Nano-drug for targeted diagnosis and treatment of atherosclerotic plaque

Abstract

The invention provides a nano-drug for targeted diagnosis and treatment of atherosclerotic plaque, the nano-particles are constructed by EGCG, formaldehyde and L-arginine, the antioxidant characteristic of EGCG is retained, and the nano-drug constructed by modifying Mn < 2 + > and DKK1 antibodies on the basis has a good relaxation value and can be used for an atherosclerotic plaque specific MRI imaging enhancer; the Cy5-labeled nano-drug can be used for an atherosclerotic plaque specific in-vivo imaging developer. The nano-drug provided by the invention has good biological safety, can delay plaque development and improve plaque stability, and is a nano-drug for precisely targeting atherosclerotic plaque and treating the atherosclerotic plaque.

Description

Technical Field

[0001] The present invention relates to a nano drug 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 killer of human health. Atherosclerosis (AS) is the main cause of CVD, accounting for more than 50% of CVD deaths. Early AS usually does not cause serious complications. As AS lesions develop, changes such as necrotic lipid core, inflammatory cell infiltration, thinning or damage of fibrous cap, and intraplaque hemorrhage appear in the plaque. The plaque becomes unstable and can rapidly progress to cause serious complications such as CVD. Therefore, identifying high-risk patients for CVD by detecting vulnerable plaques has become a top priority in the field of cardiovascular research. At present, the first choice for the treatment of AS is lifestyle intervention, followed by drug or surgical intervention. Clinical drugs including lipid-lowering drugs, cholesterol absorption inhibitors, antiplatelet drugs and angiotensin-converting enzyme inhibitors (ACEI) can alleviate the progression of AS and prevent the occurrence of atherosclerotic cardiovascular disease (ASCVD). However, these drugs cannot specifically accumulate in the AS lesion site. Although they can meet the clinical treatment requirements, there are also residual risks and certain adverse reactions. Therefore, targeted therapy has become a new direction in the current research on AS treatment.

[0003] Nanotechnology involves the cutting-edge field of the application of nanostructures in medicine and health care 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 small lesions and large blood flow in the blood vessels, which makes it difficult for conventional drugs to stay in the lesions in large quantities, bringing difficulties to the clinical treatment of AS.

[0004] The invention patent with publication number CN108498878A discloses nanoparticles prepared by a preparation strategy of polyphenol nanomedicine based on EGCG, formaldehyde and L-arginine (L-Arg), which fully retains the antioxidant properties of EGCG, and the nanoparticles are stable and reliable. The nanomedicine constructed by EGCG, formaldehyde and L-arginine retains the antioxidant properties of EGCG, but has no AS-specific targeting ability, and cannot be used for AS plaque targeted diagnosis, cannot be used for AS plaque imaging, and has non-specific distribution and other phenomena. Based on the above background, the present invention is intended to provide a nanomedicine for targeted diagnosis and treatment of atherosclerotic plaques. Summary of the invention

[0005] In order to make up for the deficiencies of the prior art, the object of the present invention is to provide a nano drug for targeted diagnosis and treatment of atherosclerotic plaques.

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

[0007] Furthermore, the components of the nanomedicine also include Cy5.

[0008] Furthermore, the particle size of the nanodrug 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 .

[0009] Furthermore, the nanomedicine is spherical, uniform in size and has good dispersibility.

[0010] In the present invention, atherosclerosis refers to plaque-related arteriosclerosis, which is the most common type of arteriosclerosis. Arteriosclerosis refers to the thickening and reduced elasticity of the arterial wall. Atherosclerosis is based on the repeated damage to the arterial wall, resulting in the deposition of lipid substances, forming plaques (also called atheromatous plaques), which are covered on the inner membrane of the arterial wall by a fibrous cap. Plaques can grow into the lumen of the artery, gradually leading to arterial stenosis.

[0011] In the present invention, nanomedicine refers to the use of nano-preparation technology to make raw materials and the like into nano-scale particles, or to combine raw materials with appropriate carrier materials to form nano-scale particles and the final pharmaceutical preparations. The size of its active ingredients or carrier particles is the primary feature of nanomedicine and an important basis for the nano-effect exhibited by the drug. The external dimensions, internal structure or surface structure of the final product or carrier material of the nanomedicine are nano-scale (about 100 nm or less), or the particle size of the final product or carrier material is below 1000 nm, and has a significant scale effect.

[0012] In the context of the present invention, Cy5 refers to a Cy dye, which is the abbreviation of cyanine, and is a compound composed of two nitrogen atoms connected by an odd number of methyl units. Cyanine compounds have the characteristics of long wavelength, adjustable absorption and emission, high extinction coefficient, good water solubility, and relatively simple synthesis. Cy series dyes are often used to label proteins, antibodies, and small molecule compounds. For the labeling of protein antibodies, the combination can be completed by a simple mixing reaction.

[0013] In the context of the present invention, Cy5 refers to a Cy dye, which is the abbreviation of cyanine, a compound composed of two nitrogen atoms connected by an odd number of methyl units. Cyanine compounds have the characteristics of long wavelength, adjustable absorption and emission, high extinction coefficient, good water solubility, and relatively simple synthesis. Cy series dyes are often used for labeling proteins, antibodies and small molecule compounds. For the labeling of protein antibodies, the combination can be completed by a simple mixing reaction.

[0014] 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.

[0015] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0016] In the present invention, pharmaceutical composition refers to a composition comprising at least one biologically active compound. The pharmaceutical composition of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or by implanted drug storage devices. The pharmaceutical composition of the present invention can contain any commonly used non-toxic pharmaceutically acceptable carrier, adjuvant or excipient. In some cases, pharmaceutical acids, bases or buffers can be used to adjust the pH of the preparation to improve the stability of the compound or its dosage form. The term parenteral as used in the present invention includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. As long as the target tissue can be reached, the pharmaceutical composition of the present invention can be administered to the receptor by any route.

[0017] In the present invention, pharmaceutically acceptable carrier refers to any drug carrier that itself 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.

[0018] The pharmaceutical composition of the present invention can also be used in combination with other drugs for treating atherosclerotic plaques. Other compounds for treating atherosclerotic plaques can be administered simultaneously with the main active ingredient (e.g., the nano drug described in the first aspect of the present invention), or even administered simultaneously in the same composition. Other therapeutic compounds can also be administered separately in a separate composition or in a dosage form different from that of the main active ingredient.

[0019] 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 and add DKK1 antibody and incubate overnight.

[0020] Furthermore, the method further comprises: adding Cy5.

[0021] 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.

[0022] Furthermore, the mass ratio of epigallocatechin gallate to L-arginine is 3.95:1.

[0023] Furthermore, the stirring and mixing condition is 30°C.

[0024] Furthermore, the stirring reaction was carried out at 30° C. for 5 min.

[0025] The addition of Mn 2+ The overnight incubation step includes: adding Mn to the nanoparticle solution 2+ Incubate overnight and purify and resuspend to obtain the Mn 2+ Solutions of nanoparticles.

[0026] Furthermore, the Mn 2+ The final concentration was 20 μg / ml.

[0027] Furthermore, the Mn 2+ Selected from MnCl2·4H2O solution.

[0028] Furthermore, the addition of Mn 2+ Incubate overnight at room temperature.

[0029] The step of adding DKK1 antibody and incubating overnight comprises: adding 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.

[0030] Furthermore, the final concentration of the DKK1 antibody is 10 μg / ml.

[0031] Furthermore, the condition for adding DKK1 antibody and incubating overnight is 4°C.

[0032] Furthermore, the purification and resuspension refers to purification and resuspension with triple-distilled water after centrifugation.

[0033] Furthermore, the centrifugation conditions are room temperature, 3.5×g, 5 min.

[0034] 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 catalyzed by nitric oxide synthase (NOS) in the body.

[0035] The full name of DKK1 is dickkopf WNT signaling pathway inhibitor 1, and its gene ID is 22943. Its protein molecular weight is about 26 KDa, and it exerts its effects through autocrine and paracrine pathways. In human disease research, DKK1 was first found to play a regulatory role in the development of colon cancer and lung cancer. To date, antibodies targeting DKK1, such as BHQ880 and DKN-01, have been clinically tested in a variety of tumor diseases.

[0036] The fourth aspect of the present invention provides a method for inhibiting the expression of DKK1 in vascular endothelial cells and / or inhibiting apoptosis of vascular endothelial cells, the method comprising treating vascular endothelial cells with the nano drug described in the first aspect of the present invention.

[0037] Furthermore, the vascular endothelial cells include HUVECs, HCMECs, HMECs, and Eahy926.

[0038] Furthermore, the vascular endothelial cells are HUVECs.

[0039] Furthermore, the cell apoptosis is oxidation-induced cell apoptosis.

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

[0041] 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 described in the first aspect of the present invention.

[0042] Furthermore, the macrophages include RAW264.7 and THP-1.

[0043] Furthermore, the macrophages are RAW264.7.

[0044] Furthermore, the inhibiting of macrophage polarization refers to inhibiting macrophage M1 polarization.

[0045] Furthermore, the inhibiting of macrophage phagocytosis of Ox-LDL also includes inhibiting the formation of foam cells.

[0046] Furthermore, the ROS generation is induced by lipopolysaccharide and interferon-γ.

[0047] Furthermore, the macrophage M1 polarization is macrophage M1 polarization induced by lipopolysaccharide and interferon γ.

[0048] In the present invention, Ox-LDL refers to oxidatively modified low-density lipoprotein, which is a product of malondialdehyde (MDA) produced by the peroxidation reaction of a large amount of polyunsaturated fatty acids in low-density lipoprotein (LDL) under the action of excessive free radicals and other oxygen-causing factors. MDA is combined with lysine residues in LDL apolipoprotein B (APOB) to undergo chemical modification. A large amount of basic and clinical research data proves that a series of pathological and physiological changes caused by Ox-LDL are the key to the formation of atherosclerosis and are closely related to the severity of the condition of patients with atherosclerosis. Ox-LDL has the biological property of being quickly phagocytosed by macrophages and smooth muscle cells, is the main factor in the formation of foam cells, has a chemotactic effect on monocytes, inhibits its migration in the lesions, causes it to accumulate in the lesions, and participates in the formation of atherosclerotic plaques. Ox-LDL also has strong cytotoxicity, changes the functional state of endothelial cells, prompts monocytes and low-density lipoproteins to enter the subendothelial membrane of the blood vessels, and accelerates the formation of lipid streaks and arteriosclerosis.

[0049] In the present invention, lipopolysaccharide (LPS) is a component of the outer wall of the cell wall of Gram-negative bacteria, mainly composed of lipids and polysaccharides. LPS is a common endotoxin, which can activate mononuclear macrophages, endothelial cells, epithelial cells, etc. through the cell signal transduction system in the body, synthesize and release a variety of cytokines and inflammatory mediators, and then cause a series of reactions in the body. It is a gold standard reagent for establishing an acute inflammation model. Interferon γ (IFN-γ) induces the production of inducible nitric oxide synthase (iNOS) in macrophages and promotes the synthesis of NO. IFN-γ can also induce the production of iNOS in microglia astrocytes, which may be related to the occurrence or protective effect of certain diseases of the central nervous system. In a specific embodiment of the present invention, lipopolysaccharide and interferon γ stimulate macrophages to produce nitric oxide (NO) through inducible nitric oxide synthase and activate stress signal cascades including the c-jun-N-terminal kinase (JNK) pathway. These events trigger an apoptotic cascade reaction, ultimately leading to death.

[0050] The sixth aspect of the present invention provides an application of the nanomedicine described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention, wherein the application comprises: 1) Application in the preparation of products for targeted diagnosis and / or treatment of atherosclerotic plaques.

[0051] 2) Application in the preparation of atherosclerotic plaque-specific MRI imaging enhancers.

[0052] 3) Application in the preparation of atherosclerotic plaque-specific in vivo imaging contrast agents.

[0053] Furthermore, the application also includes: 1) Application in inhibiting the expression of DKK1 in vascular endothelial cells and / or inhibiting the apoptosis of vascular endothelial cells.

[0054] Furthermore, the vascular endothelial cells include HUVECs, HCMECs, HMECs, and Eahy926.

[0055] Furthermore, the vascular endothelial cells are HUVECs.

[0056] Furthermore, the cell apoptosis is oxidation-induced cell apoptosis.

[0057] Furthermore, the oxidation-induced cell apoptosis is Ox-LDL-induced cell apoptosis.

[0058] 2) Application in inhibiting ROS production in macrophages, inhibiting macrophage polarization and / or inhibiting macrophage phagocytosis of Ox-LDL.

[0059] Furthermore, the macrophages include RAW264.7 and THP-1.

[0060] Furthermore, the macrophages are RAW264.7.

[0061] Furthermore, the inhibiting of macrophage polarization refers to inhibiting macrophage M1 polarization.

[0062] Furthermore, the inhibiting macrophage phagocytosis of Ox-LDL also includes inhibiting foam cell formation.

[0063] Furthermore, the ROS generation is induced by lipopolysaccharide and interferon-γ.

[0064] Furthermore, the macrophage M1 polarization is macrophage M1 polarization induced by lipopolysaccharide and interferon γ.

[0065] Advantages and beneficial effects of the present invention: (1) E / A-Mn 2+ @D NPs have good relaxation values ​​and can be used as AS plaque-specific MRI imaging enhancers; (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; (3) E / A-Mn 2+ @D NPs have good biosafety for long-term intervention; (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

[0066] Figure 1 is the particle size distribution diagram of nanomedicine. Figure 1 A in the figure is the particle size and potential distribution diagram 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 E / A-Mn 2+ @D Particle size and potential distribution of NPs.

[0067] Figure 2 E / A-Mn 2+ @D UV absorption spectrum of NPs.

[0068] Figure 3 E / A-Mn 2+ @D Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of NPs.

[0069] 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).

[0070] Figure 5 E / A-Mn 2+ @D NPs HUVECs, RAW264.7 cell toxicity test chart, Figure 5 A in the graph is different concentrations of E / A-Mn 2+ @D NPs and HUVECs cells were incubated for 12 h. Figure 5 B in the figure is different concentrations of E / A-Mn 2+ @D NPs and HUVECs cells were incubated for 24 h. Figure 5 C in the graph is different concentrations of E / A-Mn 2+ @D NPs and RAW264.7 cells were incubated 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.

[0071] Figure 6 The expression of DKK1, ICAM-1 and VCAM-1 in HUVECs cells after different treatments was detected by WB.

[0072] Figure 7 CLSM detection of E / A-Mn 2+ @D-Cy5 NPs binding to HUVECs cells in vitro.

[0073] Figure 8 E / A-Mn 2+ @D Effects of NPs on the production in RAW264.7 cells (A) and quantitative analysis (B).

[0074] Fig. 9 Figure 2 shows flow cytometry detection of M1 polarization of RAW264.7 cells after different treatments (A) and quantitative analysis (B).

[0075] Fig.10 Figure 2 shows flow cytometry detection of HUVECs apoptosis after different treatments (A) and quantitative analysis (B).

[0076] Fig.11 Observation of E / A-Mn by CLSM 2+ @D Graph showing the inhibitory effect of NPs on the phagocytosis of Ox-LDL by RAW264.7 cells.

[0077] Fig.12 Oil red O staining shows the effects of different treatments on foam cell formation.

[0078] Fig.13 Detection of E / A-Mn for WB 2+ @D Graph showing the effects of NPs on the expression of proteins related to the Wnt pathway, apoptosis, and lipophagy.

[0079] Fig.14 ApoE - / - MRI targeted imaging (AB) and in vivo imaging (CD) of the mouse abdominal aorta.

[0080] Fig.15 For 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.

[0081] Fig.16 E / A-Mn 2+ NPs and different doses of E / A-Mn 2+ @D HE, CD68, MMP-9, α-SMA, Masson, and DKK1 staining (A) and quantitative analysis (BG) of the aortic root of mice 8 weeks after NPs treatment. DETAILED DESCRIPTION

[0082] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvement by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0083] 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 be passaged 50 to 60 times in theory.

[0084] RAW 264.7 cells are derived from tumors induced by Abelson murine leukemia virus; in normal culture, the cells appear polygonal and irregular in shape, accompanied by the formation of many pseudopodia, and have a strong ability to adhere to the wall. RAW 264.7 cells are immune cells with multiple functions and are an important subject for studying phagocytosis, cellular immunity and molecular immunology.

[0085] The MTT assay, also known as the MTT colorimetric assay, is a method for detecting cell survival and growth. The detection principle is that the succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystalline formazan and deposit it in the cells, while dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve the formazan in the cells, and its light absorbance is measured at a wavelength of 490nm using an enzyme-linked immunosorbent assay, which can indirectly reflect the number of living cells. Within a certain range of cell numbers, the amount of MTT crystals formed is proportional to the number of cells. Exogenous MTT refers to Thiazolyl Blue, a cell-permeable and positively charged tetrazolium dye used to detect the reductive metabolism of cells. Thiazolyl Blue is absorbed by cells through the plasma membrane and then reduced to formaldehyde by the NAD (P) H oxidoreductase in the cells. It is often used in colorimetric assays to measure cell proliferation, cytotoxicity, and apoptosis.

[0086] The present invention is further described in detail below in conjunction with the accompanying drawings and examples. The experimental methods in the following examples are conventional methods unless otherwise specified, and the materials and reagents used in the following examples are commercially available unless otherwise specified. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Simple improvements to the present invention made according to the essence of the present invention all belong to the scope of protection claimed in the present invention.

[0087] Example 1 Preparation of Nanomedicine for Targeted Diagnosis and Treatment of Atherosclerotic Plaques 1. Experimental Materials Epigallocatechin gallate (EGCG 95%) (Dalian Meilun, MB1672-2), arginine (Arg) (B&K, 235303), aqueous formaldehyde solution (formaldehyde 37-40%, Sinopharm, 10010018), manganese chloride, tetrahydrate (McLean, M813488), Human / Mouse Dkk-1 Antibody (R&D, AF1096), Cy5 NHS eater (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 (Solera, M1020), diphenylpicrohydrazine (DPPH) (Sigma, USA, R712787), Oil Red O dye (Sigma, USA, O8010).

[0088] 2. Experimental Methods (1) Preparation of nanomedicine E / A-Mn 2+ @D NPs Weigh 22.875 mg EGCG and 7.5 μl formaldehyde aqueous solution and dissolve them in 10 ml triple distilled water, and stir to mix thoroughly at 30°C. Then add 5.79 mg L-Arg and continue to stir and react at 30°C for 5 min. Collect the nanoparticles, centrifuge at 3.5×g for 5 min at room temperature, discard the supernatant, purify with triple distilled water three times, and then resuspend the nanoparticles in 80 ml triple distilled water. Add MnCl2·4H2O solution (Mn 2+ , 2 mg / ml), mix and incubate at room temperature overnight. Collect the nanoparticles, centrifuge at 3.5×g for 5 min at room temperature, discard the supernatant, purify three times with triple distilled water, and then resuspend the nanoparticles in 10 ml triple distilled water. Add Human / Mouse Dkk-1 Antibody (1 μg / μl) to the nanoparticle solution at 10 μl / ml, mix and incubate at 4°C overnight. Collect the nanoparticles, centrifuge at 3.5×g for 5 min at room temperature, discard the supernatant, purify three times with triple distilled water, and then resuspend the nanoparticles in 10 ml triple distilled water and store in a 4°C refrigerator for use.

[0089] (2) Cytotoxicity of Nanomedicines HUVECs cells were prepared with ECM culture medium to 5 × 10 4 RAW264.7 cells were prepared into 1×10 cells / ml cell suspension using DMEM medium containing 10% FBS. 5 Cell suspension of 100 cells / ml was prepared. Overnight culture was allowed 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+ @D NP working solution, continue to culture for 12 and 24 h. Aspirate the supernatant and wash with PBS three times. Add 90 μl fresh culture medium and 10 μl MTT solution and continue to culture for 4 h. Aspirate the supernatant, add 110 μl Formazan solution to each well, and shake at low speed on a shaker for 10 min to fully dissolve the crystals. Measure the absorbance of each well at 490 nm using an enzyme-linked immunosorbent assay.

[0090] (3) Effect of nanomedicine on dickkopf-1 secretion by HUVECs stimulated by LPS Preparation of 1 × 10 5 HUVECs cell suspension was prepared at 1 μg / mL and cultured overnight to allow the cells to adhere to the wall. 1 μg / mL LPS was prepared in fresh ECM medium. After that, fresh ECM medium, 1 μg / mL LPS, 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.

[0091] (4) In vitro binding of nanodrugs to LPS-stimulated HUVECs Preparation of 1 × 10 4 HUVECs cell suspension was prepared at 100 μg / ml and cultured overnight to allow cells to adhere to the wall. Fresh ECM medium was used to stimulate cells with 1 μg / mL LPS for 24 h. 2+ -Cy5 NPs, different doses (50, 100 μg / ml) of E / A-Mn 2+ @D-Cy5 NPs were used to treat cells for 1 h. The cells were washed with PBS for 3 times and then fixed with 4% paraformaldehyde at 37°C for 15 min. The cells were washed with PBS for 3 times, the cell membranes were stained with 5 μg / ml WGA-488, the cell nuclei were counterstained with hoechst33342 at room temperature for 3 min, the cells were washed with PBS for 3 times, and finally the cells were sealed with anti-fluorescence quenching sealing agent. CLSM observation and photography were performed, and the images were processed with the dedicated software Zen.

[0092] (5) Nanomedicine inhibits the production of ROS in macrophages Preparation of 5 × 10 4 The cells were suspended in a suspension of RAW264.7 cells at 100 μg / ml and cultured overnight to allow the cells to adhere to the wall. The control group continued to be cultured 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+ @D NPs were used to treat cells. After 24 h, cells were cultured in serum-free medium containing 10 μM DCFH-DA for another 30 min. The cells were washed with PBS three times, and the ROS fluorescence intensity was quantified by microplate reader and observed and photographed by CLSM.

[0093] (6) Nanomedicine regulates macrophage polarization Preparation of 1 × 10 6 The cells were cultured overnight in a suspension of RAW264.7 cells at 1 μg / ml and allowed to adhere to the wall. The control group continued to be cultured 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+@D NPs were used to treat cells. After 24 h, single cell suspension was collected and cells were washed with PBS. Cells were incubated with FITC-labeled CD86 flow cytometry antibody (5 μg / 106 cells) and APC-labeled CD11b flow cytometry antibody (1.5 μl / 106 cells) at 4°C for 30 min. Finally, cells were resuspended in 500 μl pre-cooled PBS and tested.

[0094] (7) Nanomedicine inhibits oxidation-induced cell apoptosis Preparation of 1 × 10 5 The control group continued to be cultured 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+ @D NPs were used to treat cells. After 24 h, single cell suspension was collected and cells were washed with PBS. Cells were resuspended with 100 μl Buffer, and then 5 μl of FITC and PI were added and incubated for 15 min in the dark. Finally, 400 μl of Buffer was added to terminate the reaction and the cells were tested within 1 h.

[0095] (8) Nanomedicine inhibits macrophage phagocytosis of Ox-LDL ① CLSM observation of E / A-Mn 2+ @D NPs inhibit the phagocytosis of Dil-oxLDL by RAW264.7 cells Preparation of 5 × 10 4 The cells were cultured overnight in a suspension of RAW264.7 cells at 1 μg / ml. The control group was cultured with fresh DMEM high-glucose medium, and the model group and rescue group were first stimulated with 1 μg / ml LPS for 24 h. Then the model group continued to culture cells with 10 μg / ml Dil-oxLDL, and the rescue group was cultured with 10 μg / ml Dil-oxLDL and different doses (50, 100 μg / ml) of E / A-Mn 2+ @D NPs were used to culture cells. After 4 h, the cells were washed 3 times with PBS and then fixed with 4% paraformaldehyde at 37°C for 15 min. The cells were washed 3 times with PBS, the cell membrane was stained with 5 μg / ml WGA-488, the cell nucleus was counterstained with DAPI at room temperature for 3 min, the cells were washed 3 times with PBS, and finally the cells were sealed with anti-fluorescence quenching sealing agent. CLSM observation and photography were performed, and the images were processed with the dedicated software Zen.

[0096] ②Observation of E / A-Mn by Oil Red Staining 2+ @D NPs inhibit foam cell formation Preparation of 5 × 104 The cells were cultured overnight in a suspension of RAW264.7 cells at 1 μg / ml and allowed to adhere to the wall. The control group was cultured with fresh DMEM high-glucose medium, and the model group and rescue group were first stimulated with 1 μg / ml LPS for 24 h. Then 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, 10 μg / ml) of E / A-Mn 2+ @D NPs treated cells. After 48 h, the cells were washed 3 times with PBS and fixed with 4% paraformaldehyde at 37°C for 15 min. Washed 3 times with PBS, added Oil Red O working solution to cover the slides, and stained for 30 min at room temperature in the dark. Discarded the staining solution, washed 3 times with PBS, and then sealed with glycerol gelatin. Photographed using an electric upright microscope.

[0097] (9) Detection of Wnt pathway proteins and apoptosis and lipophagy-related proteins HUVECs and RAW264.7 cells were transferred to a cell culture medium with a density of 1×10 5 / 5×10 4 Cells were cultured in a 6-well plate with 100 cells / ml per well in a 37°C cell culture incubator overnight. Then, stimulation was given according to the different experiments described above. After stimulation, proteins were collected and the protein concentration was determined using a BCA protein detection 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 TBST solution containing 5% skim milk for 1 hour. Primary antibodies for 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 were incubated overnight on a shaker at 4°C. Finally, Western blot results were obtained using a gel imaging system. GAPDH was used as an internal reference antibody.

[0098] (10) The value of nanomedicine in targeted imaging of atherosclerotic plaques ① In vivo observation of E / A-Mn 2+ @D NPs imaging at the AS site in mice The ApoE model was successfully established - / - The 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 scanning was performed before and 24 h after administration. ApoE- / - The chest and abdomen of the mouse were placed in the scanning area. ApoE was MRI scanned using the body coil of a 9.4 T small animal MRI system (Biospec 94 / 20 USR, Bruker, Germany). - / - The abdominal aorta of the model rats was imaged by MRI. The T1WI sequence parameters were as follows: TR=1228 ms, TE=8.6 ms, FOV 31 mm×29 mm, matrix 256×256, and layer thickness 0.7 mm. The scanned images were analyzed using Image J software to measure the signal intensity SI (wall) value of the abdominal aorta wall, the signal intensity SI (muscle) value of the muscle tissue around the wall, and the background noise signal SI (noise) value outside the tissue. The CNR (contrast-to-noise) value of each layer of the image was calculated: The calculation formula is as follows: CNR = (SI (wall) -SI (muscle)) / SI (noise) According to the CNR values ​​calculated by the above method, the changes in the CNR values ​​of the images before and after the injection of the nanomedicine were compared layer by layer, and the change rate of the CNR value was calculated: The calculation formula is as follows: CNR changes % = (CNR24hpost–CNRpre) / CNRpre × 100% ② In vivo imaging observation of E / A-Mn 2+ Distribution of @D-Cy5 NPs in mouse aorta Each group of mice was injected with 10 mg / kg E / A-Mn by tail vein. 2+ @D-Cy5 NPs (the control group was injected with the same dose of saline). 2+ The mice were killed 3 and 12 hours after @D-Cy5 NPs, fully perfused, and the heart and aorta were immediately separated to the iliac artery bifurcation. After weighing, the fluorescence intensity was measured under in vivo imaging, and the average fluorescence intensity per unit volume / mass was calculated.

[0099] (11) Verification of anti-AS effect in vivo 6-8 weeks 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 anti-AS effect of nanomedicine in vivo. Samples were collected after 8 weeks of treatment, and the aorta was retained for staining and analysis of plaque area and stability.

[0100] 3. Experimental Results (1) E / A-Mn 2+ @D NPs Characterization The particle size of nanodrugs 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; E / A-Mn 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 ).

[0101] (2) Cytotoxicity of Nanomedicines 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.

[0102] (3) Effect of nanomedicine on dickkopf-1 secretion by HUVECs stimulated by LPS After 24 h of stimulation with 1 μg / ml LPS, the levels of ICAM-1 and VCAM-1 in HUVECs were significantly higher than 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 ).

[0103] (4) In vitro binding of nanodrugs to LPS-stimulated HUVECs First, HUVECs were stimulated with LPS to secrete DKK1, and the Con group and E / A-Mn 2+ -Cy5 NPs group did not detect Cy5 signal. 2+ @D-Cy5 NPs group detected different degrees of Cy5 signals, and there was a certain dose dependence. Figure 7 It shows that E / A-Mn not conjugated to Human / Mouse DKK-1 antibody 2+ -Cy5 NPs could not bind to HUVECs cells, while E / A-Mn coupled to anti-Human / Mouse DKK-1 antibody 2+ @D-Cy5 NPs can specifically bind to HUVECs cells.

[0104] (5) Nanomedicine inhibits the production of ROS in macrophages After LPS+IFN-γ stimulation, ROS generation 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.

[0105] (6) Nanomedicine regulates macrophage polarization 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 mitigating effect on M1 polarization of RAW264.7 cells after LPS+IFN-γ stimulation ( Fig. 9).

[0106] (7) Nanomedicine inhibits oxidation-induced cell apoptosis HUVECs have a low natural apoptosis rate. After stimulation with 50 µg / mL Ox-LDL, the apoptosis rate of HUVECs increased significantly to 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 ( Fig.10 ).

[0107] (8) Nanomedicine inhibits macrophage phagocytosis of Ox-LDL ① CLSM observation of E / A-Mn 2+ @D NPs inhibit the phagocytosis of Dil-oxLDL by RAW264.7 cells 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 of RAW264.7 cells in the model group was the strongest. 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 for Ox-LDL, while E / A-Mn 2+ @D NPs have a significant inhibitory effect on this phagocytic ability ( Fig.11 ).

[0108] ②Observation of E / A-Mn by Oil Red Staining 2+ @D NPs inhibit foam cell formation Fig.12 The results show that using E / A-Mn 2+ @D NPs treated cells significantly reduced intracellular lipid droplets, and 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 the formation of foam cells.

[0109] (9) Detection of Wnt pathway proteins and apoptosis and lipophagy-related proteins Western blotting detected the expression of β-catenin and p-β-catenin, reflecting the E / A-Mn 2+@D NPs activated the Wnt pathway. The results showed that the expression of β-catenin in the Model group was reduced, and the expression of E / A-Mn 2+ @D β-catenin expression increased after NPs intervention ( Fig.13 A in the figure). In order to better understand the activation of Wnt, apoptosis and lipophagy after treatment were also studied. We used Western blotting to evaluate the expression of Bax, Bcl2, Caspase3, CleavedCaspase3, Lox-1, SRA, CD36, SR-BI, ABCA1, and ABCG1. Compared with the control group, the Bax / Bcl2 and CleavedCaspase3 / Caspase3 ratios in the Model group were increased, the expression of Lox-1, SRA, and CD36 was increased, and the expression of SR-BI, ABCA1, and ABCG1 was decreased. 2+ @D After NPs intervention, the ratios of Bax / Bcl2 and Cleaved Caspase3 / Caspase3 decreased ( Fig.13 B), the expression of Lox-1, SRA, and CD36 decreased ( Fig.13 C in the figure), the expression of SR-BI, ABCA1, and ABCG1 increased ( Fig.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.

[0110] (10) The value of nanomedicine in targeted imaging of atherosclerotic plaques ① In vivo observation of E / A-Mn 2+ @D NPs imaging at the AS site in mice After injection of E / A-Mn2+ NPs or E / A-Mn 2+ @D NPs before ApoE - / - The abdominal aorta of mice showed thickening of the vascular wall and formation of intravascular plaques. 2+ @D NPs 24 hours later, the T1 signal of the abdominal aorta wall was significantly enhanced, with a statistically significant difference compared with before injection ( Fig.14 AB in the injection). While injection of E / A-Mn 2+ ApoE in NPs - / - Although the T1 signal of the mouse abdominal aorta wall was also enhanced compared with the previous one, it did not reach statistical significance ( Fig.13 AB in the figure). From the quantitative statistics of CNR, it can be seen that the injection of E / A-Mn 2+ The CNR 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-Mn 2+ NPs do not have this capability.

[0111] ② In vivo imaging observation of E / A-Mn 2+ Distribution of @D-Cy5 NPs in mouse aorta saline group C57BL6 and ApoE - / - There was no obvious Cy5 fluorescence in the mice. C57BL6 mice were injected with E / A-Mn 2 + @D-Cy5 NPs: Low-dose Cy5 fluorescence intensity was observed in some areas 3 hours after administration, and the Cy5 fluorescence intensity was enhanced after 12 hours. ApoE in AS plaque modeling was successful - / - Tail vein injection of E / A-Mn 2+ @D-Cy5 NPs also showed weak Cy5 fluorescence after 3 hours, 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), and ApoE - / - The fluorescence intensity of the aorta of mice was significantly higher than that of C57BL6 mice (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 ( Fig.14 in the CD).

[0112] (11) Verification of anti-AS effect in vivo 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. Fig.15 ). The stability of AS plaques was evaluated by staining the aortic root plaques. 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 the extracellular matrix, and increased collagen in the plaques, playing an important role in resisting the development of AS and stabilizing the plaques ( Fig.16).

[0113] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

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; Preferably, the components of the nanomedicine also include Cy5.

2. The nano drug according to claim 1, characterized in that The particle size of the nano drug 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 ; Preferably, the nanomedicine is spherical, uniform in size and has good dispersibility.

3. A pharmaceutical composition for targeted diagnosis and treatment of atherosclerotic plaques, characterized in that: The pharmaceutical composition comprises the nanodrug according to claim 1 or 2; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

4. A method for preparing the nano drug according to claim 1 or 2, 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; Preferably, the method further comprises: adding Cy5.

5. The method according to claim 4, characterized in that 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; Preferably, the mass ratio of epigallocatechin gallate to L-arginine is 3.95:1; Preferably, the stirring and mixing condition is 30°C; Preferably, the stirring reaction conditions are 30°C, 5 min; The addition of Mn 2+ The overnight incubation step includes: adding Mn to the nanoparticle solution 2+ Incubate overnight and purify and resuspend to obtain the Mn 2+ Solutions of nanoparticles; Preferably, the Mn 2+ The final concentration was 20 μg / ml; Preferably, the Mn 2+ Selected from MnCl2·4H2O solution; Preferably, the addition of Mn 2+ Overnight incubation conditions were room temperature; The step of adding DKK1 antibody and incubating overnight comprises: adding 2+ Adding DKK1 antibody to the solution of nanoparticles and incubating overnight, purifying and resuspending to obtain the nanodrug according to claim 1 or 2; Preferably, the final concentration of the DKK1 antibody is 10 μg / ml; Preferably, the condition for adding DKK1 antibody and incubating overnight is 4°C; Preferably, the purification and resuspension refers to purification and resuspension with triple distilled water after centrifugation; Preferably, the centrifugation conditions are room temperature, 3.5×g, 5 min.

6. A method for inhibiting the expression of DKK1 in vascular endothelial cells and / or inhibiting apoptosis of vascular endothelial cells, characterized in that: The method comprises treating vascular endothelial cells with the nanodrug according to claim 1 or 2; Preferably, the vascular endothelial cells include HUVECs, HCMECs, HMECs, and Eahy926; Preferably, the vascular endothelial cells are HUVECs; Preferably, the apoptosis is oxidation-induced apoptosis; Preferably, the oxidation-induced cell apoptosis is Ox-LDL-induced cell apoptosis.

7. A method for inhibiting ROS production in macrophages, inhibiting macrophage polarization and / or inhibiting macrophage phagocytosis of Ox-LDL, characterized in that: The method comprises treating macrophages with the nanodrug of claim 1 or 2; Preferably, the macrophages include RAW264.7 and THP-1; Preferably, the macrophages are RAW264.

7.

8. The method according to claim 7, characterized in that The inhibition of macrophage polarization refers to inhibition of macrophage M1 polarization; Preferably, the inhibiting macrophage phagocytosis of Ox-LDL further comprises inhibiting foam cell formation; Preferably, the ROS production is lipopolysaccharide and interferon gamma induced ROS production; Preferably, the macrophage M1 polarization is lipopolysaccharide and interferon γ-induced macrophage M1 polarization.

9. Use of the nano drug according to claim 1 or 2 or the pharmaceutical composition according to claim 3, 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.

10. The use according to claim 9, characterized in that: The application also includes: 1) Application in inhibiting the expression of DKK1 in vascular endothelial cells and / or inhibiting the apoptosis of vascular endothelial cells; Preferably, the vascular endothelial cells include HUVECs, HCMECs, HMECs, and Eahy926; Preferably, the vascular endothelial cells are HUVECs; Preferably, the apoptosis is oxidation-induced apoptosis; Preferably, the oxidation-induced apoptosis is Ox-LDL-induced apoptosis; 2) Application in inhibiting ROS production in macrophages, inhibiting macrophage polarization and / or inhibiting macrophage phagocytosis of Ox-LDL; Preferably, the macrophages include RAW264.7 and THP-1; Preferably, the macrophages are RAW264.7; Preferably, the inhibiting macrophage polarization refers to inhibiting macrophage M1 polarization; Preferably, the inhibiting macrophage phagocytosis of Ox-LDL further comprises inhibiting foam cell formation; Preferably, the ROS production is lipopolysaccharide and interferon gamma induced ROS production; Preferably, the macrophage M1 polarization is lipopolysaccharide and interferon γ-induced macrophage M1 polarization.

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