Bilirubin nano-particles for treating atherosclerosis and preparation method of bilirubin nano-particles

Abstract

The invention discloses bilirubin nanoparticles for treating atherosclerosis and a preparation method of the bilirubin nanoparticles. The bilirubin nanoparticles mainly comprise bilirubin, polyethylene glycol and selenocystamine dihydrochloride, and the whole preparation method is simple in process operation, stable in condition and good in reproducibility. The bilirubin nano-particles can be used for treating atherosclerosis and relieving inflammatory reaction, can be applied to preparation of atherosclerosis treatment drugs and inflammatory disease treatment drugs, and have wide clinical application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano-medicine and relates to a nano-particle and a preparation method and application thereof, and in particular relates to a bilirubin nano-particle for treating atherosclerosis and a preparation method thereof. Background Art

[0002] Coronary atherosclerotic heart disease, referred to as CHD, is a disease with secondary prevention treatment, mainly including antiplatelet and lipid-lowering therapy. However, based on large-scale randomized controlled trials and registration studies, among patients with CHD who receive standardized secondary prevention treatment, about 2%-10% of patients still experience major adverse cardiac events (MACE) such as cardiac death, acute myocardial infarction, and stroke each year. Therefore, it is of great clinical significance to further explore new intervention targets for secondary prevention of CHD to reduce the risk of residual MACE in patients with existing CHD.

[0003] The formation of atherosclerotic plaques is the main pathological basis of coronary heart disease. Vulnerable plaques refer to atherosclerotic plaques with a higher risk of rupture, and their main pathological features include a thin fibrous cap, a lipid-rich necrotic core, and a large number of inflammatory cell infiltrations. The rupture of vulnerable plaques and the induced acute thrombosis in the lumen are key risk factors for adverse cardiac events (MACE) such as death and myocardial infarction in patients with coronary heart disease, and reactive oxygen species (ROS) are key factors that promote the formation and rupture of vulnerable plaques. Therefore, removing excess ROS in plaques is expected to stabilize atherosclerotic plaques and reduce the risk of plaque rupture and MACE in patients. However, there is currently no effective treatment to improve the oxidative stress microenvironment in plaques.

[0004] Bilirubin is the main pigment component in bile in the human body and is also the terminal metabolite of hemoglobin in the human body. Recent studies have found that bilirubin has antioxidant properties, and slightly elevated bilirubin has cardiovascular protective effects. Based on a large coronary heart disease cohort that has been constructed, the team also found that patients with elevated bilirubin within the normal range had a significantly reduced risk of MACE by about 40% during the three-year follow-up, suggesting that bilirubin is expected to become a new drug for the treatment of coronary heart disease. However, free bilirubin in its natural state has low water solubility, is easily oxidized in the body, has poor stability in the body, is easily deposited in local tissues and causes toxicity, and is difficult to administer orally or by injection, limiting further mechanism exploration and clinical transformation applications. How to modify the structure of bilirubin in its natural state, increase its water solubility and stability, and target the microenvironmental characteristics of high levels of ROS in vulnerable plaques, so that bilirubin nanomaterials can target vulnerable plaques of atherosclerosis and remove ROS is an urgent problem to be solved. 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 bilirubin nanoparticle for atherosclerotic plaque formation and a preparation method thereof.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: A first aspect of the present invention provides bilirubin nanoparticles for treating atherosclerosis.

[0007] Furthermore, the nanoparticles contain bilirubin, polyethylene glycol and selenocystamine dihydrochloride.

[0008] Furthermore, the particle size of the nanoparticles is 100-500 nm.

[0009] Furthermore, the Zeta potential of the nanoparticles is -30~0mV.

[0010] In some embodiments, polyethylene glycol is the most commonly used polymer in biomedical applications and can be used to modify a variety of drugs. In the present invention, the inventor uses polyethylene glycol as a surface modifier, and connects the carboxyl group to the secondary amino group of bilirubin to increase the hydrophilicity of bilirubin, thereby prolonging its circulation time in the body and reducing the probability of aggregation and merging.

[0011] In the present invention, the inventors use selenocystamine dihydrochloride to react its amino group with the carboxyl group of bilirubin to form a bond, and the amino group and the carbonyl group form a Schiff base. The inflammatory acidic microenvironment of the lesion site triggers the pH-responsive Schiff base to break, and at the same time, the high level of ROS destroys the diselenide bond of the polymer core, releasing bilirubin to clear ROS, thereby increasing the ability of nanoparticles to target atherosclerotic plaques.

[0012] In a specific embodiment of the present invention, the inventors have demonstrated through animal experiments that the bilirubin nanoparticles of the present invention have the function of improving atherosclerosis and reducing plasma inflammatory indicators.

[0013] In some embodiments, the size of the nanoparticles is related to local aggregation. Larger nanoparticles (500-2000nm) tend to aggregate locally after injection, while smaller nanoparticles (20-200nm) tend to be targeted to lymph nodes and organs. The particle size of the bilirubin nanoparticles provided by the present invention is 100-500nm, the average particle size is 241.3nm, and the polydispersity index (PDI) is 0.344. This shows that the bilirubin nanoparticles of the present invention have a smaller particle size and are not easy to aggregate locally.

[0014] In the present invention, PDI represents the uniformity of the particle size distribution of the nanoparticles. The smaller the PDI value, the more uniform the size of the nanoparticles. The polydispersity index (PDI) of the nanoparticles of the present invention is 0.344, indicating that the particle size of the nanoparticles of the present invention is uniform.

[0015] The second aspect of the present invention provides a method for preparing the bilirubin nanoparticles described in the first aspect of the present invention.

[0016] Further, the method comprises the following steps: The bilirubin, methoxy polyethylene glycol carboxyl group, carboxyl group activator and organic solvent are mixed and amidated to obtain a bilirubin-polyethylene glycol complex; The bilirubin-polyethylene glycol complex is modified with selenocystamine dihydrochloride to obtain bilirubin nanoparticles.

[0017] Furthermore, the specific steps of the amidation reaction are: firstly, mixing the carboxyl group of methoxy polyethylene glycol, a carboxyl activator and an organic solvent at room temperature, then adding bilirubin to the organic solvent, and reacting under an argon atmosphere.

[0018] Furthermore, the methoxy polyethylene glycol carboxyl group, the carboxyl group activating agent and the organic solvent are mixed for 30 minutes.

[0019] Furthermore, the reaction time under argon atmosphere is 4 hours.

[0020] In the present invention, the purpose of mixing the methoxy polyethylene glycol carboxyl group, the carboxyl group activating agent and the organic solvent is to activate the carboxyl group. The mixing method is preferably stirring at room temperature, and the stirring and mixing time is preferably 30 minutes.

[0021] In the present invention, the amidation reaction is preferably carried out under an anaerobic environment. In a specific embodiment of the present invention, the amidation reaction is carried out under an argon atmosphere, and the reaction time is preferably 4 hours.

[0022] Furthermore, the carboxyl activator includes N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, DCC, HOBt, and HATU.

[0023] Preferably, the carboxyl activating agent is N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0024] Furthermore, the organic solvent is dimethyl sulfoxide.

[0025] Furthermore, the concentration ratio of the methoxypolyethylene glycol carboxyl, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and bilirubin in dimethyl sulfoxide is 1:1:1:1.

[0026] In some embodiments, the carboxyl activator can promote the dehydration condensation reaction of the carboxyl group with the nucleophilic reagent, thereby avoiding the destruction of the substrate structure by severe conditions and reducing the occurrence of side reactions. The carboxyl activator includes but is not limited to: N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, DCC, HOBt, HATU. In a specific embodiment of the present invention, the carboxyl activator used is N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC).

[0027] In the present invention, the NHS is used in combination with EDC as a catalytic enhancer to stabilize the activated ester intermediate generated by EDC and improve the reaction rate and yield.

[0028] In the present invention, the EDC is an organic compound, which is often used as an activator of carboxyl groups, and reacts with carboxyl groups to form an activated ester intermediate, and then reacts with amine groups to form an amide bond.

[0029] Furthermore, the method for obtaining the bilirubin-polyethylene glycol complex further comprises the following steps: removing impurities and undissolved bilirubin from the product obtained by the amidation reaction and then freeze-drying the product to obtain the bilirubin-polyethylene glycol complex.

[0030] Furthermore, the specific steps of removing impurities and undissolved bilirubin are: dialyzing the product obtained by the amidation reaction to remove impurities, then concentrating by rotary evaporation, dissolving the concentrated product in methanol, and centrifuging to remove undissolved bilirubin.

[0031] Preferably, the dialysis is performed using a dialysis membrane with a molecular weight cut-off value of 1000.

[0032] In the present invention, after the amidation reaction, the obtained amidation reaction solution is dialyzed and concentrated to obtain a bilirubin-polyethylene glycol complex. In the present invention, the dialysis used is operated using a dialysis membrane with a molecular weight cutoff value of 1000, and the concentration method is preferably rotary evaporation concentration. The present invention has no special requirements for the concentration method, and a concentration method well known to those skilled in the art can be used.

[0033] In the present invention, the rotary evaporation concentration is a vacuum distillation technology, which makes the liquid evenly distributed on the container wall by rotating the circular rotary bottle, and utilizes constant temperature and vacuum conditions to accelerate the evaporation of the solvent, thereby achieving the concentration and purification of the sample.

[0034] Furthermore, the specific steps of modifying selenocystamine dihydrochloride to obtain bilirubin nanoparticles are: mixing a bilirubin-polyethylene glycol complex, a carboxyl activator and water, then reacting selenocystamine dihydrochloride and sodium bicarbonate in water and adding them to the above mixture, stirring under an argon atmosphere, removing impurities and small molecular monomers and then freeze-drying to obtain bilirubin nanoparticles.

[0035] Furthermore, the carboxyl activator includes N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, DCC, HOBt, and HATU.

[0036] Preferably, the carboxyl activating agent is N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0037] Furthermore, the concentration ratio of the bilirubin-polyethylene glycol complex, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in water is 1:1:1.

[0038] Furthermore, the bilirubin-polyethylene glycol complex, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride are mixed in water and stirred at room temperature for 30 minutes.

[0039] Furthermore, the concentration ratio of selenocystamine dihydrochloride and sodium bicarbonate in water is 1:2.

[0040] Further, the selenocystamine dihydrochloride and sodium bicarbonate are reacted in water for 4 hours.

[0041] Furthermore, the stirring time under the argon atmosphere is 24 hours.

[0042] Furthermore, the method for removing impurities and small molecule monomers is to perform dialysis operation using a dialysis membrane with a molecular weight cutoff value of 1000.

[0043] The third aspect of the present invention provides use of the bilirubin nanoparticles described in the first aspect of the present invention or the bilirubin nanoparticles prepared by the method described in the second aspect of the present invention in preparing drugs for treating and / or preventing atherosclerosis.

[0044] In the present invention, the treatment and / or prevention refers to delaying the development of a disease, preventing the development of a disease, and / or reducing the severity of the symptoms that will develop or are expected to develop. Therefore, these terms include improving existing disease symptoms, preventing additional symptoms, improving or preventing potential metabolic causes of symptoms, inhibiting a disorder or disease, for example, preventing the development of a disorder or disease, alleviating a disorder or disease, regressing a disorder or disease, alleviating a condition caused by a disease or disorder, or stopping symptoms of a disease or disorder.

[0045] The fourth aspect of the present invention provides use of the bilirubin nanoparticles described in the first aspect of the present invention or the bilirubin nanoparticles prepared by the method described in the second aspect of the present invention in preparing drugs for treating and / or preventing inflammatory diseases.

[0046] Furthermore, the inflammatory diseases include but are not limited to: acute pancreatitis, rheumatoid arthritis, and diabetic wound repair.

[0047] In the present invention, the inventors found through animal experiments that the bilirubin nanoparticles can reduce the inflammatory indicators in the mouse plasma, including TNF-α, IL-6 and IL-1β, after treating the diseased mice. This shows that the bilirubin nanoparticles of the present invention have the effect of treating inflammatory diseases.

[0048] In the present invention, the TNF-α (tumor necrosis factor α) is an important inflammatory indicator, which is mainly produced by monocytes and phagocytes. It plays a variety of biological activities in the human body, including promoting cell proliferation and differentiation, immune regulation, inflammation mediation and anti-tumor effects. Abnormal values ​​of TNF-α may indicate that the patient has an inflammatory response, immune disease or tumor lesions. Specifically, an increase in TNF-α may be related to inflammation such as viral infection, bacterial infection of the respiratory system, and may also be related to immune diseases such as nephritis and lupus erythematosus, or to diabetic wound repair.

[0049] In the present invention, the IL-6 (interleukin 6) is a commonly used clinical indicator for detecting inflammation, and is mainly used to evaluate infectious diseases, inflammatory reactions and the activity and prognosis of certain diseases. Increased IL-6 indicates that the patient may have an infection. IL-6 is also involved in the pathological process of a variety of clinical diseases, including diabetes, cardiovascular disease, rheumatic diseases, etc.

[0050] In the present invention, the IL-1β (interleukin-1β) is an important pro-inflammatory cytokine that participates in regulating immune response and cell metabolism, etc. Its increase is commonly seen in a variety of diseases, including but not limited to: rheumatoid arthritis, hepatitis, respiratory tract infection, etc.

[0051] Advantages and beneficial effects of the present invention: (1) The nanoparticles provided by the present invention have uniform particle size and good stability; (2) The entire preparation method of the present invention is simple to operate, has stable conditions and good reproducibility; (3) Through a large number of experiments, the present invention has found that the bilirubin nanoparticles are effective in treating atherosclerosis, have no toxic side effects, are low-cost, and will help delay the progression of the disease. They are also highly operational and can significantly reduce the diagnosis and treatment costs of patients and medical institutions. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 is a characterization diagram of the properties of bilirubin nanoparticles; among them, Figure 1 A is the overall synthesis strategy diagram, Figure 1 B is the particle size distribution diagram of bilirubin nanoparticles, Figure 1 C is the morphology of bilirubin nanoparticles. Figure 1 D is the potential distribution diagram of the intermediate product BN-PEG and the final product BN-PEG-DAD (bilirubin nanoparticles), Figure 1 E is the UV-visible absorption peak diagram of the intermediate product BN-PEG and the final product BN-PEG-DAD in the range of 190-700 nm; Figure 2 This is a diagram of the therapeutic effect of bilirubin nanoparticles; Figure 2 A is the weight results of mice in the control group (CTRL) and bilirubin nanomaterial group (BRNP). Figure 2 B. Figure 2 C are the gross Oil Red O staining images and statistical analysis graphs of aorta of mice in different groups, Figure 2 D. Figure 2 E are Masson staining images and statistical analysis images of aortic roots of mice in different groups, Figure 2 F is the CD68 immunofluorescence staining of the aortic root of mice in different groups. Figure 2 G is the result of ELISA detection of mouse plasma inflammatory indicators. DETAILED DESCRIPTION

[0053] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and cannot be understood as limiting the present invention. It can be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

[0054] The drugs, reagents and raw materials used in the present invention are easily obtained by ordinary technicians in the field and can be obtained from commercial channels unless otherwise specified. The experimental methods in the present invention that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present invention and should not limit the scope of the present invention in any way.

[0055] Example 1 Synthesis of bilirubin nanomaterials 1. Experimental Materials 1. The names, specifications and manufacturers of the reagents used are as follows: Bilirubin, 98.0%, Aladdin Biochemical Technology Co., Ltd. Methoxypolyethylene glycol carboxyl (2000), 97.0%, Bidex Pharmaceuticals Selenocysteine ​​dihydrochloride, 97.0%, Shanghai Jizhi Biochemical Technology Co., Ltd. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 98.0%, MacLean N-Hydroxysuccinimide, 98.0%, Aladdin Biochemical Technology Co., Ltd. 2. The instruments, models and manufacturers used are as follows: Magnetic stirrer, FK-1, Fangke Instruments Freeze dryer, ZLGJ-12, Huachen Kewanda Biological Instrument Technology Co., Ltd. High-resolution transmission electron microscope (TEM), FEI Tecnai G2 F20, FEI, USA UV-Vis, UV3600, Shimadzu Dynamic light scattering (DLS), Malvern Zetasizer Nano ZS90, Malvern, UK Matrix-assisted laser desorption time-of-flight mass spectrometer (MALDI-TOF / TOF), Bruker ultraflextremeMALDI-TOF / TOF, Bruker 2. Experimental Methods 1. Preparation of PEGylated bilirubin (BN-PEG): 0.14 mmol of methoxypolyethylene glycol carboxyl (mPEG-COOH), N-hydroxysuccinimide (NHS, 0.14 mmol) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 0.14 mmol) were added to 3 mL of dimethyl sulfoxide (DMSO) at room temperature for activation for 30 min. Then bilirubin (BN, 0.14 mmol) was added to the above mixture and reacted under argon atmosphere for 4 h. The product was dialyzed (Mw = 1000) and concentrated by rotary evaporation, dissolved in methanol, and undissolved BN was removed by centrifugation. The supernatant was lyophilized to obtain BN-PEG.

[0056] 2. Further modification of selenocystamine dihydrochloride (BN-PEG-DAD): BN-PEG (0.14 mmol), N-hydroxysuccinimide (NHS, 0.14 mmol) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 0.14 mmol) were added to water and stirred at room temperature for 30 min to activate the carboxyl group. Selenocystamine dihydrochloride (DAD, 0.14 mmol) was dissolved in water, reacted with sodium bicarbonate (NaHCO3, 0.28 mmol) for 4 h, and then added to the above solution. The mixed solution was stirred for 24 h under argon atmosphere, and impurities and small molecular monomers were removed by dialysis (Mw = 1000). The final product BN-PEG-DAD was obtained by freeze drying.

[0057] 3. Experimental Results The overall synthetic strategy is shown in Figure 1 A, using the amphiphilic polymer selenocystamine dihydrochloride to connect bilirubin molecules and polyethylene glycol through amide bonds, liposomes encapsulate nanoparticles, target atherosclerotic sites by responding to pH values, expose bilirubin-diselenide nanoparticles, and the acidity and ROS enrichment of the lesion site break the bond, releasing bilirubin for treatment (A). The particle size distribution of the final product BN-PEG-DAD (i.e., bilirubin nanoparticles) is shown in Figure 1 B, the average particle size of bilirubin nanoparticles is 241.3nm, and the polydispersity index (PDI) is 0.344. Previous studies have shown that larger nanoparticles (500-2000nm) tend to aggregate locally after injection, while smaller nanoparticles (20-200nm) tend to passively target lymph nodes and organs. The nanoparticles of the present invention have a smaller particle size and are not easy to aggregate locally. PDI represents the uniformity of the nanoparticle size distribution. The smaller the PDI value, the more uniform the nanoparticle size, indicating that the particle size range of the nanoparticles of the present invention is smaller.

[0058] The morphology of bilirubin nanoparticles is shown in Figure 1 C, the particle size distribution is uniform, there is no aggregation, the nanomaterial background is clean and free of impurities, and the morphology is complete. Figure 1 D is the potential distribution diagram of the intermediate product BN-PEG and the final product bilirubin nanoparticles. The Zeta potential represents the strength of the mutual repulsion or attraction between particles. The higher the absolute value of the Zeta potential, the more stable the system. The absolute value of the Zeta potential of the nanoparticles of the present invention is 23.1, and the nanomaterial system is relatively stable. Figure 1 E is the UV-visible absorption peak of the intermediate product BN-PEG and the final product BN-PEG-DAD in the range of 190-700 nm.

[0059] Example 2 Therapeutic effects of bilirubin nanoparticles 1. Experimental Materials 1. Experimental animals and feed 1.1 6-week-old Apoe - / - male mice, Beijing Huafukang Biotechnology Co., Ltd.

[0060] 1.2 High-fat diet (21% lard, 0.15% cholesterol), D12109C, Research Diets, Inc.

[0061] 2. Injection Materials 2.1 Bilirubin nanomaterial (BRNP), as described in Example 1.

[0062] 2.2 Normal saline (0.9%, sterile), IN9000, Beijing Solebow Technology Co., Ltd.

[0063] 2.3 1 ml sterile syringe, H000341, Beijing Puli Zhicheng Biotechnology Co., Ltd.

[0064] 3. Perfusion Materials 3.1 Small animal organ perfusion system DEVOCEAN-ORGAN 10, Dingwosheng Medical Instrument Co., Ltd.

[0065] 3.2 Normal saline (0.9%, sterile), IN9000, Beijing Solebow Technology Co., Ltd.

[0066] 4. Tissue fixation and pathological staining materials 4.1 4% paraformaldehyde, LD-6001, LDBIO.

[0067] 4.2 OCT frozen section embedding medium, 4583, Merck Millipore.

[0068] 4.3 Improved Oil Red O staining kit, G1261, Beijing Solebow Technology Co., Ltd.

[0069] 4.4 Trichrome staining (Masson) kit, HT15, Sigma-Aldrich.

[0070] 5. Immunofluorescence staining materials 5.1 Recombinant Anti-CD68 antibody, ab283654, abcam.

[0071] 5.2 Anti-Alpha Skeletal Muscle Actin antibody, ab28052, abcam.

[0072] 5.3 Blocking goat serum (stock solution), SL038, Beijing Solebow Technology Co., Ltd.

[0073] 5.4 Goat anti-mouse IgG (H+L) secondary antibody, Q11011MP, Pierce.

[0074] 5.5 Goat anti-rabbit IgG (H+L) recombinant secondary antibody, SA510232, Pierce.

[0075] 5.6 Anti-fluorescence attenuation mounting medium (containing DAPI), S2110, Beijing Solebow Technology Co., Ltd.

[0076] 5.7 Sodium citrate antigen retrieval solution (1×), C1031, Beijing Solebow Technology Co., Ltd.

[0077] 6. ELISA kit 6.1 ELISA MAX™ Standard Set Human TNF-α, 430201, Biolegend. 6.2 ELISA MAX™ Standard Set Mouse TNF-α, 430901, Biolegend. 6.3 ELISA MAX™ Standard Set Human IL-6, 430501, Biolegend. 6.4 ELISA MAX™ Deluxe Set Mouse IL-6, 431304, Biolegend. 6.5 HUM IL-1B STD ABTS ELISA KIT, 900-K95, Invitrogen. 6.6 MUR. IL-1B MINI ABTS ELISA KIT, 900-M47, Invitrogen 2. Experimental Methods 1. Experimental animal grouping: 6-week-old Apoe- / - male mice were randomly divided into the following 2 groups: control group (CTRL) and bilirubin nanomaterial group (BRNP). The BRNP group was intraperitoneally injected with BRNP (20 mg / kg dissolved in 2 ml of normal saline) every other day, and the control group was intraperitoneally injected with 2 ml of normal saline every other day. The two groups of mice were fed a high-fat diet (21% lard, 0.15% cholesterol) for a total of 8 weeks to construct an atherosclerosis model, and then weighed, anesthetized mice, and killed and collected. About 1 ml of blood was drawn from the lateral wall of the left ventricle of the mouse using a 1 ml sterile syringe and injected into the EP tube. After standing at room temperature for 30 minutes, centrifuged at 3000 rpm for 15 minutes, and the serum was retained and stored in a -80 ° C refrigerator for subsequent testing. A cut was made in the right atrial appendage of the mouse, the needle tip of the infusion set was inserted into the apex, and normal saline was infused for in vivo perfusion until clear liquid flowed out of the right atrial appendage. The left and right common carotid arteries, brachiocephalic trunk, aorta, and heart tissues were collected and fixed in 4% paraformaldehyde for 48 hours. The heart, aortic root, liver, spleen, kidney and other tissues were collected, and the surrounding connective tissues were freed and placed in liquid nitrogen for quick freezing, and then stored at -80°C.

[0078] 2. Oil Red O staining: Place the tissue in 30% sucrose overnight. Embed with OCT the next day and store at -20°C. Make frozen sections of the aortic root and slice every 100um after the plaque appears. Prepare 6um thick sections and then attach the sections to the slides for Oil Red O staining. The specific steps include: wash the sections twice with distilled water, add the Oil Red O working solution to the slides and incubate for 10-15 minutes, wash twice with distilled water, and stain with hematoxylin for 2 minutes. After washing with distilled water, seal the slides with gelatin. Analyze the images with Image J software.

[0079] 3. Masson staining: Dewax the sections and immerse them in potassium dichromate solution overnight at room temperature. Then stain the sections in Weigert iron hematoxylin solution for 5 minutes and wash in running water for 5 minutes. Stain in Ponceau fuchsin staining solution for 10 minutes, and the tissue will appear bright red under a microscope. After a short rinse in distilled water, incubate the slides in phosphotungstic acid-phosphomolybdic acid solution for 1 minute, then transfer directly to aniline blue solution, stain for 2 minutes, and rinse briefly in distilled water. Then observe and take pictures using an optical microscope, export the pictures, and use Image J software for quantitative analysis of collagen.

[0080] 4. Immunofluorescence staining: Use CD68 antibody to mark macrophages in plaques. The main experimental steps include paraffin-embedded sections, baking in a 45°C oven for 1-2 hours to prevent detachment, followed by dewaxing, gradient ethanol rehydration, preparation of sodium citrate buffer to repair antigens, drop normal goat serum, block for 1 hour, incubate CD68 antibodies, and keep moisturized overnight at 4°C. After washing three times with PBS solution on the second day, incubate the slides with appropriate fluorescent-labeled secondary antibodies for 60 minutes. The light-shielded slides were washed three times in PBS, then counterstained and sealed with DAPI, photographed under a fluorescence microscope, and exported and analyzed using Image J software to analyze the area occupied by CD68-positive macrophages in the plaques.

[0081] 5. ELISA staining: Use ELISA kit to detect serum TNF-α, IL-6 and IL-1β levels. Collect serum in centrifuge tubes. After blood sample agglutination for 30 minutes, centrifuge at 1000G for 10 minutes. After aspirating serum sample, store below -20℃. Before testing, balance all reagents and samples to room temperature. Add 300μl 1× washing solution and let it soak for 30 seconds. After discarding the washing solution, pat the microplate dry on absorbent paper. After washing, add 100μl of 2-fold diluted standard to the standard well. Add 100μl of standard diluent serum sample to the blank well. Add 50μl of diluted detection antibody (1:100) to each well. Seal the plate, shake at 100 rpm, and incubate at room temperature for 2 hours. Discard the liquid and wash the plate with washing solution. Add 100μl of diluted horseradish peroxidase to each well. Labeled streptavidin (1:100). Seal the plate with a new sealing film. Shake at 100 rpm and incubate at room temperature for 45 minutes. Add 100 μl of chromogenic substrate TMB to each well, protect from light, and incubate at room temperature for 30 minutes. Add 100 μl of stop solution to each well. The color changes from blue to yellow. Use an ELISA reader for dual wavelength detection to measure the OD value at the maximum absorption wavelength of 450 nm and the reference wavelength of 570 nm. Calculate the levels of TNF-α, IL-6 and IL-1β in serum.

[0082] 3. Experimental Results The results showed that there was no significant difference in body weight between the two groups of mice ( Figure 2 A). Figure 2 BC is the gross Oil Red O staining image of the aorta, which is used to evaluate the aortic plaque area of ​​mice and perform statistical analysis; the research results show that bilirubin nanomaterials significantly reduce the atherosclerotic plaque burden of the mouse aorta. Figure 2 DE is the Masson staining image of the aorta root, which is used to evaluate the collagen fiber content in the plaque and perform statistical analysis; increased collagen fiber content can increase plaque stability and reduce the risk of rupture. The results showed that bilirubin nanomaterials significantly increased the collagen fiber content in the plaque. Figure 2 F is CD68 immunofluorescence staining of the aorta root, which is used to evaluate the content of macrophages in plaques; increased macrophage content in plaques increases plaque instability and the risk of rupture. The results show that bilirubin nanomaterials significantly reduce the content of macrophages in plaques, suggesting that BRNP can increase plaque stability. In addition, ELISA results showed that BRNP significantly reduced plasma inflammatory indicators in mice, including TNF-α, IL-6 and IL-1β ( Figure 2 G), inflammatory response is an important factor in the formation and development of atherosclerosis, suggesting that bilirubin nanomaterials play a protective role in inhibiting atherosclerosis.

[0083] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A bilirubin nanoparticle for treating atherosclerosis, characterized in that: The nanoparticles contain bilirubin, polyethylene glycol, and selenocystamine dihydrochloride.

2. The bilirubin nanoparticles according to claim 1, characterized in that The particle size of the nanoparticles is 100-500 nm; The Zeta potential of the nanoparticles is -30~0mV.

3. A method for preparing the bilirubin nanoparticles according to claim 1 or 2, characterized in that: The method comprises the following steps: The bilirubin, methoxy polyethylene glycol carboxyl group, carboxyl group activator and organic solvent are mixed and amidated to obtain a bilirubin-polyethylene glycol complex; The bilirubin-polyethylene glycol complex is modified with selenocystamine dihydrochloride to obtain bilirubin nanoparticles.

4. The method according to claim 3, characterized in that The specific steps of the amidation reaction are: firstly mixing the carboxyl group of methoxy polyethylene glycol, a carboxyl group activating agent and an organic solvent at room temperature, then adding bilirubin to the organic solvent, and reacting under an argon atmosphere; Preferably, the methoxy polyethylene glycol carboxyl group, carboxyl group activator and organic solvent are mixed for 30 minutes; Preferably, the reaction time under argon atmosphere is 4 hours.

5. The method according to claim 3 or 4, characterized in that: The carboxyl activator includes N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, DCC, HOBt, and HATU; Preferably, the carboxyl activator is N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; Preferably, the organic solvent is dimethyl sulfoxide; Preferably, the concentration ratio of methoxypolyethylene glycol carboxyl, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and bilirubin in dimethyl sulfoxide is 1:1:1:

1.

6. The method according to claim 3, characterized in that The method for obtaining the bilirubin-polyethylene glycol complex comprises the following steps: removing impurities and undissolved bilirubin from the product obtained by the amidation reaction and then freeze-drying the product to obtain the bilirubin-polyethylene glycol complex; The specific steps of removing impurities and undissolved bilirubin are: dialyzing the product obtained by the amidation reaction to remove impurities, then concentrating by rotary evaporation, dissolving the concentrated product in methanol, and centrifuging to remove undissolved bilirubin; Preferably, the dialysis is performed using a dialysis membrane with a molecular weight cut-off value of 1000.

7. The method according to claim 3, characterized in that The specific steps of modifying selenocystamine dihydrochloride to obtain bilirubin nanoparticles are: mixing a bilirubin-polyethylene glycol complex, a carboxyl activator and water, then reacting selenocystamine dihydrochloride and sodium bicarbonate in water and adding the mixture to the mixture, stirring under an argon atmosphere, removing impurities and small molecular monomers, and then freeze-drying to obtain bilirubin nanoparticles; Preferably, the carboxyl activator includes N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, DCC, HOBt, HATU; Preferably, the carboxyl activating agent is N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

8. The method according to claim 7, characterized in that The concentration ratio of the bilirubin-polyethylene glycol complex, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in water is 1:1:1; The bilirubin-polyethylene glycol complex, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride are mixed in water and stirred at room temperature for 30 minutes; The concentration ratio of selenocystamine dihydrochloride and sodium bicarbonate in water is 1:2; The selenocystamine dihydrochloride and sodium bicarbonate are reacted in water for 4 hours; The stirring time under argon atmosphere is 24h; The method for removing impurities and small molecular monomers is to perform dialysis operation using a dialysis membrane with a molecular weight cutoff value of 1000.

9. Use of the bilirubin nanoparticles according to any one of claims 1 to 2 or the bilirubin nanoparticles prepared by the method according to any one of claims 3 to 8 in preparing a drug for treating and / or preventing atherosclerosis.

10. Use of the bilirubin nanoparticles according to any one of claims 1 to 2 or the bilirubin nanoparticles prepared by the method according to any one of claims 3 to 8 in the preparation of drugs for treating and / or preventing inflammatory diseases; Preferably, the inflammatory diseases include but are not limited to: acute pancreatitis, rheumatoid arthritis, and diabetic wound repair.

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