Ultrasonic treatment was performed at 4°C for 10 min at a power of 80 W in a water bath; repeated freezing and thawing was performed 5 times at -80°C and 37°C, each for 30 min; and centrifugation was performed at 12000 rpm at 4°C for 30 min to obtain a macrophage membrane precipitate, which was denoted as or M.

[0049] S4, phosphatidylated hyaluronic acid was prepared, and the specific operation process was as follows:

[0050] 8 mg of EDC, 16 mg of NHS and 1 mg of hyaluronic acid were dissolved in PBS, and stirring was performed at a speed of 800 rpm at room temperature for 30 min to activate the carboxyl groups on the surface of the hyaluronic acid; then, 1 mg of DSPE-PEG 2000 -NH2 was added to the above solution, and stirring was performed at a speed of 800 rpm at room temperature for 24 h; the above mixture solution was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa for dialysis against deionized water for 24 h to remove free EDC, NHS and DSPE-PEG 2000-NH2. Finally, the purified material was freeze-dried by freeze-drying method, and the freeze-dried phosphatidylated hyaluronic acid was placed in a refrigerator at -80℃ for standby, denoted as HA.

[0051] S5: preparation of biomimetic PLGA composite material, 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 and 0.187 mL of biomimetic membrane dispersion were mixed, and the mixture was repeatedly extruded through a micro-extruder with a pore size of 200 nm for at least 10 times to obtain a biomimetic membrane-coated composite material loaded with curcumin and elotuzumab, denoted as: M@P@(Evol+Cur); the phosphatidylated hyaluronic acid was redissolved in PBS solution, and 750 μg of phosphatidylated hyaluronic acid solution was added to M@P@(Evol+Cur), and stirred at 37℃, 800 rpm water bath for 60 min to obtain a biomimetic PLGA composite material co-loaded with curcumin and elotuzumab, namely HA-M@P@(Evol+Cur) NPs.

[0053] Figure 1 The coomassie brilliant blue staining and western blotting diagram of HA-M@P@(Evol+Cur) NPs prepared in the application are shown in the following figure, Figure 1 wherein A is coomassie brilliant blue staining, and B is western blotting. As shown in the following figure, Figure 1 the biomimetic membrane prepared in Example 1 was analyzed for membrane characteristic protein, and the results showed that the biomimetic membrane was successfully disguised on the surface of the nano-composite and retained the characteristic proteins on the surface of the cell membrane.

[0054] Figure 2 The transmission electron microscopy diagram of HA-M@P@(Evol+Cur) NPs and empty carrier PLGANPs prepared in the application are shown in the following figure, Figure 2 wherein A is PLGANPs, and B is HA-M@P@(Evol+Cur) NPs. As shown in the following figure, Figure 2 the transmission electron microscopy diagram of HA-M@P@(Evol+Cur) NPs and empty carrier PLGANPs prepared in Example 1 was analyzed, and the results showed that uniformly dispersed spherical PLGA nanoparticles were successfully prepared, with a particle size of about 100 nm to 200 nm; the HA-modified biomimetic membrane was wrapped in the outer layer of the co-loaded curcumin and elotuzumab PLGA, showing a clear "core-shell" structure, with a particle size of about 200 nm.

[0055] Figure 3 The fusion experiment of macrophage membrane-wrapped PLGA in the application showed that the macrophage membrane successfully wrapped the PLGA nanoparticles.

[0056] Example 2

[0057] Use of a biomimetic PLGA composite 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) Method for determining the DiI-oxLDL uptake experiment and the foam cell formation experiment.

[0060] The steps of the DiI-oxLDL uptake experiment are as follows: 1.5 x 10 5 RAW264.7 cells were seeded in a 24-well plate, and 500 μL of 100 μM Hcy was added for co-incubation for 24 h; different component drugs were added for 24 h, and then 40 μg / mL DiI-oxLDL was added for 12 h; the cells were taken out, washed with PBS for 3 times, fixed with 4% paraformaldehyde for 10 min, and stained with DAPI for 30 min, and then imaged under a confocal microscope.

[0061] The different component drugs were as follows: the Control group was not treated; the Model group was added with Hcy; the Cur+Evol group was added with 3 μL of 10 μM Cur and 2.7 μL of 1.25 nM Evol after the addition of Hcy; and the HA-M@P(Evol+Cur) group was added with 18.8 μL of 60 μg / mL HA-M@P(Evol+Cur) NPs after the addition of Hcy.

[0062] The steps of the foam cell formation experiment are as follows: 1.5 x 10 5 RAW264.7 cells were seeded in a 24-well plate, and 100 μM Hcy was added for co-incubation for 24 h; different component drugs diluted with serum-free medium were added for 2 h; 80 μg / mL ox-LDL was added for 48 h; the medium was removed, and the cells were washed with PBS for 3 times, and then 3% ORO staining solution was added for staining for 30 min; and then imaged under a microscope. The different component drugs were the same as above.

[0063] The DiI-oxLDL uptake experiment and the foam cell formation experiment were used to determine the foam cell formation inhibition ability of the HA-M@P(Evol+Cur) NPs prepared in Example 1. Figure 4 To determine the DiI-oxLDL uptake inhibition and foam cell formation inhibition of the HA-M@P(Evol+Cur) NPs of the present application, Figure 4 wherein A is the DiI-oxLDL uptake, and B is the foam cell inhibition. The experimental results are shown in Figure 4 It can be seen that the HA-M@P(Evol+Cur) NPs biomimetic nano-preparation can effectively inhibit the formation of foam cells.

[0064] (2) Method for determining the regulation of macrophage polarization by HA-M@P@(Evol+Cur) NPs to reduce inflammation.

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

[0066] RAW264.7 cells were seeded in a 24-well plate containing cell slides at a density of 1×10 4 After 100 μM Hcy incubation overnight, M1 / M2 macrophages were polarized in vitro. After polarization, the cells were fixed with 4% paraformaldehyde, followed by incubation with 0.3% Triton X-100 for 30 minutes. After washing with PBS, the cells were blocked with a blocking solution containing 10% NGS and 0.3% Triton X-100 for 2 hours at room temperature. Then, the cells were incubated with one iNOS and Arg-1 respectively at 4°C overnight. Subsequently, fluorescent secondary antibodies were 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 which show that HA-M@P@(Evol+Cur) NPs can regulate the M1-M2 transformation of macrophages.

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

[0068] RAW264.7 cells were seeded in a 24-well plate and cultured for 24 h, then different components of drugs were added for co-incubation for 48 h; the culture medium was collected, and the expression of inflammatory factors was detected by ELISA kit. The experimental results are shown in Figure 5 which show that HA-M@P@(Evol+Cur) NPs can inhibit the expression of inflammatory factors.

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

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

[0071] P@Ce6 is prepared by the following method: 10 mg of PLGA is dissolved in 1 mL of dichloromethane solution to obtain a PLGA solution, 100 μL of 20 mg / mL Ce6 is added dropwise to the PLGA solution, and after being mixed thoroughly, 60 W water bath ultrasonic is used for 5 min. The above mixture is added dropwise to 10 mL of 1% PVA, and 10% power ultrasonic is used for 10 min. 100 rpm stirring is used for 12 h, and dichloromethane is volatilized. 10000 rpm centrifugation is used for 10 min, the precipitate is redissolved with PBS to prepare a Ce6-loaded PLGA dispersion, which is denoted as P@Ce6.

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

[0073] Figure 6 The blood half-life of HA-M@P@Ce6 in C57 mice in vivo is determined. As shown in Table 1, the blood circulation half-lives of P@Ce6 and HA-M@P@Ce6 are 1.313 h and 2.457 h, respectively. It is shown that the biomimetic membrane disguised nano-preparation is beneficial to prolong the blood circulation time. Figure 6

[0074] Experiment two: ApoE - / - After the mice are fed with high-fat and high-cholesterol diet for 3 months, 200 μL of P@Ce6 and HA-M@P@Ce6 with a dosage concentration of 5 mg / kg are injected into the tail vein, and the same as in experiment one, the fluorescence imaging of the aorta of the mice is performed after 12 h.

[0075] Figure 7 The targeting property of HA-M@P@Ce6 in the atherosclerotic plaque site in mice in vivo is determined. As shown in Table 2, compared with the Ce6 group, the HA-M@P@Ce6 group is significantly aggregated in the aortic plaque, which shows that the biomimetic nano-preparation prepared in example 1 can be effectively targeted to the atherosclerotic plaque site. Figure 7

[0076] (4) The HA-M@P@(Evol+Cur) NPs biomimetic nano-preparation is used for Hcy-induced atherosclerosis treatment.

[0077] ApoE - / - ​​Mice were fed with HMD+HFD diet for 3 months, and then the mice were divided into Model group, Cur group, Evol group, Cur+Evol group and HA-M@P(Evol+Cur) group. The mice were administered twice a week for 3 months, and the high-fat and high-protein diet was maintained during the administration. The tail vein was administered. Three days after the end of the treatment, the aorta of the mice was stripped for sectioning and oil red O staining to investigate the treatment effect of different treatment groups.

[0078] Table 2 Treatment grouping

[0079] Treatment group Injection drug and dosage Model group Equal volume of PBS Cur group 20 mg / kg Cur Evol group 5 mg / kg Evol Cur + Evol group 10 mg / kg Cur + 2.5 mg / kg Evol HA-M@P(Evol + Cur) group 10 mg / kg Cur + 2.5 mg / kg Evol

[0080] Figure 8 The figure is the blood vessel gross staining picture of the treatment of Hcy-induced atherosclerosis by different treatment groups of the application. As shown in Figure 8 , in the treatment scheme, the HA-M@P(Evol+Cur) treatment group, through the gross examination of the surface oil red O staining, it was shown that the aorta, thoracic artery, abdominal artery and common iliac artery contained extensive atherosclerotic plaques in the whole aorta after 12 weeks of HMD+HFD feeding. Compared with the Model group, the lipid staining of the whole aorta was significantly increased, as shown in Figure 8 , the ORO staining of the aorta observed in the mice after HA-M@P(Evol+Cur) treatment was significantly reduced. Consistent with these results, the ORO staining sections of the aortic root of each group also showed that the HA-M@P(Evol+Cur) treatment had stronger anti-atherosclerotic ability and significantly reduced homocysteine levels. It is proved that the biomimetic nanoparticles constructed by the application can effectively treat homocysteine-induced atherosclerosis.

[0081] Figure 8 In the figure, A is the ApoE - / - The representative picture of the aortic arch of the mouse is shown in A. B is the frozen section ORO staining of the aortic root of the mouse. C is the Hcy level in the serum after treatment with different drugs. - / - The representative picture of the aortic arch of the mouse is shown in A. B is the frozen section ORO staining of the aortic root of the mouse. C is the Hcy level in the serum after treatment with different drugs.

[0082] (5) The biomimetic nanoparticle preparation HA-M@P(Evol+Cur) NPs was used for in vivo biological safety inspection. The above-mentioned treatment scheme was used, and ApoE - / - After the treatment of the ApoE - / - After the treatment of the ApoE

[0083] The results are as follows Figure 9 As shown, no obvious organic lesions were observed in the organs of mice in different treatment groups, indicating that the biomimetic nanoparticles constructed in this invention have good biosafety.

[0084] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above 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 embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A biomimetic PLGA composite nanomaterial for preparing atherosclerosis treatment preparation, characterized in that, The bionic PLGA composite nanomaterial is prepared by co-loading and encapsulating curcumin and Ibritumomab tiuxetan in polylactic acid-glycolic acid copolymer, coating the polylactic acid-glycolic acid copolymer with a bionic film, and modifying the surface of the bionic film with phospholipidized hyaluronic acid. The preparation method of the bionic PLGA composite nanomaterial comprises the following steps: Curcumin is encapsulated in polylactic acid-glycolic acid copolymer to prepare a curcumin-loaded polylactic acid-glycolic acid copolymer dispersion, and the mass ratio of curcumin to polylactic acid-glycolic acid copolymer is 1:10-20; Ibritumomab tiuxetan is encapsulated in polylactic acid-glycolic acid copolymer to prepare an Ibritumomab tiuxetan-loaded polylactic acid-glycolic acid copolymer dispersion, and the mass ratio of Ibritumomab tiuxetan to polylactic acid-glycolic acid copolymer is 0.025-0.5:1; A bionic film dispersion and phospholipidized hyaluronic acid are prepared; The curcumin-loaded polylactic acid-glycolic acid copolymer dispersion, the Ibritumomab tiuxetan-loaded polylactic acid-glycolic acid copolymer dispersion, and the bionic film dispersion are mixed and extruded to prepare a bionic film-coated composite material loaded with curcumin and Ibritumomab tiuxetan, and the molar ratio of the curcumin-loaded polylactic acid-glycolic acid copolymer dispersion to the Ibritumomab tiuxetan-loaded polylactic acid-glycolic acid copolymer dispersion is 20-40:1; The phospholipidized hyaluronic acid is mixed with the bionic film-coated composite material loaded with curcumin and Ibritumomab tiuxetan, so that the phospholipidized hyaluronic acid is inserted into the surface of the bionic film-coated composite material loaded with curcumin and Ibritumomab tiuxetan, to obtain the bionic PLGA composite nanomaterial.

2. The biomimetic PLGA composite nanomaterial of claim 1, wherein, The particle size of the bionic PLGA composite nanomaterial is 100-300 nm.

3. The biomimetic PLGA composite nanomaterial of claim 1, wherein, The method for encapsulating the curcumin in polylactic acid-glycolic acid copolymer comprises any one of nano-precipitation, emulsion solvent evaporation, liquid / solvent evaporation, liquid-driven co-flow focusing, and emulsion / solvent diffusion.

4. The biomimetic PLGA composite nanomaterial of claim 1, wherein, The method for encapsulating the Ibritumomab tiuxetan in polylactic acid-glycolic acid copolymer comprises any one of spray drying, emulsion solvent evaporation, liquid / solvent evaporation, and nano-precipitation.

5. The use of the biomimetic PLGA composite nanomaterial according to claim 1, wherein, The bionic PLGA composite nanomaterial is used for preparing a preparation for treating atherosclerosis.

6. The use of the biomimetic PLGA composite nanomaterial according to claim 5, characterized in that, The atherosclerosis comprises at least one of aortic atherosclerosis, coronary atherosclerosis, cerebral atherosclerosis, and renal atherosclerosis.

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