A nanomedicine for targeted diagnosis and treatment of atherosclerotic plaques

Abstract

The present invention provides a nano drug for targeted diagnosis and treatment of atherosclerotic plaques. Nanoparticles are constructed with EGCG, formaldehyde and L-arginine to retain the antioxidant properties of EGCG. On this basis, Mn 2+ Nanomedicines constructed with DKK1 antibodies have excellent relaxivity and can be used as MRI enhancers specific for atherosclerotic plaques. Cy5-labeled nanomedicines can be used as in vivo imaging enhancers specific for atherosclerotic plaques. The nanomedicines provided by this invention have excellent biosafety, can delay plaque progression, and improve plaque stability, making them a nanomedicine that precisely targets and treats atherosclerotic plaques.

Description

Technical Field

[0001] The present invention relates to a nanomedicine for targeted diagnosis and treatment of atherosclerotic plaques, and relates to the field of precision medicine. Background Art

[0002] Cardiovascular disease (CVD) is a major threat to human health. Atherosclerosis (AS) is the primary cause of CVD, accounting for over 50% of CVD deaths. Early AS typically does not cause serious complications. However, as AS progresses, plaques develop changes such as a necrotic lipid core, inflammatory cell infiltration, thinning or rupture of the fibrous cap, and intraplaque hemorrhage. Plaques become unstable and can rapidly progress to serious complications such as CVD. Therefore, identifying patients at high risk of CVD by detecting vulnerable plaques has become a top priority in cardiovascular research. Currently, the preferred treatment for AS is lifestyle intervention, followed by medication or surgery. Clinical medications, including lipid-lowering drugs, cholesterol absorption inhibitors, antiplatelet drugs, and angiotensin-converting enzyme inhibitors (ACEIs), can mitigate AS progression and prevent the development of atherosclerotic cardiovascular disease (ASCVD). However, these drugs do not specifically target AS lesions. While they can meet clinical therapeutic needs, they also carry residual risks and are associated with adverse reactions. Therefore, targeted therapy has become a new direction in the current research on AS treatment.

[0003] Nanotechnology involves the cutting-edge application of nanostructures in medicine and healthcare for the prevention, diagnosis, and treatment of diseases. The significant progress made by nanotechnology in the diagnosis and treatment of diseases is based on its important and unique properties, such as a large surface area to mass ratio, quantum properties, and the ability to adsorb and carry substances such as drugs, probes, nucleic acids, and proteins. In the cardiovascular field, the emergence of nanotechnology-based drug delivery has also brought new hope for improving the treatment of AS. AS plaque lesions are characterized by a small lesion site and a large blood flow in the blood vessels, which makes it difficult for conventional drugs to remain in the lesion site in large quantities, bringing difficulties to the clinical treatment of AS.

[0004] Patent publication number CN108498878A discloses a strategy for preparing polyphenol nanoparticles based on EGCG, formaldehyde, and L-arginine (L-Arg). These nanoparticles fully retain the antioxidant properties of EGCG and are stable and reliable. While the nanoparticles constructed from EGCG, formaldehyde, and L-arginine retain the antioxidant properties of EGCG, they lack specific targeting for AS and cannot be used for targeted diagnosis or imaging of AS plaques, exhibiting nonspecific distribution. Based on this background, the present invention aims to provide a nanoparticle for the targeted diagnosis and treatment of atherosclerotic plaques. Summary of the Invention

[0005] In order to overcome the deficiencies of the prior art, the present invention aims to provide a nanomedicine for the targeted diagnosis and treatment of atherosclerotic plaques.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides a nano drug for targeted diagnosis and treatment of atherosclerotic plaques, the components of which include epigallocatechin gallate, formaldehyde, L-arginine, Mn 2+ and DKK1 antibodies.

[0008] Furthermore, the components of the nanomedicine also include Cy5.

[0009] Furthermore, the particle size of the nanomedicine is 215±90.3 nm, the Zeta potential is -5.60±0.42 mV, the characteristic absorption peak is at 273 nm, and the longitudinal relaxation rate r1 is 8.07 Mm -1 S -1 .

[0010] Furthermore, the nanomedicine is spherical, uniform in size and has good dispersibility.

[0011] For the purposes of this invention, atherosclerosis refers to plaque-related arteriosclerosis, the most common type of arteriosclerosis. Atherosclerosis is characterized by thickening and decreased elasticity of the arterial wall. Atherosclerosis is a condition characterized by repeated damage to the arterial wall, leading to the deposition of lipids, forming plaques (also known as atheromas), which are covered by a fibrous cap on the intima of the arterial wall. Plaques can grow into the arterial lumen, gradually leading to arterial stenosis.

[0012] For the purposes of this invention, nanomedicine refers to nanoscale particles produced by using nanofabrication techniques to produce APIs, or nanoscale particles formed by combining APIs with appropriate carrier materials, and the resulting pharmaceutical preparations. The size of the active ingredient or carrier particles is the primary characteristic of a nanomedicine and an important foundation for the nanoscale effect exhibited by the drug. The final product or carrier material of a nanomedicine typically exhibits nanoscale external dimensions, internal structures, or surface structures (approximately 100 nm or less), or a particle size of 1000 nm or less, with a significant size effect.

[0013] In the context of this invention, Cy5 refers to a Cy dye. Cy, short for cyanine, is a compound composed of two nitrogen atoms linked by an odd number of methyl units. Cyanine compounds have the characteristics of long wavelength, tunable absorption and emission, high extinction coefficient, good water solubility, and relatively simple synthesis. Cy-based dyes are commonly used to label proteins, antibodies, and small molecules. For protein and antibody labeling, binding can be achieved through a simple mixing reaction.

[0014] In the context of this invention, Cy5 refers to a Cy dye. Cy, short for cyanine, is a compound composed of two nitrogen atoms linked by an odd number of methyl units. Cyanine compounds have the characteristics of long wavelength, tunable absorption and emission, high extinction coefficient, good water solubility, and relatively simple synthesis. Cy-based dyes are commonly used to label proteins, antibodies, and small molecules. For protein and antibody labeling, binding can be achieved through a simple mixing reaction.

[0015] The second aspect of the present invention provides a pharmaceutical composition for targeted diagnosis and treatment of atherosclerotic plaques, wherein the pharmaceutical composition comprises the nanomedicine described in the first aspect of the present invention.

[0016] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0017] As used herein, a pharmaceutical composition refers to a composition comprising at least one biologically active compound. The pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. The pharmaceutical compositions of the present invention may contain any conventional non-toxic pharmaceutically acceptable carrier, excipient, or vehicle. In some cases, a pharmaceutically acceptable acid, base, or buffer may be used to adjust the pH of the formulation to enhance the stability of the formulated compound or its dosage form. The term parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The pharmaceutical compositions of the present invention may be administered to a recipient by any route that reaches the target tissue.

[0018] In the present invention, pharmaceutically acceptable carrier refers to any pharmaceutical carrier that does not induce the production of antibodies harmful to the individual receiving the composition and can be used without excessive toxicity. Suitable carriers can be large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids and amino acid copolymers. Such carriers are well known to those of ordinary skill in the art. The pharmaceutically acceptable carrier in the pharmaceutical composition can include fluids, such as water, saline, glycerol and ethanol. Auxiliary substances, such as wetting agents or emulsifiers, pH buffer substances, etc., can also be present in such vehicles.

[0019] The pharmaceutical composition of the present invention can also be used in combination with other drugs for treating atherosclerotic plaques. These other compounds can be administered simultaneously with the main active ingredient (e.g., the nanodrug described in the first aspect of the present invention), or even administered simultaneously within the same composition. The other therapeutic compound can also be administered separately, in a separate composition or in a dosage form different from that of the main active ingredient.

[0020] The third aspect of the present invention provides a method for preparing the nano drug according to the first aspect of the present invention, the method comprising the steps of: preparing nanoparticles using epigallocatechin gallate, formaldehyde, and L-arginine as raw materials, adding Mn to the nanoparticles; 2+ Incubate overnight, add DKK1 antibody and incubate overnight.

[0021] Furthermore, the method further comprises the steps of: adding Cy5.

[0022] The step of preparing nanoparticles using epigallocatechin gallate, formaldehyde and L-arginine as raw materials comprises: dissolving epigallocatechin gallate and formaldehyde in triple-distilled water, stirring and mixing, adding L-arginine, stirring and reacting, purifying and resuspending to obtain a solution of nanoparticles.

[0023] Furthermore, the mass ratio of epigallocatechin gallate to L-arginine is 3.95:1.

[0024] Furthermore, the stirring and mixing condition is 30°C.

[0025] Furthermore, the stirring reaction conditions are 30° C. and 5 min.

[0026] The addition of Mn 2+ The overnight incubation step includes: adding Mn 2+ Incubate overnight and purify and resuspend to obtain the Mn 2+ Solutions of nanoparticles.

[0027] Furthermore, the Mn 2+ The final concentration was 20 μg / ml.

[0028] Furthermore, the Mn 2+ Selected from MnCl2·4H2O solution.

[0029] Furthermore, the addition of Mn 2+ Incubate overnight at room temperature.

[0030] The step of adding DKK1 antibody and incubating overnight comprises: 2+ DKK1 antibody is added to the solution of nanoparticles and incubated overnight. After purification and resuspending, the nanomedicine according to the first aspect of the present invention is obtained.

[0031] Furthermore, the final concentration of the DKK1 antibody was 10 μg / ml.

[0032] Furthermore, the DKK1 antibody was added and incubated overnight at 4°C.

[0033] Furthermore, the purification and resuspension refers to purification and resuspension with triple-distilled water after centrifugation.

[0034] Furthermore, the centrifugation conditions are room temperature, 3.5×g, 5 min.

[0035] L-arginine (L-Arg) is the substrate for the synthesis of NOS. Nitric oxide (NO) is closely related to the occurrence and development of AS and is produced in the body by nitric oxide synthase (NOS).

[0036] DKK1 (Dickkopf WNT signaling pathway inhibitor 1) has a gene ID of 22943. Its molecular weight is approximately 26 kDa, and it exerts its effects through both autocrine and paracrine pathways. DKK1 was first discovered in human disease research to regulate the development of colon and lung cancer. To date, antibodies targeting DKK1, such as BHQ880 and DKN-01, have been clinically tested in various tumor diseases.

[0037] The fourth aspect of the present invention provides a method for inhibiting DKK1 expression in vascular endothelial cells and / or inhibiting apoptosis of vascular endothelial cells, the method comprising treating vascular endothelial cells with the nanomedicine according to the first aspect of the present invention.

[0038] Furthermore, the vascular endothelial cells include HUVECs, HCMECs, HMECs, and Eahy926.

[0039] Furthermore, the vascular endothelial cells are HUVECs.

[0040] Furthermore, the cell apoptosis is oxidation-induced cell apoptosis.

[0041] Furthermore, the oxidation-induced cell apoptosis is Ox-LDL-induced cell apoptosis.

[0042] The fifth aspect of the present invention provides a method for inhibiting ROS production in macrophages, inhibiting macrophage polarization and / or inhibiting macrophage phagocytosis of Ox-LDL, the method comprising treating macrophages with the nanodrug according to the first aspect of the present invention.

[0043] Furthermore, the macrophages include RAW264.7 and THP-1.

[0044] Furthermore, the macrophages are RAW264.7.

[0045] Furthermore, the inhibiting of macrophage polarization refers to inhibiting macrophage M1 polarization.

[0046] Furthermore, the inhibiting of macrophage phagocytosis of Ox-LDL also includes inhibiting foam cell formation.

[0047] Furthermore, the ROS generation is induced by lipopolysaccharide and interferon γ.

[0048] Furthermore, the macrophage M1 polarization is macrophage M1 polarization induced by lipopolysaccharide and interferon γ.

[0049] In the present invention, Ox-LDL refers to oxidatively modified low-density lipoprotein (LDL). This is the product of peroxidation of the numerous polyunsaturated fatty acids within low-density lipoprotein (LDL) under the influence of excessive free radicals and other oxygen-generating factors, ultimately producing malondialdehyde (MDA). MDA then chemically modifies the LDL apolipoprotein B (APOB) by binding to lysine residues. Numerous basic and clinical research data demonstrate that the series of pathophysiological changes triggered by Ox-LDL are key to the development of atherosclerosis and are closely related to the severity of atherosclerosis. Ox-LDL has the biological property of being rapidly phagocytosed by macrophages and smooth muscle cells, making it a major factor in the formation of foam cells. It also has a chemotactic effect on monocytes, inhibiting their migration within lesions and causing them to accumulate there, contributing to the formation of atherosclerotic plaques. Ox-LDL also exhibits strong cytotoxicity, altering the functional state of endothelial cells, encouraging monocytes and low-density lipoprotein to enter the subintimal layer of blood vessels, and accelerating the formation of lipid streaks and arteriosclerosis.

[0050] In the present invention, lipopolysaccharide (LPS) is a component of the outer cell wall of Gram-negative bacteria, primarily composed of lipids and polysaccharides. LPS is a common endotoxin that, through cell signaling pathways, activates monocytes, macrophages, endothelial cells, and epithelial cells, leading to the synthesis and release of various cytokines and inflammatory mediators, thereby inducing a series of responses in the body. It is a gold standard reagent for establishing acute inflammatory models. Interferon-γ (IFN-γ) induces the production of inducible nitric oxide synthase (iNOS) in macrophages, promoting NO synthesis. IFN-γ also induces iNOS production in microglia and astrocytes, potentially implicated in the development or protection of certain central nervous system diseases. In specific embodiments of the present invention, LPS and IFN-γ stimulate macrophages to produce nitric oxide (NO) via inducible nitric oxide synthase and activate stress signaling cascades, including the c-jun-N-terminal kinase (JNK) pathway. These events trigger an apoptotic cascade, ultimately leading to death.

[0051] The sixth aspect of the present invention provides a use of the nanomedicine according to the first aspect of the present invention or the pharmaceutical composition according to the second aspect of the present invention, wherein the use comprises:

[0052] 1) Application in the preparation of products for targeted diagnosis and / or treatment of atherosclerotic plaques.

[0053] 2) Application in the preparation of atherosclerotic plaque-specific MRI imaging enhancers.

[0054] 3) Application in the preparation of atherosclerotic plaque-specific in vivo imaging contrast agents.

[0055] Furthermore, the application also includes:

[0056] 1) Application in inhibiting DKK1 expression in vascular endothelial cells and / or inhibiting apoptosis in vascular endothelial cells.

[0057] Furthermore, the vascular endothelial cells include HUVECs, HCMECs, HMECs, and Eahy926.

[0058] Furthermore, the vascular endothelial cells are HUVECs.

[0059] Furthermore, the cell apoptosis is oxidation-induced cell apoptosis.

[0060] Furthermore, the oxidation-induced cell apoptosis is Ox-LDL-induced cell apoptosis.

[0061] 2) Application in inhibiting ROS production in macrophages, inhibiting macrophage polarization and / or inhibiting macrophage phagocytosis of Ox-LDL.

[0062] Furthermore, the macrophages include RAW264.7 and THP-1.

[0063] Furthermore, the macrophages are RAW264.7.

[0064] Furthermore, the inhibiting of macrophage polarization refers to inhibiting macrophage M1 polarization.

[0065] Furthermore, the inhibiting of macrophage phagocytosis of Ox-LDL also includes inhibiting foam cell formation.

[0066] Furthermore, the ROS generation is induced by lipopolysaccharide and interferon γ.

[0067] Furthermore, the macrophage M1 polarization is macrophage M1 polarization induced by lipopolysaccharide and interferon γ.

[0068] Advantages and beneficial effects of the present invention:

[0069] (1) E / A-Mn 2+ @D NPs have good relaxivity and can be used as AS plaque-specific MRI imaging enhancers;

[0070] (2) Cy5-labeled E / A-Mn 2+ @D NPs(E / A-Mn 2+ @D-Cy5 NPs) can be used as an AS plaque-specific in vivo imaging developer;

[0071] (3) E / A-Mn 2+ @D NPs long-term intervention has good biosafety;

[0072] (4) E / A-Mn 2+ @D Long-term intervention with NPs can delay the development of AS plaques and improve plaque stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 is the particle size distribution diagram of nanomedicine, Figure 1 A in the figure is the particle size and potential distribution of E / A NPs. Figure 1 B in the equation is E / A-Mn 2+ Particle size and potential distribution of NPs, Figure 1 C in the equation is E / A-Mn 2+ @D Particle size and potential distribution of NPs.

[0074] Figure 2 E / A-Mn2+ @D UV absorption spectrum of NPs.

[0075] Figure 3 E / A-Mn 2+ Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of @D NPs.

[0076] Figure 4 For different concentrations (Mn 2+ )E / A-Mn 2+ @D T1-weighted imaging of NPs (A) and its linear relationship with 1 / T1 value (B).

[0077] Figure 5 E / A-Mn 2+ @D NPs HUVECs, RAW264.7 cell cytotoxicity test chart, Figure 5 A in the equation is different concentrations of E / A-Mn 2+ @D NPs were incubated with HUVECs cells for 12 h. Figure 5 B in the figure represents different concentrations of E / A-Mn 2+ @D NPs were incubated with HUVECs cells for 24 h. Figure 5 C in the equation is different concentrations of E / A-Mn 2+ @D NPs were incubated with RAW264.7 cells for 12 h. Figure 5 D in the figure is different concentrations of E / A-Mn 2+ @D NPs were incubated with RAW264.7 cells for 24 h.

[0078] Figure 6 The expression of DKK1, ICAM-1 and VCAM-1 in HUVECs cells after different treatments was detected by WB.

[0079] Figure 7 CLSM detection of E / A-Mn 2+ @D-Cy5 NPs bind to HUVECs cells in vitro.

[0080] Figure 8 E / A-Mn 2+ @D NPs’ effects on RAW264.7 cells (A) and quantitative analysis (B).

[0081] Figure 9 Figure 3 shows flow cytometry detection of M1 polarization in RAW264.7 cells after different treatments (A) and quantitative analysis (B).

[0082] Figure 10 Figure 2 shows the flow cytometry detection of HUVECs apoptosis after different treatments (A) and quantitative analysis (B).

[0083] Figure 11 CLSM observation of E / A-Mn 2+ @D Graph showing the inhibitory effect of NPs on the phagocytosis of Ox-LDL by RAW264.7 cells.

[0084] Figure 12 Oil red O staining shows the effects of different treatments on foam cell formation.

[0085] Figure 13 For WB detection of E / A-Mn 2+ @D Graph showing the effects of NPs on the expression of proteins related to the Wnt pathway, apoptosis, and lipophagy.

[0086] Figure 14 ApoE - / - MRI targeted imaging (AB) and in vivo imaging (CD) of the mouse abdominal aorta.

[0087] Figure 15 E / A-Mn 2+ NPs and different doses of E / A-Mn 2+ @D Oil red O staining of mouse aortic plaques after NPs treatment.

[0088] Figure 16 E / A-Mn 2+ NPs and different doses of E / A-Mn 2+ HE, CD68, MMP-9, α-SMA, Masson, and DKK1 staining (A) and quantitative analysis (BG) of the aortic root of mice 8 weeks after D NPs treatment. DETAILED DESCRIPTION

[0089] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art and do not constitute a limitation of the present invention in any way.

[0090] HUVECs cells refer to human umbilical vein endothelial cells (HUVECs), which are commonly used cell models in vascular endothelial cell experiments. They have the potential of stem cells and can theoretically be passaged 50 to 60 times.

[0091] RAW 264.7 cells are derived from an Abelson murine leukemia virus-induced tumor. In normal culture, the cells exhibit polygonal and irregular shapes, with numerous pseudopodia, and possess a strong ability to adhere to surrounding surfaces. RAW 264.7 cells are immune cells with diverse functions, making them an important target for studies of phagocytosis, cellular immunity, and molecular immunology.

[0092] The MTT assay, also known as the MTT colorimetric assay, is a method for measuring cell viability and growth. The assay works by reducing exogenous MTT to water-insoluble, blue-purple crystalline formazan by succinate dehydrogenase in the mitochondria of living cells, which then deposits within the cell. This reduction occurs in dead cells, but not in dead cells. Dimethyl sulfoxide (DMSO) dissolves the formazan in cells, and its absorbance at 490 nm is measured using an enzyme-linked immunosorbent assay (ELISA), providing an indirect indicator of viable cell count. Within a certain cell population range, the amount of MTT crystals formed is proportional to the cell number. Exogenous MTT refers to thiazolyl blue, a cell-permeable, positively charged tetrazolium dye used to monitor cellular reductive metabolism. Thiazolyl blue is taken up by cells across the plasma membrane and then reduced to formaldehyde by intracellular NAD(P)H oxidoreductase. It is frequently used in colorimetric assays to measure cell proliferation, cytotoxicity, and apoptosis.

[0093] The present invention will be further described in detail below with reference to the accompanying drawings and examples. The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified. The following examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Simple modifications to the present invention based on the essence of the present invention fall within the scope of protection claimed in the present invention.

[0094] Example 1 Preparation of Nanomedicine for Targeted Diagnosis and Treatment of Atherosclerotic Plaques

[0095] 1. Experimental Materials

[0096] Epigallocatechin gallate (EGCG 95%) (Dalian Meilun, MB1672-2), arginine (Arg) (J&K, 235303), formaldehyde solution (formaldehyde 37-40%, Sinopharm, 10010018), manganese chloride tetrahydrate (McLean, M813488), human / mouse Dkk-1 antibody (R&D, AF1096), Cy5 NHS inhibitor (Xi'an Ruixi, RH-5029), APC-labeled CD11b flow cytometry antibody (BioLegend, USA, 101212), BODIPY dye (Invitrogen, USA, D3922), FITC Annexin V Apoptosis Detection Kit I (BD, USA, 556547), MTT kit (Solarbold, M1020), diphenyl picrohydrazine (DPPH) (Sigma, USA, R712787), and Oil Red O dye (Sigma, USA, O8010).

[0097] 2. Experimental Methods

[0098] (1) Preparation of nanomedicine E / A-Mn 2+ @D NPs

[0099] 22.875 mg of EGCG and 7.5 μl of formaldehyde solution were weighed and dissolved in 10 ml of triple-distilled water. The mixture was stirred at 30°C to mix thoroughly. 5.79 mg of L-Arg was then added and the reaction was continued at 30°C with stirring for 5 min. The nanoparticles were collected and centrifuged at 3.5 × g for 5 min at room temperature. The supernatant was discarded and the nanoparticles were purified three times with triple-distilled water. The nanoparticles were then resuspended in 80 ml of triple-distilled water. MnCl2·4H2O solution (MnCl2·4H2O) was added to the nanoparticle solution at 10 μl / ml. 2+ , 2 mg / ml), mix well, and incubate at room temperature overnight. Collect the nanoparticles, centrifuge at 3.5 × g for 5 minutes at room temperature, discard the supernatant, and purge three times with triple-distilled water. Then resuspend the nanoparticles in 10 ml of triple-distilled water. Add 10 μl / ml of Human / Mouse Dkk-1 Antibody (1 μg / μl) to the nanoparticle solution, mix well, and incubate at 4°C overnight. Collect the nanoparticles, centrifuge at 3.5 × g for 5 minutes at room temperature, discard the supernatant, purge three times with triple-distilled water, and then resuspend the nanoparticles in 10 ml of triple-distilled water and store in a refrigerator at 4°C until use.

[0100] (2) Cytotoxicity of nanomedicines

[0101] HUVECs were prepared into 5 × 10 4RAW264.7 cells were prepared into 1×10 cells / ml cell suspension using DMEM medium containing 10% FBS. 5 Cell suspension of 100 cells / ml was cultured overnight to allow cells to adhere to the wall. The original culture medium was discarded and different concentrations of E / A-Mn prepared from fresh culture medium were added. 2+ Add 5 μl of DNP working solution and continue incubation for 12 and 24 hours. Aspirate the supernatant and wash three times with PBS. Add 90 μl of fresh culture medium and 10 μl of MTT solution and continue incubation for 4 hours. Aspirate the supernatant and add 110 μl of Formazan dissolving solution to each well. Shake on a shaker at low speed for 10 minutes to fully dissolve the crystals. Measure the absorbance of each well at 490 nm using an enzyme-linked immunosorbent assay (ELISA).

[0102] (3) Effect of nanomedicine on dickkopf-1 secretion by HUVECs stimulated by LPS

[0103] Prepare 1×10 5 A HUVECs cell suspension of 1 μg / mL was prepared and cultured overnight to allow the cells to adhere. 1 μg / mL LPS was prepared in fresh ECM medium. Fresh ECM medium, 1 μg / mL LPS, and different doses (50, 100 μg / ml) of E / A-Mn 2+ @DNPs and 1 μg / mL LPS were treated with cells for 24 h. WB verification of E / A-Mn 2+ @D Effect of NPs on DKK1 secretion from HUVECs stimulated by LPS.

[0104] (4) In vitro binding of nanomedicine to LPS-stimulated HUVECs

[0105] Prepare 1×10 4 A suspension of HUVECs at 100 μg / ml was prepared and cultured overnight to allow the cells to adhere. The cells were stimulated with 1 μg / mL LPS in fresh ECM medium for 24 h. 2+ -Cy5 NPs, different doses (50, 100 μg / ml) of E / A-Mn 2+ Cells were treated with @D-Cy5 NPs for 1 hour. The cells were washed three times with PBS and fixed with 4% paraformaldehyde for 15 minutes at 37°C. The cells were washed three times with PBS. The cell membranes were stained with 5 μg / ml WGA-488, and the nuclei were counterstained with Hoechst 33342 for 3 minutes at room temperature. The cells were washed three times with PBS and mounted with anti-fluorescence quenching mounting medium. CLSM images were obtained and processed using Zen software.

[0106] (5) Nanomedicine inhibits the production of ROS in macrophages

[0107] Prepare 5×10 4 The cells were suspended in a suspension of RAW264.7 cells at a concentration of 100 μg / ml and cultured overnight to allow the cells to adhere. The control group continued to culture with fresh DMEM high-glucose medium, the model group was treated with 100 ng / ml LPS + 100 UI / ml IFN-γ, and the rescue group was treated with 100 ng / ml LPS + 100 UI / ml IFN-γ and different doses (50, 100 μg / ml) of E / A-Mn 2+ Cells were treated with @D NPs. After 24 hours, cells were cultured in serum-free medium containing 10 μM DCFH-DA for an additional 30 minutes. The cells were washed three times with PBS. ROS fluorescence intensity was quantified using a microplate reader and observed and photographed using CLSM.

[0108] (6) Nanomedicine regulates macrophage polarization

[0109] Prepare 1×10 6 The cells were suspended in a suspension of RAW264.7 cells at a concentration of 1 μg / ml and cultured overnight to allow the cells to adhere. The control group continued to culture with fresh DMEM high-glucose medium, the model group was treated with 1 μg / ml LPS + 20 ng / ml IFN-γ, and the rescue group was treated with 1 μg / ml LPS + 20 ng / ml IFN-γ and different doses (50, 100 μg / ml) of E / A-Mn 2+ Cells were treated with D NPs. After 24 hours, single-cell suspensions were collected and washed with PBS. Cells were incubated with FITC-labeled CD86 flow cytometry antibodies (5 μg / 10⁶ cells) and APC-labeled CD11b flow cytometry antibodies (1.5 μl / 10⁶ cells) at 4°C for 30 minutes. Finally, cells were resuspended in 500 μl of ice-cold PBS and analyzed.

[0110] (7) Nanomedicine inhibits oxidation-induced cell apoptosis

[0111] Prepare 1×10 5 The control group continued to culture with fresh ECM medium, the model group was treated with 50 μg / mL Ox-LDL, and the rescue group was treated with 50 μg / mL Ox-LDL and different doses (50, 100 μg / ml) of E / A-Mn 2+ Treat cells with D NPs. After 24 hours, collect single-cell suspensions and wash with PBS. Resuspend cells in 100 μl of buffer, then add 5 μl each of FITC and PI and incubate in the dark for 15 minutes. Finally, add 400 μl of buffer to terminate the reaction and analyze within 1 hour.

[0112] (8) Nanomedicine inhibits macrophage phagocytosis of Ox-LDL

[0113] ① CLSM observation of E / A-Mn 2+ @D NPs inhibit the phagocytosis of Dil-oxLDL by RAW264.7 cells

[0114] Prepare 5×10 4 The cells were suspended in a suspension of RAW264.7 cells at 1 μg / ml and cultured overnight to allow the cells to adhere. The control group continued to culture with fresh DMEM high-glucose medium, while the model group and rescue group were first stimulated with 1 μg / ml LPS for 24 hours. The model group was then cultured with 10 μg / ml Dil-oxLDL, while the rescue group was treated with 10 μg / ml Dil-oxLDL and different doses (50 and 100 μg / ml) of E / A-Mn. 2+ Cells were incubated with @D NPs. After 4 hours, the cells were washed three times with PBS and fixed with 4% paraformaldehyde for 15 minutes at 37°C. The cells were washed three times with PBS, the cell membranes were stained with 5 μg / ml WGA-488, and the nuclei were counterstained with DAPI for 3 minutes at room temperature. The cells were washed three times with PBS and mounted with anti-fluorescence quenching mounting medium. CLSM images were obtained and processed using the dedicated software Zen.

[0115] ②Observation of E / A-Mn by Oil Red Staining 2+ @D NPs inhibit foam cell formation

[0116] Prepare 5×10 4 The cells were suspended in a suspension of RAW264.7 cells at 1 μg / ml and cultured overnight to allow the cells to adhere. The control group continued to be cultured in fresh DMEM high-glucose medium, while the model and rescue groups were first stimulated with 1 μg / ml LPS for 24 hours. The model group was then treated with 50 μg / ml Ox-LDL, while the rescue group was treated with 50 μg / ml Ox-LDL and different doses (50 and 10 μg / ml) of E / A-Mn. 2+ @D NPs treated cells. After 48 hours, cells were washed three times with PBS and fixed with 4% paraformaldehyde at 37°C for 15 minutes. Washed three times with PBS, the slides were covered with Oil Red O working solution, and stained for 30 minutes at room temperature in the dark. The staining solution was discarded, the slides were washed three times with PBS, and then mounted with glycerol-gelatin. Images were taken using a motorized upright microscope.

[0117] (9) Detection of Wnt pathway proteins and apoptosis and lipophagy-related proteins

[0118] HUVECs and RAW264.7 cells were transferred to a cell culture medium with a density of 1×10 5 / 5×10 4Cells were plated in 6-well plates at 100 cells / ml per well and cultured overnight in a 37°C cell culture incubator. Stimulation was then administered as described above for the various experiments. After stimulation, protein was collected and concentrations were determined using a BCA protein assay kit. Equal amounts of protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes, which were then blocked in 5% skim milk in TBST for 1 hour. The membranes were incubated overnight at 4°C on a shaker with primary antibodies against β-catenin, p-β-catenin, GSK-3β, p-GSK-3β, Wnt3a, Bax, Bcl2, Caspase3, Cleaved Caspase3, Lox-1, SRA, CD36, SR-BI, ABCA1, and ABCG1. Western blot analysis was performed using a gel imaging system. GAPDH was used as a loading control antibody.

[0119] (10) The value of nanomedicine in targeted imaging of atherosclerotic plaques

[0120] ① In vivo observation of E / A-Mn 2+ @D NPs imaging at the AS site in mice

[0121] The successfully modeled ApoE - / - Mice were randomly divided into two groups and treated with 6 μmol / kg Mn 2+ E / A-Mn was injected into the tail vein at a dose of 2+ NPs and E / A-Mn 2+ @D NPs, and T1WI sequence scans were performed before and 24 h after administration. ApoE - / - The chest and abdomen of the mouse were placed in the scanning area. ApoE was detected using the body coil of a 9.4 T small animal MRI system (Biospec 94 / 20 USR, Bruker, Germany). - / - MRI imaging of the abdominal aorta of the model rats was performed. T1WI sequence parameters were as follows: TR = 1228 ms, TE = 8.6 ms, FOV 31 mm × 29 mm, matrix 256 × 256, and slice thickness 0.7 mm. The scanned images were analyzed using Image J software to measure the signal intensity (SI) of the abdominal aorta wall, the signal intensity (SI) of the surrounding muscle tissue, and the background noise (SI) outside the tissue. The CNR (contrast-to-noise) value for each slice was calculated:

[0122] The calculation formula is as follows: CNR = (SI (wall) -SI (muscle)) / SI (noise)

[0123] According to the CNR values calculated by the above method, the changes in the CNR values of the images before and after the nanomedicine injection were compared layer by layer, and the change rate of the CNR value was calculated:

[0124] The calculation formula is as follows: CNR changes % = (CNR24hpost–CNRpre) / CNRpre × 100%

[0125] ② In vivo imaging observation of E / A-Mn 2+ Distribution of @D-Cy5 NPs in mouse aorta

[0126] Each group of mice was injected with 10 mg / kg E / A-Mn into the tail vein. 2+ @D-Cy5 NPs (the control group was injected with the same dose of saline). 2+ Mice were sacrificed 3 and 12 hours after D-Cy5 NPs administration and fully perfused. The heart and aorta were immediately isolated up to the iliac bifurcation. After weighing, fluorescence intensity was measured under in vivo imaging, and the mean fluorescence intensity per unit volume / mass was calculated.

[0127] (11) Verification of anti-AS effect in vivo

[0128] 6-8 week old male ApoE - / - 40 mice were fed with normal diet for 1 week and high-fat diet for 4 weeks, and then randomly divided into saline group, E / A-Mn group, and 2+ NPs group, low dose E / A-Mn 2+ @D NPs group, high dose E / A-Mn 2+ @D NPs group. Used to study the in vivo anti-AS effects of nanomedicines. Aorta samples were collected 8 weeks after treatment, and staining was performed to analyze plaque area and stability.

[0129] 3. Experimental Results

[0130] (1) E / A-Mn 2+ @D NPs Characterization

[0131] The particle size of the nanodrug was measured by Malvern particle size analyzer, and the particle size of E / A NPs was 195.4±66.8 nm, and the Zeta potential was -13.60±1.05 mV; 2+ The particle size of NPs was 216.5±71.4 nm, and the Zeta potential was 0.73±0.54 mV; E / A-Mn 2+The particle size of @D NPs was 215±90.3 nm and the Zeta potential was -5.60±0.42 mV ( Figure 1 ). Figure 2 UV-visible absorption spectrum shows E / A-Mn 2+ @D NPs have an obvious characteristic absorption peak near 273 nm, which is basically consistent with the characteristic absorption peak of free EGCG aqueous solution, proving that EGCG is successfully encapsulated. 2+ @D NPs were morphologically characterized. Figure 3 It was found that E / A-Mn 2+ @D NPs have a spherical morphology, uniform size and good dispersion. The E / A-Mn 2+ The longitudinal relaxation rate r1 of @D NPs is 8.07 Mm -1 S -1 ( Figure 4 ).

[0132] (2) Cytotoxicity of nanomedicines

[0133] Different concentrations of E / A-Mn 2+ @D NPs were co-incubated with RAW264.7 and HUVECs cells for 12 and 24 h. Even at a concentration of 300 μg / ml, the survival rate of cells in each group did not decrease significantly compared with the control group ( Figure 5 ). Proof E / A-Mn 2+ @D NPs have good safety and very low toxicity to RAW264.7 and HUVECs cells.

[0134] (3) Effect of nanomedicine on dickkopf-1 secretion by HUVECs stimulated by LPS

[0135] After HUVECs were stimulated with 1 μg / ml LPS for 24 h, the levels of ICAM-1 and VCAM-1 in the cells were significantly increased compared with those in the control group, indicating that the HUVECs inflammation model was successfully induced. 2+ After treatment with @D NPs, the expression levels of ICAM-1, VCAM-1 and DKK1 decreased significantly, indicating that E / A-Mn 2+ @D NPs can effectively inhibit the expression of DKK1 in HUVECs cells ( Figure 6 ).

[0136] (4) In vitro binding of nanomedicine to LPS-stimulated HUVECs

[0137] First, HUVECs were stimulated with LPS to secrete DKK1, and the Con group and E / A-Mn2+ -Cy5 NPs group did not detect Cy5 signal. 2+ Different degrees of Cy5 signals were detected in the @D-Cy5 NPs group, and there was a certain dose dependence. Figure 7 Indicates that E / A-Mn is not conjugated to Human / Mouse DKK-1 antibody 2+ -Cy5 NPs could not bind to HUVECs cells, while E / A-Mn coupled with anti-Human / Mouse DKK-1 antibody 2+ @D-Cy5 NPs can specifically bind to HUVECs cells.

[0138] (5) Nanomedicine inhibits the production of ROS in macrophages

[0139] After LPS+IFN-γ stimulation, ROS production in RAW264.7 cells increased significantly, while E / A-Mn 2+ @D NPs treatment can effectively alleviate the production of ROS in RAW264.7 cells ( Figure 8 ), and there is a certain dose dependence.

[0140] (6) Nanomedicine regulates macrophage polarization

[0141] RAW264.7 macrophages have a low M1 polarization rate. After LPS+IFN-γ stimulation, the M1 polarization rate of RAW264.7 cells increased significantly, reaching more than 60%. 2+ After treatment with @D NPs, the M1 polarization rate of RAW264.7 cells in the low-dose group decreased to about 45%, and the M1 polarization rate of RAW264.7 cells in the high-dose group decreased to 30%. 2+ @D NPs have a significant alleviating effect on the M1 polarization of RAW264.7 cells after LPS+IFN-γ stimulation ( Figure 9 ).

[0142] (7) Nanomedicine inhibits oxidation-induced cell apoptosis

[0143] HUVECs have a low natural apoptosis rate. After stimulation with 50 μg / mL Ox-LDL, the apoptosis rate of HUVECs increased significantly, reaching about 27%. 2+ After treatment with @D NPs, the apoptosis rate of HUVECs in the low-dose group decreased to about 20%, and the apoptosis rate of HUVECs in the high-dose group decreased to about 14%. 2+@D NPs have a significant alleviating effect on HUVECs apoptosis after stimulation with 50 μg / mL Ox-LDL ( Figure 10 ).

[0144] (8) Nanomedicine inhibits macrophage phagocytosis of Ox-LDL

[0145] ① CLSM observation of E / A-Mn 2+ @D NPs inhibit the phagocytosis of Dil-oxLDL by RAW264.7 cells

[0146] Blue fluorescence represents the cell nucleus, green fluorescence represents the cell membrane labeled with WGA-488, and red fluorescence represents Ox-LDL labeled with Dil. The red fluorescence intensity was the strongest in the RAW264.7 cells of the model group. 2+ After treatment with @D NPs, the red fluorescence intensity in the cells was significantly reduced. The above results suggest that inflammatory cells have a strong phagocytic ability to Ox-LDL, while E / A-Mn 2+ @D NPs have a significant inhibitory effect on this phagocytic ability ( Figure 11 ).

[0147] ②Observation of E / A-Mn by Oil Red Staining 2+ @D NPs inhibit foam cell formation

[0148] Figure 12 The results show that using E / A-Mn 2+ After the cells were treated with @D NPs, the intracellular lipid droplets were significantly reduced, and the E / A-Mn 2 + @D The higher the concentration of NPs, the more obvious the reduction of intracellular lipid droplets, suggesting that E / A-Mn 2+ @D NPs have the ability to inhibit foam cell formation.

[0149] (9) Detection of Wnt pathway proteins and apoptosis and lipophagy-related proteins

[0150] Western blotting detected the expression of β-catenin and p-β-catenin, which reflected the expression of E / A-Mn 2+ @D NPs activate the Wnt pathway. The results showed that the expression of β-catenin in the Model group was reduced, and E / A-Mn 2+ @D NPs intervention increased β-catenin expression ( Figure 13To better understand Wnt activation, apoptosis and lipophagy were also investigated after treatment. Western blotting was used to assess the expression of Bax, Bcl2, Caspase3, Cleaved Caspase3, Lox-1, SRA, CD36, SR-BI, ABCA1, and ABCG1. Compared with the control group, the Bax / Bcl2 and Cleaved Caspase3 / Caspase3 ratios were increased in the Model group, as were the expression of Lox-1, SRA, and CD36, and the expression of SR-BI, ABCA1, and ABCG1. E / A-Mn 2+ @D NPs intervention reduced the ratio of Bax / Bcl2 and Cleaved Caspase3 / Caspase3 ( Figure 13 B), the expression of Lox-1, SRA, and CD36 decreased ( Figure 13 C in the figure), the expression levels of SR-BI, ABCA1, and ABCG1 increased ( Figure 13 C in E / A-Mn 2+ @D NPs intervention delayed cell apoptosis and inhibited lipid phagocytosis by inhibiting macrophage lipid influx and increasing lipid efflux.

[0151] (10) The value of nanomedicine in targeted imaging of atherosclerotic plaques

[0152] ① In vivo observation of E / A-Mn 2+ @D NPs imaging at the AS site in mice

[0153] After injection of E / A-Mn2+ NPs or E / A-Mn 2+ @D NPs before ApoE - / - The abdominal aorta of mice showed thickening of vascular wall and formation of intravascular plaque. 2+ @D NPs 24 hours after injection, the T1 signal of the abdominal aorta wall was significantly enhanced, with a statistical difference compared with before injection ( Figure 14 AB in). While injection of E / A-Mn 2+ ApoE in NPs - / - Although the T1 signal of the abdominal aorta wall of mice was enhanced compared with the previous one, it did not reach statistical significance ( Figure 13 From the quantitative statistics of CNR, it can be seen that the injection of E / A-Mn 2+ The CNR of mice injected with NPs decreased by 7.3%, while that of mice injected with E / A-Mn 2+ @D NPs decreased the CNR of mice by 35%, indicating that E / A-Mn 2+ @D NPs have better targeting ability to AS lesions, while E / A-Mn2+ NPs do not have this capability.

[0154] ② In vivo imaging observation of E / A-Mn 2+ Distribution of @D-Cy5 NPs in mouse aorta

[0155] saline group C57BL6 and ApoE - / - There was no obvious Cy5 fluorescence in the mice. 2 + @D-Cy5 NPs showed low-dose Cy5 fluorescence intensity in some areas 3 hours after administration, and the Cy5 fluorescence intensity increased after 12 hours. - / - Injection of E / A-Mn into the tail vein of mice 2+ @D-Cy5 NPs also showed weak Cy5 fluorescence 3 hours later, and the Cy5 fluorescence intensity increased significantly after 12 hours. Compared with C57BL6 mice at the same time point, the Cy5 fluorescence of both groups increased at 3 hours but there was no difference (5.613±0.427×105 VS 5.324±0.076×105, p / sec / cm2 / sr / uw / cm2 / mg, p=0.525). - / - The fluorescence intensity of the mouse aorta was significantly higher than that of the C57BL6 mouse (8.758±0.639×105VS 5.950±0.130×105, p / sec / cm2 / sr / uw / cm2 / mg, p=0.0026), indicating that E / A-Mn 2+ @D-Cy5 NPs have specific targeting ability to aortic AS plaques ( Figure 14 CD in the ).

[0156] (11) Verification of anti-AS effect in vivo

[0157] After 8 weeks of high-fat feeding and different treatments, the model group, E / A-Mn 2+ NPs group, low dose E / A-Mn 2+ @D NPs group, high dose E / A-Mn 2+ The average aortic plaque areas in the D NPs group were 55%, 43%, 42%, and 20%, respectively. Figure 15 By staining the aortic root plaques, the stability of AS plaques was evaluated. The results showed that nanomedicine intervention reduced inflammation in the plaques, increased the content of smooth muscle cells in the plaques, reduced the degradation of extracellular matrix, and increased collagen in the plaques, playing an important role in resisting the development of AS and stabilizing the plaques ( Figure 16 ).

[0158] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention can be implemented over a wide range under equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides embodiments, it will be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any variations, uses, or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the disclosed scope of this application.

Claims

1. A nanomedicine for targeted diagnosis and treatment of atherosclerotic plaques, characterized in that: Its components include epigallocatechin gallate, formaldehyde, L-arginine, Mn 2+ and DKK1 antibodies.

2. The nanomedicine according to claim 1, characterized in that The components of the nanomedicine also include Cy5.

3. The nanomedicine according to claim 1, wherein The particle size of the nanomedicine is 215±90.3 nm, the Zeta potential is -5.60±0.42 mV, the characteristic absorption peak is at 273 nm, and the longitudinal relaxation rate r1 is 8.07 Mm -1 S -1 .

4. The nanomedicine according to claim 1, characterized in that The nano drug is spherical, uniform in size and good in dispersibility.

5. A pharmaceutical composition for targeted diagnosis and treatment of atherosclerotic plaques, characterized in that: The pharmaceutical composition comprises the nanodrug according to any one of claims 1 to 4.

6. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition further includes a pharmaceutically acceptable carrier.

7. A method for preparing the nanomedicine according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: preparing nanoparticles using epigallocatechin gallate, formaldehyde and L-arginine as raw materials, adding Mn 2+ Incubate overnight, add DKK1 antibody and incubate overnight.

8. The method according to claim 7, characterized in that The method further comprises the steps of adding Cy5.

9. The method according to claim 7, characterized in that The steps of preparing nanoparticles using epigallocatechin gallate, formaldehyde, and L-arginine as raw materials include: dissolving epigallocatechin gallate and formaldehyde in triple-distilled water, stirring and mixing, adding L-arginine, stirring and reacting, purifying and resuspending to obtain a solution of nanoparticles; The addition of Mn 2+ The overnight incubation step includes: adding Mn 2+ Incubate overnight and purify and resuspend to obtain the Mn 2+ Solutions of nanoparticles; The step of adding DKK1 antibody and incubating overnight comprises: 2+ DKK1 antibody is added to the solution of the nanoparticles and incubated overnight, and the nanomedicine according to any one of claims 1 to 4 is obtained after purification and resuspending.

10. The method according to claim 9, characterized in that The mass ratio of epigallocatechin gallate to L-arginine is 3.95:

1.

11. The method according to claim 9, characterized in that The stirring and mixing condition is 30°C.

12. The method according to claim 9, wherein The stirring reaction conditions are 30° C. and 5 min.

13. The method according to claim 9, wherein The Mn 2+ The final concentration was 20 μg / ml.

14. The method according to claim 9, characterized in that The Mn 2+ Selected from MnCl2·4H2O solution.

15. The method according to claim 9, characterized in that The addition of Mn 2+ Incubate overnight at room temperature.

16. The method according to claim 9, wherein The final concentration of the DKK1 antibody was 10 μg / ml.

17. The method according to claim 9, characterized in that The conditions for adding DKK1 antibody and incubating overnight were 4°C.

18. The method according to claim 9, characterized in that The purification and resuspension refers to purification and resuspension with triple-distilled water after centrifugation.

19. The method according to claim 18, wherein The centrifugation conditions are room temperature, 3.5×g, 5 min.

20. Use of the nanomedicine according to any one of claims 1 to 4 or the pharmaceutical composition according to any one of claims 5 to 6, characterized in that: The applications include: 1) Application in the preparation of products for targeted diagnosis and / or treatment of atherosclerotic plaques; 2) Application in the preparation of atherosclerotic plaque-specific MRI imaging enhancers; 3) Application in the preparation of atherosclerotic plaque-specific in vivo imaging contrast agents.

21. The use according to claim 20, characterized in that The diagnosis and / or treatment of atherosclerotic plaques is achieved by inhibiting DKK1 expression in vascular endothelial cells, inhibiting apoptosis of vascular endothelial cells, inhibiting ROS generation in macrophages, inhibiting macrophage polarization and / or inhibiting macrophage phagocytosis of Ox-LDL.

22. The use according to claim 21, characterized in that The vascular endothelial cells include HUVECs, HCMECs, HMECs, and Eahy926.

23. The use according to claim 21, characterized in that The vascular endothelial cells are HUVECs.

24. The use according to claim 21, characterized in that The cell apoptosis is oxidation-induced cell apoptosis.

25. The use according to claim 24, characterized in that The oxidation-induced cell apoptosis is Ox-LDL-induced cell apoptosis.

26. The use according to claim 21, characterized in that The macrophages include RAW264.7 and THP-1.

27. The use according to claim 21, characterized in that The macrophages are RAW264.

7.

28. The use according to claim 21, characterized in that The inhibition of macrophage polarization refers to the inhibition of macrophage M1 polarization.

29. The use according to claim 21, characterized in that The inhibition of macrophage phagocytosis of Ox-LDL also includes inhibition of foam cell formation.

30. The use according to claim 21, characterized in that The ROS production is induced by lipopolysaccharide and interferon γ.

31. The use according to claim 28, characterized in that The macrophage M1 polarization is induced by lipopolysaccharide and interferon γ.

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