A biomimetic nanoliposome preparation for inhibiting PCSK9 and its preparation method and application

Abstract

The present invention relates to a bionic nanoliposome for inhibiting PCSK9 and a preparation method and application thereof. The bionic nanoliposome for inhibiting PCSK9 comprises an antibody component, a lipid membrane material and a bionic cell membrane, wherein the antibody is eloumab; the lipid membrane material comprises soybean lecithin, cholesterol, and DSPE‑PEG2000‑NH2; and the bionic cell membrane is a macrophage membrane. In the present invention, eloumab is encapsulated in the nanoliposome, the bionic cell membrane is disguised in the outer layer of the nanoliposome encapsulating eloumab, and DSPE‑PEG2000‑NH2 is modified on the surface of the bionic cell membrane. The present application can deliver the liposome-encapsulated eloumab in a targeted sustained-release manner, and prolong the blood residence time, thereby prolonging the drug action time, effectively reducing the expression level of PCSK9, increasing the expression level of LDLR, reducing LDL expression, and alleviating the progression of atherosclerosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a bionic nanoliposome preparation for inhibiting PCSK9, and a preparation method and application thereof. Background Art

[0002] At present, arteriosclerotic cardiovascular disease (ASCVD) has surpassed infectious diseases to become the world's number one killer. Abnormal blood lipid metabolism is an important risk factor in the development of coronary heart disease, among which elevated low-density lipoprotein cholesterol (LDL-C) levels occupy a core position. The "China Cardiovascular Health and Disease Report 2019" pointed out that the number of people with dyslipidemia in my country has increased significantly, while the awareness, treatment and control rates are still at a low level overall. In the 10-year high-risk population for atherosclerotic cardiovascular disease (ASCVD), the LDL-C treatment rate was 5.5% and the compliance rate was 25.5%; while in the extremely high-risk population, the LDL-C treatment rate was only 14.5%, and the compliance rate was even lower at only 6.8%. The 2020 "Chinese Expert Consensus on Blood Lipid Management in Patients with Ultra-High-Risk Atherosclerotic Cardiovascular Disease" pointed out that for ultra-high-risk ASCVD patients, intensive lipid-lowering therapy can achieve a lower LDL-C target value or even control it below 1.0mmol / L, which can bring significant cardiovascular benefits.

[0003] Evolocumab is a new type of biological monoclonal antibody preparation, with the trade name Repatha. It is a human monoclonal immunoglobulin G2 (IgG2) that targets human proprotein convertase subtilisin / kexin type 9 (PCSK9), binds to PCSK9, and inhibits the binding of circulating PCSK9 to the low-density lipoprotein receptor (LDLR), thereby preventing PCSK9-mediated LDLR degradation, allowing LDLR to recirculate to the surface of liver cells, resulting in an increase in the number of LDLRs that can clear low-density lipoprotein in the blood, thereby lowering LDL-C levels. Eloumab was approved for marketing by the National Medical Products Administration in 2018, becoming the first and only PCSK9 inhibitor approved in China for the treatment of homozygous familial hypercholesterolemia in adults or adolescents aged 12 years and above; it was approved for use in adult patients with atherosclerotic cardiovascular disease (ASCVD) in 2019; in the same year, it was approved again for broader indications, becoming the first PCSK9 inhibitor approved in China for use in adult patients with primary hypercholesterolemia (including heterozygous familial hypercholesterolemia) and mixed dyslipidemia.

[0004] While evolocumab shows good therapeutic prospects, its adverse reactions are also increasingly concerned. In clinical research reports, the common side effects of the drug are upper respiratory tract infection, influenza, gastroenteritis, and nasopharyngitis, followed by local injection site reactions and allergic reactions. The most common adverse reaction that led to the discontinuation of Repatha and had a higher incidence than the placebo group was myalgia.

[0005] Given the adverse reactions caused by the high dosage of systemic evolocumab and the limitations of its administration method, how to reduce the dosage without affecting the efficacy and reduce side effects is the key to the current use of this drug to treat atherosclerosis.

[0006] The development of nanotechnology has brought hope for solving this medical problem. Nanoliposomes are liposomes that use a phospholipid bilayer biomembrane structure to improve bioavailability and maintain their original performance by encapsulating active substances. In addition, due to their small size and surface effects, they can also enhance the contact between substances and cells and improve targeting. However, after entering the body, due to the effects of various factors such as albumin, opsonins, and antibodies in the blood, the liposomes rupture, the encapsulated drugs leak rapidly, and are quickly recognized and absorbed by the reticuloendothelial system, limiting its "missile" function. For this reason, polyethylene glycol (PEG) modification is needed to avoid the surveillance of the reticuloendothelial phagocytic system, increase the stability of liposomes in the blood, and prolong the half-life of the drug. On the other hand, by encapsulating the macrophage membrane, the characteristic proteins on the cell membrane can help the liposomes escape the clearance of the body's immune system, and at the same time achieve the effect of using inflammatory chemotaxis to treat atherosclerosis. Summary of the invention

[0007] In view of this, the present invention provides a bionic nanoliposome preparation for inhibiting PCSK9 and a preparation method and application thereof. The bionic nanoliposome preparation for inhibiting PCSK9 can deliver liposome-entrapped evolocumab in a targeted sustained-release manner, prolong the blood residence time, reduce the dosage of the drug, and effectively reduce the expression level of PCSK9, thereby alleviating the progression of atherosclerosis.

[0008] The invention provides a bionic nanoliposome preparation for inhibiting PCSK9, comprising an antibody, a lipid membrane material and a bionic cell membrane.

[0009] Preferably, the antibody is evolocumab; the lipid membrane material includes soybean lecithin, cholesterol, and DSPE-PEG2000-NH2; and the bionic cell membrane is a macrophage membrane.

[0010] The present invention also provides a method for preparing a biomimetic nanoliposome preparation for inhibiting PCSK9, comprising the following steps:

[0011] S1. Preparation of bionic cell membrane;

[0012] S2. Preparing nanoliposomes that inhibit PCSK9 by reverse evaporation method: quantitatively weighing soybean lecithin, cholesterol, and DSPE-PEG2000-NH2, mixing and dissolving in a mixture of chloroform, ether and ultrapure water; adding eloumab to the lipid solution, and performing water bath ultrasound to form a W / O type emulsion; placing the obtained emulsion in a rotary evaporator to evaporate under reduced pressure to remove the organic solvent, and after reaching a colloidal state, adding a hydration medium, and continuing to distill under reduced pressure to remove the remaining organic solvent; rotating and hydrating until the gel on the bottle wall falls off, and filtering through a 0.22 μm microporous filter membrane to obtain nanoliposomes that inhibit PCSK9; and separating the unencapsulated free antibodies by dialysis;

[0013] S3. Mixing the PCSK9-inhibiting nanoliposome solution with the PBS solution of the bionic cell membrane and stirring to obtain the PCSK9-inhibiting bionic nanoliposome preparation.

[0014] Preferably, in S1, the specific preparation process of the bionic cell membrane is: when the RAW264.7 cells in the cell culture dish are fully grown, the cells are scraped with a cell scraper and then centrifuged; the collected cells are washed twice with PBS (pH7.4) at 4°C, and then suspended in a membrane protein extractant A containing PMSF (1mM); the mixture is lysed in an ice bath for 30-60min, and sonicated in a 4°C water bath for 10-15min; the mixture is repeatedly frozen and thawed at 37°C and -80°C for 4 times, each time for 20-30min; the supernatant is then centrifuged and centrifuged to obtain the macrophage membrane.

[0015] Preferably, the mass ratio of the lecithin to the antibody is 1-5:1; the mass ratio of the lecithin to the DSPE-PEG2000-NH2 is 10-20:6.

[0016] Preferably, the hydration medium is a phosphate buffer containing 2% Tween-80.

[0017] Preferably, the dialysis is performed using a dialysis bag with a molecular weight cutoff of 200-300 kDa for 24-72 hours.

[0018] Preferably, the mass ratio of the lecithin to the macrophage membrane is 10:1; the stirring speed is 800-1000 rpm, and the time is 1-2 hours.

[0019] Preferably, in S2, the average particle size of the PCSK9-inhibiting nanoliposomes is 160-180 nm; and the encapsulation efficiency of the PCSK9-inhibiting nanoliposomes for the antibody active ingredient is 90%.

[0020] The present invention also provides an application of a bionic nanoliposome preparation for inhibiting PCSK9, which is used for preparing a drug for preventing and / or treating atherosclerosis.

[0021] The beneficial effects of the present invention are:

[0022] 1. Since the structure of nanoliposomes is similar to that of cell membranes, both of which are composed of substances such as phospholipids and cholesterol, are non-toxic and have high safety, the present invention selects them as carriers for encapsulating drugs without causing toxic / side effects on the human body.

[0023] 2. The biomimetic nanoliposome preparation constructed by the present invention is loaded with evolocumab, which inhibits the binding of PCSK9 to low-density lipoprotein receptor (LDLR), so that LDLR can be recirculated to the surface of liver cells, thereby reducing LDL-C levels to treat atherosclerosis.

[0024] 3. The present invention modifies the liposome surface with PEG and macrophage membrane to avoid the surveillance of the reticuloendothelial phagocytic system, increase the stability in the blood, prolong the drug circulation half-life, and maximize the effect of preventing and treating atherosclerosis.

[0025] 4. The present invention solves the problems of large clinical dosage, short in vivo half-life and drug toxicity and side effects, and provides new theoretical support for the development of new drugs for the prevention and treatment of atherosclerosis and related clinical prevention and treatment, which has important scientific significance, practical value and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments and in conjunction with the accompanying drawings, wherein

[0027] Figure 1 Electron microscopic images of drug-loaded nanoliposomes wrapped in nanoliposomes and biomimetic membranes;

[0028] Figure 2 This is a protein immunoblot of the biomimetic cell membrane;

[0029] Figure 3 Particle size and potential diagram of Lipo, Lipo@E and (Lipo+M)@E;

[0030] Figure 4 The drug uptake and lysosomal escape abilities of Lipo, Lipo@E, and (Lipo+M)@E;

[0031] Figure 5 is the cumulative release of Evol from Lipo@E and (Lipo+M)@E;

[0032] Figure 6 Western Blot results showing that (Lipo+M)@E reduces PCSK9 expression;

[0033] Figure 7 The IF situation of (Lipo+M)@E reducing PCSK9 expression;

[0034] Figure 8 is the blood half-life of (Lipo+M)@E in mice;

[0035] Fig. 9 The targeting of (Lipo+M)@E to atherosclerotic plaques in mice;

[0036] Fig.10 The gross Oil Red O staining of blood vessels in different treatment groups for atherosclerosis;

[0037] Fig.11 The Oil Red O staining of the aortic root, arch and abdominal aorta of mice under different treatment groups;

[0038] Fig.12 The expression of PCSK9 in serum of mice with atherosclerosis after treatment in different treatment groups.

[0039] Fig.13 H&E staining of the heart, liver, spleen, lung and kidney of mice with atherosclerosis treated with different treatment groups. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with the accompanying drawings and specific preferred embodiments, but the protection scope of the present invention is not limited thereby.

[0041] In the following examples, unless otherwise specified, the raw materials and instruments used are commercially available, and the methods used are conventional methods in the art.

[0042] A bionic nanoliposome preparation for inhibiting PCSK9 comprises an antibody, a lipid membrane material and a bionic cell membrane. The antibody is evolocumab; the lipid membrane material comprises soybean lecithin, cholesterol, and DSPE-PEG2000-NH2; and the bionic cell membrane is a macrophage membrane.

[0043] A method for preparing a biomimetic nanoliposome preparation for inhibiting PCSK9 comprises the following steps:

[0044] S1. Prepare biomimetic cell membranes. The specific process is as follows: when the RAW264.7 cells in the cell culture dish are full, scrape the cells with a cell scraper and centrifuge; wash the collected cells twice with PBS (pH7.4) at 4°C, and then suspend them in membrane protein extractant A containing PMSF (1mM); lyse the mixture in an ice bath for 30-60min, and ultrasonicate in a 4°C water bath for 10-15min. Repeat freezing and thawing at 37°C and -80°C for 4 times, each time for 20-30min; then centrifuge to obtain the supernatant, and continue centrifugation to obtain macrophage membranes.

[0045] S2. Prepare nanoliposomes that inhibit PCSK9 by reverse evaporation method: quantitatively weigh soybean lecithin, cholesterol, and DSPE-PEG2000-NH2, mix and dissolve in a mixture of chloroform, ether and ultrapure water; add eloumab to the lipid solution, perform water bath ultrasound, and form a W / O type emulsion; place the obtained emulsion in a rotary evaporator and evaporate under reduced pressure to remove the organic solvent, and after reaching a colloidal state, add a hydration medium, and continue to distill under reduced pressure to remove the remaining organic solvent; rotate and hydrate until the gel on the bottle wall falls off, and pass through a 0.22μm microporous filter membrane to obtain nanoliposomes that inhibit PCSK9; use dialysis to separate unencapsulated free antibodies; the average particle size of the nanoliposomes that inhibit PCSK9 is 160-180nm; the encapsulation rate of the nanoliposomes that inhibit PCSK9 for the active ingredients of the antibody is: 90%.

[0046] S3. Mixing the PCSK9-inhibiting nanoliposome solution with the PBS solution of the bionic cell membrane and stirring to obtain the PCSK9-inhibiting bionic nanoliposome preparation.

[0047] The mass ratio of the lecithin to the antibody is 1-5:1; the mass ratio of the lecithin to the DSPE-PEG2000-NH2 is 10-20:6. The hydration medium is a phosphate buffer containing 2% Tween-80. The dialysis is performed using a dialysis bag with a molecular weight cutoff of 200-300 kDa for 24-72 hours. The mass ratio of the lecithin to the macrophage membrane is 10:1; the stirring speed is 800-1000 rpm, and the time is 1-2 hours.

[0048] The bionic nanoliposome preparation is used for preparing medicine for preventing and / or treating atherosclerosis.

[0049] Example 1

[0050] (1) Preparation of (Lipo+M)@E biomimetic nanoparticles

[0051] a. Preparation of biomimetic cell membrane (M)

[0052] The preparation of macrophage (RAW264.7 cell) membrane was carried out according to the instructions of the membrane protein extraction preparation kit. When the RAW264.7 cells in the cell culture dish were full, the cells were washed three times with PBS, scraped with a cell scraper, and centrifuged at 800rpm for 5min. The collected cells were washed twice with PBS (pH7.4) at 4°C, and then suspended in membrane protein extractant A containing PMSF (1mM). The mixture was lysed in an ice bath for 30min, and the probe was sonicated at 4°C for 10min (80W, on 0.3s, off 0.5s). Then it was frozen and thawed repeatedly at 37°C and -80°C for 5 times, each time for 20-30min. Then it was centrifuged at 4°C, 800rpm for 10min. The supernatant was taken and centrifuged at 4°C, 13000rpm for 30min to obtain the macrophage membrane.

[0053] b. Preparation of Lipo@E, in this application, Lipo refers to a separate liposome already available in the prior art, used as a control example;

[0054] Weigh 20 mg of soybean lecithin, 9 mg of cholesterol, and 6 mg of DSPE-PEG2000-NH2 and dissolve them in 13 ml of solvent, including 4 ml of chloroform, 6 ml of ether, and 3 ml of ultrapure water; dilute eloumab to a 10 mg / ml solution, slowly add it to the lipid solution with a syringe, and ultrasonicate it in a water bath for 5 minutes to form a W / O type emulsion. The obtained emulsion is required not to be stratified within half an hour. The obtained emulsion is placed in a rotary evaporator at 37°C and evaporated under reduced pressure to remove the organic solvent. After reaching a colloidal state, 30 ml of hydration medium is added, and the remaining organic solvent is continued to be distilled under reduced pressure. Rotate and hydrate until the gel on the bottle wall falls off, and pass through a 0.22 μm microporous filter membrane to obtain nanoliposomes that inhibit PCSK9. The unencapsulated free antibody is separated by dialysis.

[0055] c. Preparation of (Lipo+M)@E bionic nanoformulation;

[0056] The macrophage membrane obtained above was dissolved into 1 mg / ml, and the Lipo@E solution was mixed with the bionic cell membrane (M) after water bath ultrasonic treatment at a mass ratio of 10:1, and stirred in a water bath at 800 rpm for 1 hour at 37°C to obtain the (Lipo+M)@E bionic nanoformulation;

[0057] (2) Characterization of (Lipo+M)@E biomimetic nanoformulation

[0058] like Figure 1 As shown, the Lipo, Lipo@E and (Lipo+M)@E biomimetic nanoparticles prepared in this example were subjected to DLS particle size analysis. The results showed that uniformly dispersed spherical Lipo materials and (Lipo+M)@E biomimetic nanoparticles were successfully prepared, with particle sizes of about 100nm and 200nm, respectively.

[0059] like Figure 2 As shown, the bionic cell membrane prepared in this example was subjected to membrane characteristic protein analysis, and the results showed that the bionic membrane was successfully prepared.

[0060] like Figure 3 As shown, the particle size potential analysis of Lipo, Lipo@E and (Lipo+M)@E was performed by DLS, and the results showed that the average particle size of (Lipo+M)@E was 180nm and the potential condition was good.

[0061] like Figure 5 As shown in the figure, compared with Lipo@E, the cumulative release of Evol is reduced after being wrapped by the bionic cell membrane, indicating that the prepared bionic nanoformulation achieves sustained release.

[0062] Example 2

[0063] A biomimetic nanoliposome preparation for inhibiting PCSK9, which is different from Example 1 in that when preparing the nanoliposomes encapsulating evolocumab, the mass ratio of phospholipid to antibody is 1:1.

[0064] Example 3

[0065] A biomimetic nanoliposome preparation for inhibiting PCSK9, which is different from Example 1 in that when preparing the nanoliposomes encapsulating evolocumab, the mass ratio of phospholipid to antibody is 5:1.

[0066] Example 4

[0067] A biomimetic nanoliposome preparation for inhibiting PCSK9, which is different from Example 1 in that the mass ratio of lecithin to DSPE-PEG2000-NH2 is 10:6.

[0068] Example 5

[0069] A biomimetic nanoliposome preparation for inhibiting PCSK9, which is different from Example 1 in that dialysis is performed using a dialysis bag with a molecular weight cutoff of 200-300 kDa for 36 hours.

[0070] Example 6

[0071] A bionic nanoliposome preparation for inhibiting PCSK9 is different from Example 1 in that when preparing the bionic macrophage membrane-wrapped nanoliposome-encapsulated drug, the mass ratio of phospholipid to macrophage membrane is 5:1.

[0072] Example 7

[0073] The present invention provides a bionic nanoliposome preparation for inhibiting PCSK9 and its application in the prevention and treatment of atherosclerosis. The (Lipo+M)@E bionic nano preparation prepared in Example 1 is used as an example to illustrate the effect.

[0074] like Figure 4 As shown, blank liposomes and nanoliposomes wrapped by biomimetic macrophage membranes were labeled with Dil, added to smooth muscle cells, and lysosomes were labeled with lysosome probes. The uptake of biomimetic nanoliposomes and their lysosomal escape ability were observed using confocal microscopy. The results showed that the uptake of (Lipo+M)@E by smooth muscle cells was concentration-dependent, and the uptake of lysosomes was effectively avoided, protecting eloumab from damage by the slightly acidic environment.

[0075] The above results indicate that the (Lipo+M)@E biomimetic nanoliposome preparation prepared in Example 1 can escape clearance by the immune system in the circulation and escape uptake by lysosomes after entering cells.

[0076] Example 8

[0077] like Figure 6 As shown, each group of nanomedicines prepared in Example 1 was added to LPS-intervention smooth muscle cells, and the effect of (Lipo+M)@E on PCSK9 expression was observed by protein immunoblotting. The results showed that (Lipo+M)@E significantly reduced the expression of PCSK9.

[0078] like Figure 7 As shown, each group of nanomedicines prepared in Example 1 was added to LPS-intervention smooth muscle cells, and the effect of (Lipo+M)@E on PCSK9 expression was observed by immunofluorescence. The results showed that (Lipo+M)@E significantly reduced the expression of PCSK9.

[0079] Example 9

[0080] The (Lipo+M)@E bionic nanoliposome preparation prepared in Example 1 was used to measure the blood half-life and in vivo targeting of the (Lipo+M)@E bionic nanoliposome preparation in mice by detecting the fluorescence intensity through in vivo imaging of small animals.

[0081] Experiment 1: Take 250ul Lipo (4mg / ml), 40ul dihydrochlorin (Ce6) (1mg / ml), add 1710ul double distilled water, place the mixture at 4℃800-1000rpm and stir overnight to obtain Lipo@Ce6. Take 250ul Lipo (4mg / ml), 40ul dihydrochlorin (Ce6) (1mg / ml), 50ul macrophage membrane, add 1660ul double distilled water, place the mixture at 4℃800-1000rpm and stir overnight to obtain (Lipo+M)@Ce6. Prepare 1mg / ml Ce6 for control experiment.

[0082] Ce6, Lipo@Ce6 and (Lipo+M)@Ce6 prepared in Experiment 1 were injected into C57BL / 6 mice via the tail vein, 200 μl per mouse, and blood samples were collected from the eye sockets at different time points for fluorescence intensity measurement.

[0083] The results are as follows Figure 8 As shown, the fluorescence of the (Lipo+M)@Ce6 group was significantly prolonged compared with the Ce6 group and the Lipo@Ce6 group, indicating that the bionic nanoliposomes significantly prolonged the half-life in vivo.

[0084] Experiment 2: After ApoE- / - mice were fed a high-fat diet for one month, 200 μL of 10 mg / kg ICG, Lipo@ICG, and (Lipo+M)@ICG were injected into the tail vein. Twelve hours later, the aorta of the mice was taken for fluorescence imaging.

[0085] Among them, Lipo@ICG is prepared according to the following method:

[0086] Weigh 20 mg of soybean lecithin, 9 mg of cholesterol, and 6 mg of DSPE-PEG2000-NH2, mix and dissolve in 13 ml of solvent, the solvent includes 4 ml of chloroform, 6 ml of ether and 3 ml of ultrapure water; stir at room temperature for 15 minutes to fully dissolve, then add to a round-bottom flask and remove the organic solvent by reduced pressure evaporation at 37°C in a rotary evaporator, add the ICG solution to the above round-bottom flask, stir in water at 55°C for 4 hours, and ultrasonicate the probe in an ice bath for 10 minutes to obtain Lipo@ICG.

[0087] (Lipo+M)@ICG was prepared according to the following method:

[0088] The Lipo@ICG solution was mixed with the PBS solution containing macrophage membranes and the mixture was placed at 4°C. Fig. 9 As shown, compared with the ICG and Lipo@ICG groups, the (Lipo+M)@ICG group significantly aggregated in the aortic arch and abdominal aorta, indicating that the (Lipo+M)@E bionic nanoformulation prepared in Example 1 can effectively target the site of atherosclerotic plaques.

[0089] Example 10

[0090] The (Lipo+M)@E bionic nano preparation prepared in Example 1 is used for the early prevention and treatment of atherosclerosis.

[0091] After ApoE- / - mice were fed a high-fat diet for one month, they were given 5 mg / kg twice a week for 2 consecutive months, during which they maintained a high-fat diet; all were administered by tail vein injection, and the treatment groups were divided into Evol alone group, Lipo@E group and (Lipo+M)@E group. Three days after the end of the treatment, the aorta of the mice was stripped and macroscopic Oil Red O staining was performed to examine the therapeutic effects of the different treatment groups.

[0092] The results are as follows Fig.10 As shown, the (Lipo+M)@E treatment group significantly reduced the formation of atherosclerotic plaques in the treatment regimen, indicating that the bionic nanoformulation constructed by the present invention can effectively prevent the progression of early atherosclerosis.

[0093] Embodiment 11

[0094] Using the treatment scheme adopted in Example 10, after the treatment of ApoE- / - mice was completed, the frozen sections of the aortic root, arch and abdominal aorta of each group of mice were stained with Oil Red O, and the lipid staining was observed under an inverted microscope.

[0095] like Fig.11 As shown in the figure, the Oil Red O staining of the aortic root and arch of the abdominal aorta in the (Lipo+M)@E group was significantly reduced compared with the Evol group and the Lipo@E group, indicating that (Lipo+M)@E effectively removes lipids in various parts.

[0096] Example 12

[0097] Using the treatment scheme adopted in Example 10, after the treatment of ApoE- / - mice was completed, the serum of mice with different treatment components under different treatment schemes was collected to examine the expression of PCSK9 in the serum between different treatment groups.

[0098] The results are as follows Fig.12 As shown, the expression level of PCSK9 in serum of (Lipo+M)@E group was significantly lower than that of Evol group and Lipo@E group, indicating that (Lipo+M)@E effectively reduced the expression of PCSK9 in blood.

[0099] Example 12

[0100] Using the treatment regimen adopted in Example 10, after the treatment of ApoE- / - mice was completed, the main organs (heart, liver, spleen, lung and kidney) of mice with different treatment components under different treatment regimens were collected for H&E staining to examine the biological safety of different treatment groups.

[0101] The results are as follows Fig.13 As shown, no obvious organic lesions were found in the organs of mice in different treatment groups, indicating that the bionic nano-preparation constructed by the present invention has good biosafety.

[0102] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a biomimetic nanoliposome preparation for inhibiting PCSK9, characterized in that: The bionic nanoliposome preparation for inhibiting PCSK9 comprises antibodies, lipid membrane materials and bionic cell membranes; The antibody is evolocumab; the lipid membrane material includes soybean lecithin, cholesterol, and DSPE-PEG2000-NH2; the bionic cell membrane is a macrophage membrane; The preparation method of the biomimetic nanoliposome preparation for inhibiting PCSK9 comprises the following steps: S1, preparing a bionic cell membrane; in S1, the specific preparation process of the bionic cell membrane is: when the RAW264.7 cells in the cell culture dish are full, the cells are scraped off with a cell scraper and then centrifuged; the collected cells are washed twice with PBS with a pH of 7.4 at 4°C, and then suspended in a membrane protein extractant A containing 1 mM PMSF; the mixture is lysed in an ice bath for 30-60 minutes, and ultrasonicated in a 4°C water bath for 10-15 minutes; the mixture is repeatedly frozen and thawed at 37°C and -80°C for 4 times, each time for 20-30 minutes; then the supernatant is centrifuged and centrifuged again to obtain a macrophage membrane; S2. Prepare nanoliposomes that inhibit PCSK9 by reverse phase evaporation method: quantitatively weigh soybean lecithin, cholesterol, and DSPE-PEG2000-NH2, mix and dissolve in a mixture of chloroform, ether and ultrapure water; add eloumab to the lipid solution, and perform water bath sonication to form a W / O type emulsion; place the obtained emulsion in a rotary evaporator to evaporate under reduced pressure to remove the organic solvent, add a hydration medium after reaching a colloidal state, and continue to distill under reduced pressure to remove the remaining organic solvent; rotate and hydrate until the gel on the bottle wall falls off, and filter through a 0.22µm microporous filter membrane to obtain nanoliposomes that inhibit PCSK9; use dialysis to separate the unencapsulated free antibodies; S3. Mixing the PCSK9-inhibiting nanoliposome solution with the PBS solution of the bionic cell membrane and stirring the mixture, thereby obtaining the PCSK9-inhibiting bionic nanoliposome preparation.

2. The method for preparing the biomimetic nanoliposome preparation for inhibiting PCSK9 according to claim 1, characterized in that: The mass ratio of the lecithin to the antibody is 1-5:1; the mass ratio of the lecithin to the DSPE-PEG2000-NH2 is 10-20:

6.

3. The method for preparing the biomimetic nanoliposome preparation for inhibiting PCSK9 according to claim 1, characterized in that: The hydration medium is a phosphate buffer containing 2% Tween-80.

4. The method for preparing the biomimetic nanoliposome preparation for inhibiting PCSK9 according to claim 1, characterized in that: The dialysis is performed using a dialysis bag with a molecular weight cut-off of 200-300 kDa for 24-72 hours.

5. The method for preparing the biomimetic nanoliposome preparation for inhibiting PCSK9 according to claim 1, characterized in that: The mass ratio of the lecithin to the macrophage membrane is 10:1; the stirring speed is 800-1000 rpm, and the time is 1-2 hours.

6. The method for preparing the biomimetic nanoliposome preparation for inhibiting PCSK9 according to claim 1, characterized in that: In S2, the average particle size of the nanoliposomes inhibiting PCSK9 is 160-180 nm; the encapsulation rate of the nanoliposomes inhibiting PCSK9 for the antibody active ingredient is 90%.

7. An application of a biomimetic nanoliposome preparation for inhibiting PCSK9 as claimed in any one of claims 1 to 6, characterized in that: Used for preparing medicine for preventing and / or treating atherosclerosis.

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