Nanometer diagnosis and treatment agent capable of monitoring AS disease course

Abstract

The invention discloses a nano diagnosis and treatment agent capable of monitoring the AS disease course. The diagnosis and treatment agent is prepared from phospholipid, auxiliary lipid, antioxidant nano enzyme, a fluorescent probe compound, a loaded drug and an LDL adsorption functional polymer. According to the AS nano diagnosis and treatment agent, efficient endothelial barrier penetration is achieved by taking LDL, a traditional passive penetration mode depending on the ELVIS effect is broken through, and the plaque penetration efficiency of a fluorescent probe and a treatment medicine is improved; a disease course-related plaque specific imaging system is constructed by taking the migration rate of LDL to the interior and subcutaneous tissues as a dynamic indication, and accurate monitoring of the AS development stage is realized; the antioxidant nano-enzyme with mimic enzyme activity is adopted as a core, active oxygen is removed, oxidation of a key pathogenic factor LDL is inhibited, and the function of slowing down AS progress is achieved. The invention provides a new strategy for realizing the integration of disease course monitoring and diagnosis and treatment of the AS.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and in particular relates to a nano-diagnostic and therapeutic agent capable of monitoring the course of AS. Background Art

[0002] Atherosclerosis (AS) is a chronic inflammatory disease characterized by endothelial cell dysfunction, inflammatory responses, lipid metabolism disorders, and oxidative stress, primarily affecting the cardiovascular and cerebrovascular systems. Notably, AS develops insidiously and suddenly without warning, often leading to late-stage disease detection, with plaques on the verge of rupture and a high risk of acute cardiovascular events such as ischemic stroke and coronary heart disease. Therefore, developing a precise diagnostic and treatment approach for AS that combines disease progression monitoring with treatment is crucial.

[0003] In the prior art, molecular imaging technology, as a non-invasive diagnostic technology, can detect abnormalities before the disease has organic lesions, providing an effective means for the diagnosis of AS. Among them, fluorescence imaging is widely used in the diagnosis of AS due to its high sensitivity. For example, Chinese patent CN116836188A discloses a fluorescent probe for detecting oxidized low-density lipoprotein (ox-LDL), which is delivered into macrophages for early detection of AS. Another example is Chinese patent CN111956610B, which discloses a nano-drug carrier for the diagnosis and treatment of atherosclerosis, which is composed of an active oxygen-responsive dicarbonyl-bonded fluorescent molecule and a cyclodextrin loaded with drugs in the lumen. It can enter the plaque with the help of the extravasation of damaged blood vessels, and responsively release fluorescent molecules and drugs, thereby achieving the diagnosis and treatment of AS lesions. Despite the presence of extravasation through leaky vasculature and inflammatory cell-mediated sequestration (ELVIS) at the plaque site, macrophages reside beneath the endothelial barrier, and the plaque structure is complex, covered by a fibrous cap composed of a collagen-rich matrix and proliferating smooth muscle cells. Therefore, the ELVIS effect at the plaque site significantly limits the penetration efficiency of fluorescent probes that rely solely on passive diffusion, significantly limiting diagnostic sensitivity. Furthermore, because the size of the endothelial gap is not positively correlated with the course of AS, passive diffusion-based fluorescent probe imaging is difficult to accurately reflect the progression of the disease and cannot achieve real-time monitoring of the AS course.

[0004] Endothelial cell dysfunction is the initiating link in the development and progression of AS. Circulating low-density lipoprotein (LDL) accumulates at the lesion site, continuously infiltrates into the subendothelial matrix, and is oxidatively modified by subendothelial reactive oxygen species (ROS) to form ox-LDL. Ox-LDL stimulates endothelial cells to secrete large amounts of adhesion molecules (such as VCAM-1) and chemokines (such as MCP-1), recruiting circulating monocytes to the site of injury. These monocytes migrate and infiltrate the subendothelium, further differentiating into macrophages. Macrophages continuously phagocytize accumulated ox-LDL to form foam cells. Excessive lipid accumulation triggers cell necrosis, ultimately forming fibrous plaques. Furthermore, studies have shown that LDL crosses the endothelial barrier primarily through active transport mechanisms during the pathological process of AS. The above research background suggests that nano-diagnostic and therapeutic agents can be delivered across the endothelium by riding on LDL, improving the migration efficiency across the endothelial barrier through active transport that is independent of the ELVIS effect. At the same time, combined with the characteristics that LDL migration and accumulation under the vascular endothelium are positively correlated with the disease progression of AS, dynamic monitoring of the AS disease progression can be achieved.

[0005] In current research, strategies to treat AS by scavenging ROS and alleviating inflammation have received widespread attention. For example, small molecule antioxidant drugs such as probucol, vitamin C, and vitamin E, however, due to their nonspecific distribution and rapid clearance in the body, they struggle to achieve effective concentrations locally in plaques, limiting their therapeutic efficacy in treating AS. The emergence of antioxidant nanozymes with ROS-scavenging capabilities offers new avenues for the treatment of AS. These functional nanozymes possess activities similar to those of natural antioxidant enzymes (such as SOD, CAT, and GPx). They possess highly efficient ROS-scavenging abilities, can improve the oxidative stress microenvironment in plaques, inhibit LDL oxidation, and slow AS progression.

[0006] The present invention prepares a nano-diagnostic agent capable of monitoring the course of AS by screening a series of LDL adsorption functional polymers, assembling them with antioxidant nanozymes, phospholipids, auxiliary lipids, fluorescent probe complexes and drugs with anti-AS functions. The nano-diagnostic agent uses LDL adsorption functional polymers and auxiliary lipids to ride on LDL to achieve active transport across the endothelial barrier and improve the efficiency of plaque penetration; uses the fluorescent probe complex to perform imaging diagnostic functions, uses the migration rate of LDL to the subendothelium as a monitoring marker, and establishes plaque-specific imaging directly related to the course of AS; uses antioxidant nanozymes to clear ROS in the microenvironment, blocks the vicious cycle of "oxidative stress-inflammatory response", intervenes in LDL, a key pathogenic factor of AS, and inhibits LDL oxidation; and synergizes antioxidant nanozymes with anti-AS drugs to achieve multi-effect treatment of AS. In summary, the present invention proposes an innovative integrated strategy for monitoring the course of AS and diagnosis and treatment. Summary of the Invention

[0007] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art and provide a nano-diagnostic and therapeutic agent that can monitor the course of AS. The nano-diagnostic and therapeutic agent penetrates the endothelial barrier independently of the ELVIS effect and has high penetration efficiency. The nano-diagnostic and therapeutic agent uses the subendothelial mobility of LDL as a marker to achieve course-related plaque imaging. It utilizes antioxidant nanoenzymes to inhibit LDL oxidation and cooperates with anti-AS drugs to achieve a therapeutic effect on AS while imaging.

[0008] Another object of the present invention is to provide an application of the highly efficient penetrating nano-diagnostic and therapeutic agent.

[0009] The present invention achieves the above-mentioned object through the following scheme:

[0010] In the first aspect, a nano-diagnostic and therapeutic agent capable of detecting the course of AS is provided, wherein the nano-diagnostic and therapeutic agent is composed of 10 to 30 parts by weight of phospholipids, 20 to 40 parts by weight of auxiliary lipids, 5 to 15 parts by weight of antioxidant nanoenzymes, 0.1 to 10 parts by weight of fluorescent probe complex A-PEG-B, 0 to 10 parts by weight of loaded drugs, and 30 to 50 parts by weight of LDL adsorption functional polymer DSPE-PEG-C; in the fluorescent probe complex, A is a fluorescent dye, PEG is polyethylene glycol 2000, and B is a phospholipid; in the LDL adsorption functional polymer DSPE-PEG-C, C is an LDL adsorption material, and DSPE refers to 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine.

[0011] Specifically, diagnosis and treatment refers to diagnosis, auxiliary diagnosis, monitoring, auxiliary monitoring, screening or auxiliary screening, treatment and / or auxiliary treatment, etc., all of which are within the scope of protection of the present invention.

[0012] Furthermore, the nano-diagnostic and therapeutic agent consists of 15 to 25 parts by weight of phospholipids, 25 to 35 parts by weight of auxiliary lipids, 8 to 12 parts by weight of antioxidant nanoenzymes, 0.5 to 2 parts by weight of fluorescent probe complex A-PEG-B, 0 to 5 parts by weight of loaded drugs, and 30 to 40 parts by weight of LDL adsorption functional polymer DSPE-PEG-C.

[0013] Furthermore, the phospholipid content in the nano-diagnostic and therapeutic agent may be 15 to 18 parts by weight, 17 to 22 parts by weight, or 19 to 23 parts by weight.

[0014] Furthermore, the auxiliary lipid in the nano-diagnostic and therapeutic agent can be 25 to 28 parts by weight, 27 to 32 parts by weight, or 29 to 35 parts by weight.

[0015] Furthermore, the antioxidant nanozyme in the nano-diagnostic and therapeutic agent is 8 to 11 parts by weight or 9 to 12 parts by weight.

[0016] Furthermore, the content of the fluorescent probe complex A-PEG-B in the nano-diagnostic and therapeutic agent is 0.8 to 1.1 parts by weight or 0.9 to 1.5 parts by weight.

[0017] Furthermore, the drug loaded in the nano-diagnostic and therapeutic agent can be 0.1 to 2 parts by weight, 2 to 4 parts by weight, or 2.5 to 5 parts by weight.

[0018] Furthermore, the LDL adsorption functional polymer DSPE-PEG-C in the nano-diagnostic and therapeutic agent is 35 to 38 parts by weight or 37 to 39 parts by weight.

[0019] Furthermore, the nano-diagnostic and therapeutic agent consists of 20 parts by weight of phospholipids, 29 parts by weight of auxiliary lipids, 10 parts by weight of antioxidant nanoenzymes, 1 part by weight of fluorescent probe complex A-PEG-B, 2.5 parts by weight of loaded drugs, and 37.5 parts by weight of LDL adsorption functional polymer DSPE-PEG-C.

[0020] Furthermore, the phospholipids constituting the nano-diagnostic and therapeutic agent are any one or more of soybean lecithin (SPC), 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycerol-3-phosphatidylethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine (DPPE), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycerol-3-phosphatidylglycerol (DSPG), 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylglycerol (DPPG), and 1,2-dioleoyl-sn-glycerol-3-phosphatidylglycerol (DOPG).

[0021] Furthermore, the auxiliary lipid is any one or more of cholesterol, 1,2-distearoyl-sn-glycerol-3-phosphatidylethanolamine-polyethylene glycol (DSPE-PEG), 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine-polyethylene glycol (DPPE-PEG), and 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol (DOPE-PEG).

[0022] Furthermore, the antioxidant nanozyme is any one of manganese dioxide nanozyme, cuprous oxide nanozyme, cerium oxide nanozyme, polydopamine (PDA), polytannic acid (PTA), Prussian blue nanozyme, and graphene nanozyme.

[0023] The present invention uses the efficient scavenging ability of antioxidant nanoenzymes on ROS to inhibit the oxidation of LDL in the lesion microenvironment, block the vicious cycle of "oxidative stress-inflammatory response", and achieve the purpose of treating AS.

[0024] Furthermore, in the fluorescent probe complex A-PEG-B, A is a fluorescent dye, PEG is polyethylene glycol 2000, and B is a phospholipid. The molar ratio of A, PEG, and B in the fluorescent probe complex A-PEG-B is 1:1:1.

[0025] Furthermore, A is any one of pentamethine cyanine dye (Cy5), heptamethine cyanine dye (Cy7), indocyanine green (ICG), fluorescein isothiocyanate (FITC), dihydrochlorin (Ce6), and rhodamine B (RB); B is any one of DSPE, DPPE, and DOPE.

[0026] Furthermore, the loaded drug is a drug with anti-AS function, such as atorvastatin, probucol, rapamycin, curcumin, vitamin E, aspirin, etc., which can be selected according to actual needs; the present invention combines antioxidant nanozymes with anti-AS drugs to achieve multi-effect treatment of AS.

[0027] Furthermore, in the LDL adsorption functional polymer DSPE-PEG-C, PEG is polyethylene glycol 2000 and C is an LDL adsorption ligand; the LDL adsorption ligand is an anionic adsorption ligand or a hydrophobic adsorption ligand or an immune adsorption ligand, such as heparin, glutathione, tryptophan, aspartic acid, glutamic acid, phosphatidylethanolamine, and ApoB-100 antibody.

[0028] The present invention utilizes LDL adsorption ligands to achieve riding on LDL, promoting the nano-diagnostic and therapeutic agent to actively cross the endothelial barrier and improve the efficiency of plaque penetration; and further, along with LDL, it is recognized and phagocytosed by macrophages / foam cells, exerting anti-AS therapeutic effects.

[0029] Furthermore, the LDL adsorption functional polymer DSPE-PEG-C is prepared by amide reaction: DSPE-PEG 2000 -COOH, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS) were dissolved in anhydrous dimethyl sulfoxide (DMSO) in a molar ratio of 1:4:7 and activated under magnetic stirring at room temperature for 1 hour. Then, an excess of LDL adsorption ligand was added, and the reaction was continued under magnetic stirring at room temperature for 24 hours. The crude product was transferred to a dialysis bag (molecular weight cutoff of 3500Da) and dialyzed with deionized water for 24 hours to remove free EDC, NHS, DMSO, etc., and then lyophilized to obtain the final product DSPE-PEG-C.

[0030] In a second aspect, the present invention provides a method for preparing the nano-diagnostic and therapeutic agent described above, the method specifically comprising the following steps:

[0031] (1) Phospholipids, auxiliary lipids, fluorescent probe complexes, loaded drugs and antioxidant nanozymes were dissolved in chloroform, magnetically stirred at room temperature for 0.5 to 5 hours, transferred to a round-bottom flask, and vacuum evaporated at 37 to 60°C for 0.5±0.1 hours to form a uniform film; deionized water was added to the round-bottom flask, and the mixture was placed in a rotary evaporator for 0.5±0.1 hours. Ultrasonic probe was used at a power of 100 to 400 W for 5 to 10 minutes, and the mixture was extruded through a membrane 1 to 10 times to make the particle size uniform;

[0032] (2) The solution obtained in step (1) was mixed with DSPE-PEG-C, and the mixture was incubated at 37° C. and 100 rpm for 4 to 12 hours to obtain the nano-diagnostic and therapeutic agent.

[0033] In a third aspect, the present invention provides the use of the nano-diagnostic and therapeutic agent in the preparation, treatment and monitoring of the course of atherosclerotic cardiovascular disease.

[0034] Preferably, the product is a drug or a system carrying the drug;

[0035] Preferably, the atherosclerotic cardiovascular disease is any one of atherosclerosis, acute coronary syndrome, peripheral arterial atherosclerosis, and coronary heart disease.

[0036] In a fourth aspect, the present invention provides a method for monitoring the course of atherosclerotic cardiovascular disease, which is achieved by administering the aforementioned nano-diagnostic and therapeutic agent to a patient.

[0037] The nano-diagnostic and therapeutic agent exhibits superior LDL binding ability, antioxidant capacity, transendothelial barrier transport efficiency and macrophage foaming inhibition function, and shows imaging diagnostic ability in AS model mice that is positively correlated with the course of the disease.

[0038] Beneficial effects:

[0039] Compared with existing technologies, this invention innovatively proposes a design strategy for nanodiagnostic and therapeutic agents. This strategy significantly improves plaque penetration efficiency by actively transporting LDL across the endothelial barrier. It also leverages the synergistic effects of antioxidant nanoenzymes and anti-AS drugs to achieve multi-effect treatments for AS. Finally, it leverages the migration and accumulation of LDL to enable precise imaging and diagnosis of the AS disease progression. The nanodiagnostic and therapeutic agents described in this invention are characterized by their high plaque penetration and correlation with the disease course, providing a new approach and direction for the precise diagnosis and treatment of AS and promising application prospects.

[0040] Compared with the existing technology, the present invention has the following advantages: the antioxidant nanozyme in the diagnostic and therapeutic agent has good mimetic enzyme activity, can reduce the oxidation of LDL, synergize with anti-AS drugs, and effectively treat AS; the LDL adsorption functional polymers and auxiliary lipids in the diagnostic and therapeutic agent can enable the diagnostic and therapeutic agent to ride on LDL and cross the endothelial barrier through active transport, thereby improving the efficiency of fluorescent probes and therapeutic drugs penetrating plaques and playing a role in precise imaging diagnosis and treatment; the nanodiagnostic and therapeutic agent described in the present invention uses the mobility of LDL as an indicator to perform specific imaging of plaques, and is relevant to the course of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without inventiveness and labor.

[0042] in:

[0043] Figure 1 The particle size diagram and encapsulation efficiency diagram of the nano-diagnostic agent prepared in the embodiment; wherein, Figure 1 Figure A is the particle size diagram; Figure 1 Figure B is the encapsulation efficiency diagram;

[0044] Figure 2 The anti-LDL oxidation ability of the nano-diagnostic and therapeutic agent prepared in the example;

[0045] Figure 3 The adsorption performance of the nano-diagnostic agent prepared in the example on LDL;

[0046] Figure 4 The transendothelial transport behavior of the nano-theranostic agent prepared in Example 2, where # represents p≤0.001 vs. Comparative Example 2;

[0047] Figure 5 The ability of the nano-diagnostic and therapeutic agent prepared in the example to inhibit macrophage foaming in vitro, where * represents p ≤ 0.001 vs. PBS, and # represents p ≤ 0.001 vs. Comparative Example 2;

[0048] Figure 6 The antioxidant effect of the nanodiagnostic and therapeutic agent prepared in the example in the oxidative damage cell model, where * represents p ≤ 0.001 vs. PBS, and # represents p ≤ 0.001 vs. Comparative Example 2;

[0049] Figure 7 The imaging diagnostic ability of the nano-diagnostic and therapeutic agent prepared in the example in atherosclerosis model mice, where * represents p ≤ 0.05 vs. Comparative Example 2;

[0050] Figure 8 The blood lipid levels of the nanodiagnostic and therapeutic agent prepared in the example after treatment in atherosclerosis model mice, where * represents p≤0.001 vs. Saline, # represents p≤0.01 vs. Comparative Example 2; Figure 8 Figure A shows the TG levels in atherosclerosis model mice after treatment; Figure 8 Panel B shows the TCHO level in atherosclerosis model mice after treatment; Figure 8 Figure C shows the LDL-C level in atherosclerosis model mice after treatment; Figure 8 Figure D shows the HDL-C level in atherosclerosis model mice after treatment;

[0051] Figure 9This is the immunofluorescence quantitative level of ROS in the aorta after treatment with the nano-diagnostic and therapeutic agent prepared in the example in atherosclerosis model mice, where * represents p≤0.05 vs. Saline, and # represents p≤0.001 vs. Comparative Example 4. DETAILED DESCRIPTION

[0052] The technical solutions provided by the present invention are described in detail below with reference to specific embodiments. It should be noted that the following embodiments are only used to further explain the present invention and are not intended to limit the scope of protection thereof.

[0053] Example 1

[0054] The LDL adsorption functional polymer DSPE-PEG-tryptophan was prepared by the following steps: 10 μmol of DSPE-PEG 2000 -COOH, 40 μmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), and 70 μmol of N-hydroxysuccinimide (NHS) were dissolved in 2 mL of anhydrous dimethyl sulfoxide (DMSO) and activated with magnetic stirring at room temperature for 1 hour; then 40 μmol of tryptophan was added, and the reaction was continued with magnetic stirring at room temperature under nitrogen protection for 24 hours. The crude product was transferred to a dialysis bag (molecular weight cutoff of 3500 Da) and dialyzed with deionized water for 24 hours to remove free EDC, NHS, DMSO, etc., and then lyophilized to obtain the final product DSPE-PEG-tryptophan.

[0055] 4 mg of DSPE, 5.8 mg of DPPE-PEG, 0.2 mg of DPPE-PEG-Cy5, 0.5 mg of curcumin, and 2 mg of cuprous oxide nanozyme were accurately weighed and dissolved in 2 mL of chloroform. The mixture was magnetically stirred at room temperature for 1 h, transferred to a round-bottom flask, and vacuum evaporated at 55 ° C for 0.5 h to form a uniform film. 3 mL of deionized water was added to the round-bottom flask, placed in a rotary evaporator for 0.5 h, ultrasonicated at 200 W power for 5 min by probe ultrasound, and extruded through a membrane twice to make the particle size uniform. The resulting solution was mixed with 7.5 mg of DSPE-PEG-tryptophan and incubated at 37 ° C at 100 rpm for 8 h to obtain the nano-diagnostic and therapeutic agent.

[0056] Example 2

[0057] The rest is the same as Example 1, except that heparin is used instead of tryptophan to prepare DSPE-PEG-heparin.

[0058] 4 mg of DOPE, 1 mg of cholesterol, 4.8 mg of DSPE-PEG, 0.2 mg of DSPE-PEG-ICG, 0.5 mg of atorvastatin, and 2 mg of Prussian blue nanozyme were accurately weighed and dissolved in 2 mL of chloroform. The mixture was magnetically stirred at room temperature for 0.5 h, transferred to a round-bottom flask, and vacuum evaporated at 45 ° C for 0.5 h to form a uniform film. 3 mL of deionized water was added to the round-bottom flask, and the mixture was placed in a rotary evaporator for 0.5 h. The mixture was ultrasonicated at 100 W power for 10 min using a probe and extruded through a membrane 5 times to make the particle size uniform. The resulting solution was mixed with 7.5 mg of DSPE-PEG-heparin and incubated at 37 ° C at 100 rpm for 12 h to obtain the nano-diagnostic and therapeutic agent.

[0059] Example 3

[0060] Other aspects were the same as in Example 1, except that phosphatidylethanolamine (PE) was used instead of tryptophan to prepare DSPE-PEG-PE.

[0061] 4 mg of DPPC, 5.8 mg of DOPE-PEG, 0.2 mg of DOPE-PEG-FITC, 0.5 mg of atorvastatin, and 2 mg of cerium oxide nanozyme were accurately weighed and dissolved in 2 mL of chloroform. The mixture was magnetically stirred at room temperature for 2 h, transferred to a round-bottom flask, and vacuum evaporated at 60 ° C for 0.5 h to form a uniform film. 3 mL of deionized water was added to the round-bottom flask, placed in a rotary evaporator for 0.5 h, ultrasonicated at 300 W power for 5 min, and extruded through a membrane 5 times to make the particle size uniform. The resulting solution was mixed with 7.5 mg of DSPE-PEG-PE and incubated at 37 ° C at 100 rpm for 6 h to obtain the nano-diagnostic and therapeutic agent.

[0062] Example 4

[0063] The rest is the same as Example 1, except that ApoB-100 antibody is used instead of tryptophan to prepare DSPE-PEG-ApoB-100 antibody.

[0064] 4 mg DPPC, 1 mg cholesterol, 4.5 mg DPPE-PEG, 0.5 mg DPPE-PEG-RB, 0.5 mg rapamycin and 2 mg cerium oxide nanozyme were accurately weighed and dissolved in 2 mL of chloroform, magnetically stirred at room temperature for 3 h, transferred to a round-bottom flask, and vacuum evaporated at 60 ° C for 0.5 h to form a uniform film. 3 mL of deionized water was added to the round-bottom flask, placed in a rotary evaporator for 0.5 h, ultrasonicated at 400 w power for 5 min by probe ultrasound, and extruded through the membrane twice to make the particle size uniform. The resulting solution was mixed with 7.5 mg DSPE-PEG-ApoB-100 antibody and incubated at 37 ° C at 100 rpm for 8 h to obtain the nano-diagnostic and therapeutic agent.

[0065] Example 5

[0066] Accurately weigh 5mg SPC, 4.5mg DSPE-PEG, 0.5mg DSPE-PEG-ICG, 0.5mg rapamycin, 0.5mg curcumin and 2mg polydopamine and dissolve them in 2mL chloroform. Stir magnetically at room temperature for 2h, transfer to a round-bottom flask, and evaporate under reduced pressure for 0.5h at 55°C to form a uniform film. Add 3mL of deionized water to the round-bottom flask, place it on a rotary evaporator for 0.5h, ultrasonicate it at a power of 400w through the probe for 5min, and extrude it through the membrane twice to make the particle size uniform. The resulting solution is mixed with 7.5mg DSPE-PEG-tryptophan obtained in Example 1 and incubated at 37°C at 100rpm for 10h to obtain the nano-diagnostic and therapeutic agent.

[0067] Example 6

[0068] Accurately weigh 4mg DPPC, 4mg cholesterol, 1.8mg DSPE-PEG, 0.2mg DSPE-PEG-Ce6, 0.5mg rapamycin and 2mg manganese dioxide nanozyme and dissolve them in 2mL chloroform, stir magnetically at room temperature for 4h, transfer to a round-bottom flask, and vacuum evaporate for 0.5h at 60°C to form a uniform film. 3mL of deionized water was added to the round-bottom flask, placed in a rotary evaporator for 0.5h, ultrasonicated at 400w power by probe for 5min, and extruded through the membrane 5 times to make the particle size uniform. The resulting solution was mixed with 7.5mg DSPE-PEG-heparin obtained in Example 2 and incubated at 37°C at 100rpm for 6h to obtain the nano-diagnostic and therapeutic agent.

[0069] Comparative Example 1

[0070] Accurately weigh 5 mg of DSPE, 4.8 mg of DPPE-PEG, 0.2 mg of DPPE-PEG-Cy5, and 1 mg of probucol and dissolve them in 2 mL of chloroform. Stir magnetically at room temperature for 1 h, transfer them to a round-bottom flask, and evaporate them under reduced pressure for 0.5 h at 55 ° C to form a uniform film. Add 3 mL of deionized water to the round-bottom flask, place it on a rotary evaporator for 0.5 h, ultrasonically ultrasonicate it for 5 min at a power of 200 w using a probe, and extrude it through the membrane 5 times to make the particle size uniform. The resulting solution was mixed with 7.5 mg of DSPE-PEG-PE obtained in Example 3, and incubated at 37 ° C at 100 rpm for 6 h to obtain the nano-diagnostic and therapeutic agent.

[0071] Comparative Example 2

[0072] 4 mg of DPPC, 1 mg of cholesterol, 4.5 mg of DSPE-PEG, 0.5 mg of DSPE-PEG-ICG, 1 mg of rapamycin, and 2 mg of Prussian blue nanozyme were accurately weighed and dissolved in 2 mL of chloroform. The mixture was magnetically stirred at room temperature for 2 hours, transferred to a round-bottom flask, and vacuum evaporated at 60°C for 0.5 hours to form a uniform film. 3 mL of deionized water was added to the round-bottom flask, and the mixture was placed on a rotary evaporator for 0.5 hours. Ultrasonic probe ultrasound was used at 400 W for 5 minutes, and the mixture was extruded through a membrane five times to uniformize the particle size, thereby obtaining the nanodiagnostic and therapeutic agent.

[0073] Comparative Example 3

[0074] Accurately weigh 6.5 mg of SPC, 3 mg of DSPE-PEG, 0.5 mg of DSPE-PEG-FITC, and 2 mg of copper oxide nanozyme and dissolve them in 2 mL of chloroform. Stir magnetically at room temperature for 0.5 h, transfer them to a round-bottom flask, and evaporate them under reduced pressure for 0.5 h at 45 ° C to form a uniform film. Add 3 mL of deionized water to the round-bottom flask, place it on a rotary evaporator for 0.5 h, ultrasonically ultrasonicate it for 10 min at a power of 100 w through a probe, and extrude it through the membrane 3 times to make the particle size uniform. The resulting solution was mixed with 7.5 mg of DSPE-PEG-tryptophan obtained in Example 1 and incubated at 37 ° C at 100 rpm for 4 h to obtain the nano-diagnostic and therapeutic agent.

[0075] Comparative Example 4

[0076] 7mg DPPC, 1mg cholesterol, 1.5mg DSPE-PEG, 0.5mg DSPE-PEG-ICG, 0.5mg rapamycin and 2mg silica nanoparticles were accurately weighed and dissolved in 2mL chloroform, magnetically stirred at room temperature for 2h, transferred to a round-bottom flask, and vacuum evaporated at 60°C for 0.5h to form a uniform film. 3mL of deionized water was added to the round-bottom flask, placed in a rotary evaporator for 0.5h, ultrasonicated at 400w power by probe for 5min, and extruded through the membrane 5 times to make the particle size uniform. The resulting solution was mixed with 7.5mg DSPE-PEG-PE obtained in Example 3, incubated at 37°C at 100rpm for 6h to obtain the nano-diagnostic and therapeutic agent. The silica nanoparticles are nanoparticles that do not have antioxidant function.

[0077] Comparative Example 5

[0078] Accurately weigh 7 mg DOPE, 2.5 mg DSPE-PEG, 0.5 mg DSPE-PEG-Ce6, 1 mg curcumin and 1 mg polydopamine and dissolve them in 2 mL chloroform. Stir magnetically at room temperature for 1 hour, transfer to a round-bottom flask, and evaporate under reduced pressure at 60 ° C for 1 hour to form a uniform film. Add 3 mL of deionized water to the round-bottom flask, place it on a rotary evaporator for 0.5 hours, ultrasonically ultrasonicate it at 200w power for 5 minutes, and extrude it through the membrane 5 times to make the particle size uniform. The resulting solution was mixed with 7.5 mg DSPE-PEG-heparin from Example 2 and incubated at 37 ° C at 100 rpm for 4 hours to obtain the nano-diagnostic and therapeutic agent.

[0079] Experimental Example 1: Determination of particle size distribution and drug encapsulation efficiency of nano-diagnostic agents

[0080] 1 mL of the nano-diagnostic and therapeutic agent obtained in Examples 1-4 and Comparative Examples 1-5 was accurately measured and placed in a 10 mL volumetric flask, diluted to the scale with water, and the particle size of the nano-diagnostic and therapeutic agent was measured.

[0081] The ultrafiltration method was used to determine the encapsulation efficiency of the loaded drug in the nano-diagnostic agent. 0.5 mL of the nano-diagnostic agent was added to the inner tube of an ultrafiltration tube with a molecular weight cutoff of 100 kDa, centrifuged at 2000 rpm for 30 min, and the preparation in the inner tube was resuspended with 0.5 mL of deionized water. 0.2 mL was added to 1.8 mL of methanol, vortexed for 3 min to break the emulsion, and the encapsulated drug concentration C1 was detected and calculated by HPLC. In addition, 0.2 mL of the nano-diagnostic agent before ultrafiltration centrifugation was taken, and the total concentration of the drug C0 was detected after breaking the emulsion using the same method, and the encapsulation efficiency EE% of the nano-diagnostic agent was calculated.

[0082]

[0083] Depend on Figure 1 It can be seen that the particle size distribution of Examples 1-4 and Comparative Examples 1-4 is uniform, all around 20 nm, and their particle sizes are all within the range of being able to take LDL to achieve active transendothelial transport (particle size is below 40 nm). However, the particle size of the nano-diagnostic agent prepared in Comparative Example 5 is about 56 nm, which is too large to be conducive to taking LDL to achieve transendothelial transport, so this prescription is not used for the functional evaluation of subsequent nano-diagnostic agents. Except for Comparative Example 3, which has no loaded drug, the drug encapsulation efficiency of Examples 1-4 and Comparative Examples 1, 2, 4, and 5 is all above 85%, which can encapsulate drugs well.

[0084] Experimental Example 2 Anti-LDL Oxidation Ability of Nano-Therapeutic Agents

[0085] The anti-LDL oxidation ability of the nano-diagnostic agent was investigated using a malondialdehyde (MDA) assay kit. The oxidation product of LDL, MDA, can condense with thiobarbituric acid to form a red product with a maximum absorption peak at 532 nm. 1 mL of LDL solution (2 mg / mL) was mixed with 1 mL of the nano-diagnostic agent obtained in Examples 1-4 and Comparative Examples 1-4, respectively, and then 50 μM CuSO4 solution and 150 μM H2O2 were added to start oxidation. The mixture was incubated at 37 ° C. on a shaker at 120 rpm, and samples were taken at 0 h and 24 h, respectively, to measure the MDA content.

[0086] like Figure 2 As shown, the nano-diagnostic and therapeutic agents obtained in Examples 1-4 and Comparative Examples 2 and 3 can effectively inhibit the oxidation of LDL within 24 hours, while Comparative Example 1 can reduce the oxidation of LDL to a certain extent, and the LDL co-incubated with Comparative Example 4 is largely oxidized within 24 hours. It proves that although small molecule antioxidants can have a certain protective effect on LDL, their antioxidant effect is weak; in contrast, antioxidant nanozymes show strong anti-LDL oxidation function, reflecting the advantages of antioxidant nanozymes.

[0087] Experimental Example 3 Adsorption performance of nano-diagnostic and therapeutic agents on LDL

[0088] Microthermophoresis (MST) detects the interaction between molecules by measuring the change in the swimming speed of molecules in the test solution in a temperature gradient field, and reflects the magnitude of the intermolecular binding force by calculating the dissociation constant Kd.

[0089] LDL solution (2mg / mL) and 1.5mol / L (pH 9.0) carbonate buffer were mixed in a volume ratio of 9: 1, 0.2mg of FITC powder was accurately weighed and added to the mixed solution, and the reaction was stirred in the dark for 12h at 4°C. The solution was then transferred to a dialysis bag (molecular weight cutoff of 8-14kDa), dialyzed overnight at 4°C to remove free FITC, and the solution in the bag was collected to obtain FITC-LDL. 16 microcentrifuge tubes were taken, and the nano-diagnostic agents obtained in Examples 1-4 and Comparative Examples 1-4 were used as mother liquors. 16 different aqueous solutions of preparations with a concentration gradient were prepared using a doubling dilution method, each with a volume of 10μL; 10μL of FITC-LDL was then added sequentially and mixed with a pipette. 16 MST standard capillaries were taken, each inserted into a microcentrifuge tube, and the sample was drawn to a height of more than two-thirds of the capillary. Place the sample-loaded capillaries on the sample loading platform in order, open the operating software, select the blue light channel, set the excitation power to 20%, and the MST power to medium for testing.

[0090] like Figure 3As shown, the Kd of the nano-therapeutic agent obtained in Example 1 and LDL is 33.0 μM, the Kd of the nano-therapeutic agent obtained in Example 2 and LDL is 20.6 μM, the Kd of the nano-therapeutic agent obtained in Example 3 and LDL is 1.6 μM, the Kd of the nano-therapeutic agent obtained in Example 4 and LDL is 0.7 μM, the Kd of the nano-therapeutic agent obtained in Comparative Example 1 and LDL is 1.5 μM, the Kd of the nano-therapeutic agent obtained in Comparative Example 3 and LDL is 118 μM, and the Kd of the nano-therapeutic agent obtained in Comparative Example 4 and LDL is 45.1 μM, indicating that the nano-therapeutic agents obtained in Examples 1-4 and Comparative Examples 1, 3, and 4 can all bind to LDL and have the potential to ride LDL across the endothelium. However, the Kd values ​​measured in Comparative Examples 3 and 4 are significantly higher than those in Examples 1-4, and the values ​​are larger, indicating that the binding force of the two to LDL is weak, which may be because the ratio of their auxiliary lipids affects their binding ability to LDL. In addition, the Kd between the nanodiagnostic agent obtained in Comparative Example 2 and LDL was not measured, indicating that it had no LDL binding ability, reflecting the importance of LDL adsorption functional polymer modification.

[0091] Experimental Example 4: Transport of Nanotherapeutic Agents Across the Endothelium by LDL

[0092] Complete culture medium was added to the 24-well plate, and sterile forceps were used to insert the Transwell chamber (polyester membrane pore size 0.4 μm, diameter 6.5 mm, cell growth area 0.33 cm 2 ) were inserted into the plate. HUVEC cells in the logarithmic growth phase were cultured at 2×10 5 ELVIS cells / mL were seeded on the polyester membrane of the Transwell chamber (500 μL / well), which was placed in a cell culture incubator for 48 h to form a dense endothelial cell monolayer to construct a non-ELVIS effector endothelial cell model.

[0093] The fluorescence intensity of the nanotherapeutic agents obtained in Examples 1-4 and Comparative Examples 1-4 was adjusted to the same, and the transcellular transport behavior of the nanotherapeutic agents after riding on LDL was investigated using the non-ELVIS effect endothelial cell model constructed above. Different nanotherapeutic agents and LDL were added to the upper chamber of the Transwell, where the concentration of LDL was 50 μg / mL, and incubated with HUVEC cells in the Transwell chamber for 4 hours. The culture medium in the lower chamber was collected, and the fluorescence intensity was measured by fluorescence spectrophotometer.

[0094] like Figure 4As shown, the nano-diagnostic agents obtained in Examples 1-4 and Comparative Example 1 can all achieve trans-endothelial transport in the presence of LDL, and their transcellular transport capacity is consistent with the results of binding capacity; the transcellular transport amount of the nano-diagnostic agents obtained in Comparative Examples 3 and 4 is relatively small, because the ratio of auxiliary lipids is related to the ability to achieve transcellular transport by riding on LDL; in contrast, the nano-diagnostic agent obtained in Comparative Example 2 can hardly achieve transcellular transport, which is related to the absence of LDL adsorption functional polymer components in the preparation. It can be concluded that the nano-diagnostic agent can actively cross the endothelial barrier by riding on LDL, which is beneficial to improve the efficiency of the nano-diagnostic agent in penetrating plaques and deliver the fluorescent probe and loaded drug to the subendothelial space to play the role of imaging diagnosis and treatment.

[0095] Experimental Example 5: Nano-therapeutic agent inhibits macrophage foaming

[0096] THP-1 macrophages were cultured at a rate of 2 × 10 5 The cells were seeded at a density of 100 ng / mL into a 24-well plate (0.5 mL / well) and cultured in complete medium containing 100 ng / mL PMA for 24 h to allow the cells to adhere. The Transwell chamber seeded with intact HUVEC monolayer cells cultured for 48 h was assembled with a 24-well plate seeded with THP-1 macrophages to construct an endothelial-macrophage co-culture Transwell model.

[0097] H2O2 (800 μM) was added to the lower chamber of the Transwell for pre-incubation for 2 h, and then different nano-diagnostic and therapeutic agents and LDL were added to the Transwell chamber. A group with PBS and LDL was set up as a control and incubated together for 24 h.

[0098] Lipid droplets in THP-1 macrophages were stained with 0.3% Oil Red O (ORO) staining solution and observed under an inverted fluorescence microscope to assess its ability to inhibit macrophage foam cell formation. Specifically, cells were fixed with 4% paraformaldehyde for 20 minutes, washed three times with PBS, and then permeabilized with 60% isopropanol for 15 minutes to facilitate Oil Red O staining. 0.3% ORO was then added and incubated in the dark for 1 hour, followed by three washes with PBS and finally infiltration with PBS. The cells were observed and photographed under a brightfield inverted fluorescence microscope, and quantified using ImageJ.

[0099] like Figure 5As shown, the smaller the Oil Red O staining area, the lower the lipid content in the cell, that is, the stronger the ability of the preparation to inhibit macrophage foaming. Examples 1-4 showed a strong ability to inhibit macrophage foaming, and were proportional to their transendothelial cell transport ability; however, the ability of Comparative Examples 1, 3, and 4 to inhibit macrophage foaming was significantly weaker than that of Examples 1-4. This is because the small molecule antioxidant in Comparative Example 1 has a weak ability to inhibit LDL oxidation, the transcellular transport amount of Comparative Example 3 is relatively small, and the silica nanoparticles in Comparative Example 4 have no antioxidant function; and Comparative Example 2 failed to play an anti-macrophage foaming role because it could not achieve transcellular transport. This test example shows that the nanoformulation needs to first cross the endothelial barrier before it can enter the subendothelial space to clear ROS, and further be taken up by macrophages and exert an anti-AS effect.

[0100] Experimental Example 6 Antioxidant Effect of Nanotherapeutic Agents in Oxidative Damage Cell Model

[0101] Referring to Experimental Example 5, an endothelial-macrophage co-culture Transwell model was constructed. After pre-stimulating the macrophages in the lower chamber with 800 μM H2O2 for 2 hours, LDL and different nano-therapeutic agents were added to the Trasnwell chamber. The group with PBS and LDL was used as the model group, and the group without H2O2 and LDL was used as the control group. After incubation for 24 hours, 10 μL CCK-8 solution was added to each well and the cells were incubated for another 1.5 hours. The absorbance at 450 nm was measured using a microplate reader, and the cell survival rate was calculated.

[0102] like Figure 6 As shown, H2O2 stimulation can cause macrophage damage, and cell viability drops to about 40%. Examples 1-4 all show excellent effects in protecting macrophages from oxidative damage, increasing cell viability to more than 80%; while Comparative Examples 1, 3, and 4 also have certain functions in protecting macrophages, their protective abilities are significantly weaker than those of Examples 1-4; in contrast, the cell viability after treatment in Comparative Example 2 is still very low (only 46%), because it cannot achieve transendothelial transport, and no drugs or antioxidant nanozymes are enriched around macrophages, and it cannot play a protective role on macrophages.

[0103] Test Example 7

[0104] Male ApoE knockout mice with a C57BL / 6J genetic background were randomly divided into two groups (n=3) and fed a high-fat diet for 8 weeks and 12 weeks, respectively, to establish early-stage AS models (8 weeks) and late-stage AS models (12 weeks). Comparative Example 2 and Example 2 were injected into the tail vein, respectively. Twelve hours after administration, the mice were dissected, the aortic tree was removed, and images were taken using an in vivo imaging device for quantitative analysis.

[0105] like Figure 7As shown, there was no significant difference in the accumulation of comparative example 2 in the aorta between the early model and the late model. In contrast, the accumulation of example 2 in the aorta in the late model was significantly higher than that in the early model, showing an imaging diagnostic effect that was positively correlated with the course of AS.

[0106] Test Example 8

[0107] The above-mentioned AS late model (12 weeks) mice were randomly divided into 5 groups (n=6). 0.1 mL of normal saline, Comparative Example 2, Comparative Example 4, Example 2 and Example 4 were injected into the tail vein respectively, and the drug dosage was 1 mg / kg and the antioxidant nanozyme dosage was 2 mg / kg. The administration frequency was 2 times a week for a total of one month. After the administration, fresh blood was collected from the orbits of each group of mice, and the blood was centrifuged at 3000 rpm for 10 minutes at 4 ° C. The upper serum was collected and the contents of four blood lipids, triglycerides TG, total cholesterol TCHO, LDL-C and HDL-C, were detected by a fully automatic blood biochemical analyzer.

[0108] like Figure 8 As shown, both Example 2 and Example 4 can reduce TG, TCHO, LDL-C and increase HDL-C, and have good blood lipid regulation effects, while Comparative Example 2 and Comparative Example 4 have poor blood lipid regulation effects. This is because the nano-diagnostic and therapeutic agent described in Comparative Example 2 cannot achieve effective plaque penetration by riding on LDL, and there is no antioxidant nanoenzyme in Comparative Example 4, and the loaded rapamycin alone cannot achieve an ideal blood lipid regulation effect.

[0109] Test Example 9

[0110] The above-mentioned AS late model (12 weeks) mice were randomly divided into 5 groups (n=6). 0.1 mL of normal saline, comparative example 1, comparative example 2, example 2 and example 4 were injected into the tail vein respectively, and the drug loading dose was 1 mg / kg, the antioxidant nanozyme dose was 2 mg / kg, and the administration frequency was 2 times a week for a total of one month. After the administration, the mouse aorta was collected for sectioning, and the ROS content in the aorta was analyzed by immunofluorescence, observed and photographed under an upright fluorescence microscope, and quantitative analysis was performed using Image J.

[0111] like Figure 9 As shown, Example 2 and Example 4 can effectively reduce the ROS level in the aorta of atherosclerotic mice after treatment, while Comparative Examples 2 and Comparative Examples 4 have poor ROS scavenging effects. This is attributed to the fact that the diagnostic and therapeutic agent in Comparative Example 2 cannot achieve effective plaque penetration. Even if it contains antioxidant nanozymes, it still cannot reach the subintima of the plaque to exert an antioxidant effect. The diagnostic and therapeutic agent described in Comparative Example 4 cannot play a role in scavenging ROS due to the lack of antioxidant nanozymes.

[0112] The above data show that the nanodiagnostic and therapeutic agent of the present invention has the ability to efficiently transport across endothelial cells by riding on LDL, and can synergize antioxidant nanoenzymes and anti-AS drugs to achieve anti-AS therapeutic effects, showing diagnostic imaging characteristics positively correlated with the course of disease in atherosclerosis model mice.

[0113] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the appended claims.

Claims

1. A nano-diagnostic agent, characterized in that: The nanodiagnostic and therapeutic agent consists of 10 to 30 parts by weight of phospholipids, 20 to 40 parts by weight of auxiliary lipids, 5 to 15 parts by weight of antioxidant nanoenzymes, 0.1 to 10 parts by weight of fluorescent probe complex A-PEG-B, 0 to 10 parts by weight of loaded drugs, and 30 to 50 parts by weight of LDL adsorption functional polymer DSPE-PEG-C; in the fluorescent probe complex, A is a fluorescent dye, PEG is polyethylene glycol 2000, and B is a phospholipid; in the LDL adsorption functional polymer DSPE-PEG-C, C is an LDL adsorption ligand.

2. The nanodiagnostic agent according to claim 1, characterized in that The nano-diagnostic and therapeutic agent consists of 15-25 parts by weight of phospholipids, 25-35 parts by weight of auxiliary lipids, 8-12 parts by weight of antioxidant nanoenzymes, 0.5-2 parts by weight of fluorescent probe complex A-PEG-B, 0-5 parts by weight of loaded drugs, and 30-40 parts by weight of LDL adsorption functional polymer DSPE-PEG-C.

3. The nanodiagnostic agent according to claim 1, characterized in that The phospholipids in the nanodiagnostic and therapeutic agent are any one or more of soybean lecithin, 1,2-distearoyl-sn-glycerol-3-phosphocholine, 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine, 1,2-dioleoyl-sn-glycerol-3-phosphocholine, 1,2-distearoyl-sn-glycerol-3-phosphatidylethanolamine, 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine, 1,2-distearoyl-sn-glycerol-3-phosphatidylglycerol, 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylglycerol, and 1,2-dioleoyl-sn-glycerol-3-phosphatidylglycerol.

4. The nano-diagnostic agent according to claim 1, characterized in that The auxiliary lipid is any one or more of cholesterol, 1,2-distearoyl-sn-glycerol-3-phosphatidylethanolamine-polyethylene glycol, 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine-polyethylene glycol, and 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol.

5. The nano-diagnostic agent according to claim 1, characterized in that The antioxidant nanozyme is any one of manganese dioxide nanozyme, cuprous oxide nanozyme, cerium oxide nanozyme, polydopamine, polytannic acid, Prussian blue nanozyme, and graphene nanozyme.

6. The nano-diagnostic agent according to claim 1, characterized in that The molar ratio of A, PEG and B in the fluorescent probe complex A-PEG-B is 1:0.5~2:0.5~2, preferably 1:1:1; Preferably, A is any one of pentamethine cyanine dye, heptamethine cyanine dye, indocyanine green, fluorescein isothiocyanate, dihydrochlorin, and rhodamine B; Preferably, B is any one of 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine, and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.

7. The nano-diagnostic agent according to claim 1, characterized in that The loaded drug is a drug with anti-AS function; preferably, the drug is any one or more of atorvastatin, probucol, rapamycin, curcumin, vitamin E, and aspirin.

8. The nano-diagnostic agent according to claim 1, characterized in that In the LDL adsorption functional polymer DSPE-PEG-C, C is an anionic adsorption ligand, a hydrophobic adsorption ligand, or an immune adsorption ligand; Preferably, C is selected from any one of heparin, glutathione, tryptophan, aspartic acid, glutamic acid, phosphatidylethanolamine, and ApoB-100 antibody.

9. The method for preparing the nano-diagnostic and therapeutic agent according to claims 1 to 8, characterized in that: Prepared by the following method: (1) Dissolve phospholipids, auxiliary lipids, fluorescent probe complexes, loaded drugs and antioxidant nanozymes in chloroform, stir magnetically at room temperature for 0.5-5 h, transfer to a reaction vessel, and evaporate under reduced pressure at 37-60 °C for 0.5±0.1 h to form a uniform film; add deionized water to the reaction vessel, place it in a rotary evaporator for 0.5±0.1 h, sonicate with a probe at a power of 100-400 W for 5-10 min, and extrude through a membrane 1-10 times to make the particle size uniform; (2) The obtained solution was mixed with DSPE-PEG-C and incubated at 37°C and 100 rpm for 4 to 12 h to obtain the nano-diagnostic and therapeutic agent.

10. Use of the nano-diagnostic agent according to any one of claims 1 to 8 in the preparation of a product for treating and monitoring the course of atherosclerotic cardiovascular disease; Preferably, the product is a drug or a system carrying the drug; Preferably, the atherosclerotic cardiovascular disease is any one of atherosclerosis, acute coronary syndrome, peripheral arterial atherosclerosis, and coronary heart disease.

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