Macrophage membrane coated bionic carrier-free nano-drug as well as preparation method and application thereof

Abstract

The invention discloses a bionic carrier-free nano-drug coated with a macrophage membrane as well as a preparation method and application of the bionic carrier-free nano-drug. The preparation method of the bionic carrier-free nano-drug comprises the following steps: S1, preparing PR-coated PBNPs: preparing a carrier-free self-assembled nano-drug PR-coated PBNPs from puerarin PR and probucol PB by adopting a nano-precipitation method; and S2, preparing MPR (at) PBNPs: coating the surface of the PR (at) PBNPs with a macrophage membrane, so as to obtain the bionic carrier-free nano-drug MPR (at) PBNPs. According to the nano-drug disclosed by the invention, due to the coating of a macrophage membrane, MPR-coated PBNPs can be promoted to be enriched to an AS diseased region; in the plaque region, the MPR-coated PBNPs releases PR and PB drugs, so that the oxidative stress and apoptosis of endothelium and macrophages are effectively relieved; in addition, endothelial cells can be inhibited from absorbing lipid and foam macrophages can be prevented from being formed, so that the atherosclerosis can be efficiently and safely treated.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a macrophage membrane-coated bionic carrier-free nanomedicine, a preparation method thereof, and an application thereof. Background Art

[0002] Atherosclerosis (AS) is a chronic inflammatory disease characterized by lipid accumulation and plaque formation within the arterial wall. It primarily affects critical blood vessels such as the coronary, carotid, and cerebral arteries, leading to high-risk cardiovascular diseases such as coronary heart disease and stroke, posing a serious threat to human health. Currently, clinical treatments for AS primarily include medication, interventional therapy, and surgery. However, these traditional treatments have limitations. For example, medications can have low drug utilization and adverse reactions with long-term use; interventional therapy with vascular stents can lead to restenosis and late in-stent thrombosis; and surgery is invasive, traumatic, and potentially associated with complications. Therefore, developing new methods and technologies to treat AS is of great value and research significance.

[0003] Nanodrugs developed using nanotechnology can alter the physicochemical properties, pharmacodynamics, and pharmacokinetic characteristics of drugs. Compared with traditional drug delivery methods, nanodrugs exhibit significant advantages in improving therapeutic efficacy and reducing side effects. Therefore, nanodrugs hold great potential in the treatment of AS. The development of novel targeted nanodrugs for the treatment of AS has become a hot topic. However, current nanodrugs developed for the treatment of AS still have several shortcomings, such as complex preparation, the need for nanocarriers, low drug loading, and poor specific targeting. Traditional Chinese medicine self-assembled nanoparticles (TCM-SANs) are a novel nanodrug delivery technology that leverages the TCM-SAN properties of active ingredients from traditional Chinese medicines to form nanoparticles that do not require additional carriers. A significant advantage of TCM-SANs is their high drug loading, which enables the effective encapsulation and delivery of more drug ingredients. Furthermore, since nanocarriers are not required, this approach avoids potential adverse reactions caused by nanocarriers and simplifies the preparation process.

[0004] Numerous studies have shown that oxidative stress and inflammatory responses are key factors in the development of AS, leading to endothelial cell dysfunction, lipid oxidation, foam cell formation, and plaque instability, thereby promoting the progression and deterioration of AS. Therefore, given the central role of oxidative stress and inflammation in AS, anti-inflammatory and antioxidant strategies are ideal strategies for treating AS. Puerarin (PR) is an important bioactive isoflavone compound extracted from the traditional Chinese medicine Pueraria lobata. Studies have shown that puerarin has antioxidant, anti-inflammatory, and apoptosis-reducing effects, and has a good protective effect against vascular dysfunction. It has been clinically used in China and other Asian countries to treat cardiovascular disease, hypertension, and diabetes. In addition, the intrinsic amphiphilic nature of PR gives it the ability to self-assemble into micelles or micelle-like nanostructures in aqueous environments, a property that can be exploited to encapsulate other hydrophobic drugs. Probucol (PB) is an FDA-approved antioxidant with excellent anti-AS function.

[0005] Cell membrane-coated nanoparticle technology is a novel biomimetic strategy for nanomedicines. By coating nanomedicines with membranes extracted from specific cells, these nanomedicines are endowed with unique biological properties. This technology primarily relies on proteins on cell membranes, which can recognize specific cells or tissues in the body, thereby enhancing the targeting of the coated nanomedicine. Research has shown that macrophages play a crucial role in the pathogenesis of AS, participating not only in the inflammatory response at the site of AS lesions but also in lipid metabolism. Numerous characteristic proteins on the surface of macrophage membranes can recognize AS lesions. Summary of the Invention

[0006] The purpose of the present invention is to address the above problems and provide a macrophage membrane-coated bionic carrier-free nanomedicine and its preparation method and application.

[0007] In order to achieve its purpose, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention provides a method for preparing a macrophage membrane-coated biomimetic carrier-free nanomedicine, comprising the following steps:

[0009] S1. Preparation of PR@PBNPs

[0010] Puerarin PR and probucol PB were prepared into carrier-free self-assembled nanoparticles PR@PBNPs by nanoprecipitation method;

[0011] S2. Preparation of MPR@PBNPs

[0012] PR@PBNPs are mixed with macrophage membranes, and the macrophage membranes are coated on the surface of PR@PBNPs to prepare the biomimetic carrier-free nanomedicine MPR@PBNPs.

[0013] Preferably, S1, preparing PR@PBNPs: dissolving puerarin, probucol and DSPE-PEG-COOH in an organic solvent and mixing them evenly, dropping the obtained mixed solution into pure water, and then transferring it to a dialysis bag for dialysis. After dialysis, the liquid in the dialysis bag is collected to obtain a PR@PBNPs solution; the molecular weight of PEG in the DSPE-PEG-COOH is 1000-3000 (preferably 1500-2500).

[0014] Preferably, in step S1, the amounts of puerarin, probucol, DSPE-PEG-COOH, organic solvent and pure water are in the ratio of 4-6 mg:8-12 mg:1 mg:0.8-1.2 mL:3-5 mL; the organic solvent is selected from DMF or DMSO;

[0015] Preferably, the dialysis time in step S1 is 5 to 14 hours (preferably 7 to 14 hours), and the molecular weight cutoff (MWCO) of the dialysis bag is 3000 to 4000 Da, preferably MWCO 3500 Da.

[0016] Preferably, S2, preparing MPR@PBNPs: mixing and evenly mixing the PR@PBNPs solution and the macrophage membrane in a mass ratio of 7 to 11:1, and then ultrasonicating in an ultrasonic water bath to coat the macrophage membrane on the PR@PBNPs to obtain macrophage membrane-coated bionic carrier-free nanodrug MPR@PBNPs; the mass ratio of the raw materials puerarin and probucol to the macrophage membrane in step S1 is 4 to 6:8 to 12:1.

[0017] Preferably, after the PR@PBNPs and macrophage membranes are mixed, they are vortexed every 7 to 12 minutes, and vortexed 2 to 3 times in total to fully mix the two; the ultrasonic time is 2.5 to 4 minutes, and the ultrasonic conditions are 40 to 44 kHz and 90 to 110 W power.

[0018] Preferably, the method for obtaining the macrophage membrane is as follows: culturing macrophages until the cell confluence reaches 80%-90%, collecting the cells and centrifuging to obtain a cell pellet, adding a buffer to resuspend the cells, placing the cell suspension in liquid nitrogen and repeatedly freezing and thawing 4-5 times, and grinding to fully break the cells, centrifuging the ground cell suspension in a centrifuge at low temperature and low speed for 7-12 minutes, and then taking the supernatant and centrifuging it at high speed for 15-20 minutes to obtain a macrophage membrane pellet.

[0019] The second aspect of the present invention provides a macrophage membrane-coated biomimetic carrier-free nanomedicine, which is prepared by any of the preparation methods described above.

[0020] The third aspect of the present invention provides the use of the above-mentioned macrophage membrane-coated biomimetic carrier-free nanomedicine in the preparation of a drug for treating atherosclerosis.

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

[0022] The present invention combines the self-assembly strategy of traditional Chinese medicine with the biomimetic technology of macrophage membrane coating to construct a highly effective and safe biomimetic Chinese herbal co-assembled nanodrug (MPR@PBNPs) for the treatment of AS. First, PR and PB were prepared into carrier-free self-assembled nanodrugs using a nanoprecipitation method. Macrophage membranes were then coated on the surface of PR@PBNPs using membrane coating technology, and further prepared into biomimetic carrier-free nanodrugs (MPR@PBNPs). In vitro experiments showed that MPR@PBNPs can effectively reduce oxidative stress and apoptosis of endothelial and macrophage cells; and can inhibit the uptake of lipids by endothelial cells and the formation of foamy macrophages.

[0023] The present invention prepares PR and PB into PR@PBNPs using TCM-SAN (Traditional Chinese Medicine-based Self-assembled Nanoparticles) technology, thereby achieving carrier-free and efficient loading of PR and PB. The PR@PBNPs are then coated with macrophage membranes to prepare MPR@PBNPs, which can better target and accumulate in AS lesions and improve the therapeutic effect. This overcomes the problems of complex preparation, the need for nanocarriers, and low drug loading in current nanomedicines for the treatment of AS. MPR@PBNPs injected intravenously can promote the enrichment of MPR@PBNPs in AS lesions due to the coating of macrophage membranes. In the plaque area, MPR@PBNPs release PR and PB drugs, effectively reducing oxidative stress and apoptosis of endothelial cells and macrophages; and can inhibit the uptake of lipids by endothelial cells and the formation of foamy macrophages, thereby achieving efficient and safe treatment of AS. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Are: (A) hydrodynamic diameter, (B) zeta potential and (C) PDI of MM, RR@PBNPs and MPR@PBNPs (n=3, mean±SD): TEM images of PR@PBNPs (D), MM (E), MPR@PBNPs (F) (scale bar: 100 nm): (G) Coomassie brilliant blue analysis of protein components of Raw264.7, MM, MPR@PBNPs and RR@PBNPs: (H) Western blot detection of CD47, CD68, integrin α4 and integrin β1 in Raw264.7, MM, MPR@PBNPs and RR@PBNPs.

[0025] Figure 2 Figures 1 and 2 show: (A) CLSM images of PR@PBNPs or MPR@PBNPs phagocytosed by normal and activated endothelial cells (scale bar: 10 μm); (B) Flow cytometric quantification and (C) corresponding fluorescence intensity analysis of nanoparticle uptake (n = 3, mean ± SD); (D) CLSM visualization (scale bar: 10 μm) and (E) flow cytometric images; and (F) quantitative analysis of DiD-labeled nanoparticles internalized by activated Raw264.7 macrophages (n = 3, mean ± SD).

[0026] Figure 3 The following are: (A) Fluorescence images of ROS in HUVECs after treatment with different drugs (Scale bar: 100 μm): (B) Flow cytometry detection of intracellular ROS generation levels after incubation of treatment groups with HUVECs; (C) Quantitative analysis of flow cytometry data (n=3, mean±SD): (D) Fluorescence images of live / dead cell staining of HUVECs in different treatment groups (Scale bar: 100 μm): (E) Flow cytometric profiles of live / dead cell staining of HUVECs after treatment: (F) Quantitative analysis of HUVECs viability (n=3, mean±SD): (G) Oil red O staining images of intracellular lipid droplet deposition after co-incubation of different treatment groups with HUVECs: (Scale bar: 100 μm); (H) Quantitative analysis of Oil red O staining images of intracellular lipid droplet deposition after co-incubation of different treatment groups with HUVECs (n=3).

[0027] Figure 4 Figure 3: (A) Cellular uptake of PR@PBNPs and MPR@PBNPs by normal and LPS-activated Raw264.7 cells; (B) Flow cytometric analysis of nanoparticle internalization by normal and activated macrophages; (C) Fluorescence intensity of internalized nanoparticles (n=3, mean±SD); (D) DCFH-DA fluorescence imaging of intracellular ROS levels (scale bar: 100 μm); (E) Flow cytometric quantification of ROS generation by macrophages; (F) Statistical analysis of ROS fluorescence intensity (n=3, mean±SD); (G) LPS stimulation Apoptosis images of macrophages treated with free PR-PB, PR@PBNPs or MPR@PBNPs under stimulation (scale bar: 100 μm): (H) Live / dead cell profiles by flow cytometry after treatment: (I) Quantitative analysis of cell viability (n=3, mean±SD): (J) Oil red O staining of intracellular lipid droplet deposition after co-incubation of different treatment groups with Raw264.7 cells (scale bar: 100 μm): (K) Quantitative analysis of Oil red O staining of intracellular lipid droplet deposition after co-incubation of different treatment groups with Raw264.7 cells (n=3).

[0028] Figure 5In vivo therapeutic evaluation of MPR@PBNPs: (A) Intravenous injection of DiD-PR@PBNPs or DiD-MPR@PBNPs into ApoE - / - Fluorescence imaging of the aorta of model mice after 24 hours; (B) Intravenous injection of DiD-PR@PBNPs or DiD-MPR@PBNPs into ApoE - / - Fluorescence imaging of the heart, liver, spleen, lung, and kidney of model mice after 24 hours; (C) Quantitative analysis of fluorescence signals of the aorta (n=3); *P<0.05; (D) Quantitative analysis of fluorescence signals of major organs of mice (n=3); (E) Quantification of aortic lesion area (n=3, mean±SD); (F) ApoE expression in different treatment groups after treatment - / - Representative images of aorta stained with Oil Red O in model mice; (G) Semiquantitative lipid deposition analysis (n=3, mean±SD); (H) Aortic root histochemistry: ORO (lipid), α-SMA (smooth muscle cells), toluidine blue (necrotic core), CD68 (macrophages), Masson's trichrome (collagen) (scale bar: 100 μm); (I) Smooth muscle cell proliferation / migration analysis (n=3, mean±SD); (J) Ratio of necrotic core to plaque (n=3, mean±SD); (K) Macrophage infiltration area (n=3, mean±SD); (L) Expression of collagen and MMP-9 in plaques (n=3, mean±SD); Statistical significance: *P≤0.05, **P≤0.01, ns indicates no significant difference.

[0029] Figure 6 The following are: (A) H&E-stained sections of major organs for histopathological evaluation (scale: 100 μm); (B) blood lipids: HDL (high-density lipoprotein), LDL (low-density lipoprotein), TG (triglyceride), and CHO (total cholesterol); (C) routine blood tests: platelets (PLT), white blood cells (WBC), red blood cells (RBC), and hemoglobin (HGB); (D) liver and kidney function indicators: AST (aspartate aminotransferase), ALT (alanine aminotransferase), CREA (creatinine), and urea (blood urea nitrogen); all data are mean ± SD (n = 3). DETAILED DESCRIPTION

[0030] The present invention will be further described below with reference to the embodiments, but the present invention is not limited thereto.

[0031] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0032] Example 1

[0033] 1. Main reagents and materials

[0034] Puerarin (CAS No. 3681-99-0) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Probucol (CAS No. 23288-49-5) was purchased from Shanghai MacLean Biochemical Co., Ltd. (Shanghai, China). DiD, also known as 1,1'-octadecanediyl-3,3,3',3'-tetramethyl-dicarbonitrogen-4-chlorobenzenesulfonate (CAS No. 127274-91-3), was purchased from Beijing Baisi Biotechnology Co., Ltd. (Beijing, China). CCK-8 assay kit (Cell Counting Kit-8) was purchased from APExBIO Technology LLC (Shanghai, China). Anti-CD68 polyclonal antibody and anti-integrin β1 polyclonal antibody were purchased from Wuhan Protein Technology Biotechnology Co., Ltd. (Wuhan). Rabbit anti-integrin α4 and rabbit anti-CD47 antibodies were purchased from Cell Signaling Technology, USA. Hoechst 33342, reactive oxygen species (ROS) detection kit, calcein-AM / PI double staining kit, and LysoTracker Green ddd-26 were purchased from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). OilRed O (ORO) staining kit was purchased from Solarbio Technology Co., Ltd. (Beijing, China). TNF-α was purchased from Beijing Zhongnuo Biotechnology Co., Ltd. (Beijing, China). Lipopolysaccharide (LPS) and tert-butyl peroxide (tBHP) were purchased from Sigma-Aldrich (USA). Oxidized low-density lipoprotein (ox-LDL) was purchased from Guangzhou Yuanyi Biotechnology Co., Ltd. (Guangzhou, China). DSPE-PEG 2000 -COOH (distearyl-phosphatidylethanolamine-polyethylene glycol-carboxyl) was purchased from Beijing Bailingwei Technology Co., Ltd. Mouse macrophage cell line Raw264.7 cells were purchased commercially.

[0035] 2. Methods

[0036] (1) Preparation of PR@PBNPs

[0037] PR@PBNPs were prepared by nanoprecipitation method. 5 mg puerarin (PR), 10 mg probucol (PB) and 1 mg DSPE-PEG were accurately weighed. 2000-COOH was dissolved in 1 mL of N,N-dimethylformamide (DMF) solution and mixed thoroughly. 1 mL of the resulting mixed solution was uniformly added to 4 mL of pure water (500 rpm). The mixture was transferred to a dialysis bag (MWCO 3500) and dialyzed overnight to remove unbound free drug and the organic solvent DMF. Finally, the solution was collected to obtain the nanodrug PR@PBNPs. To prepare DiD-labeled nanoparticles, 3 μL of a 1 mg / mL DiD solution was added to the organic phase DMF to prepare DiD-labeled PR@PBNPs (DiD-PR@PBNPs). The remaining steps were the same as those for the preparation of PR@PBNPs.

[0038] (2) Acquisition of macrophage membranes

[0039] After RAW264.7 cells were cultured in a cell culture flask until about 90% fusion, the cells were collected and centrifuged in a centrifuge tube (2000 rpm, 5 min) to obtain a cell pellet. Buffer was added to the cell pellet to resuspend the cells. Subsequently, the cell suspension was placed in liquid nitrogen and repeatedly frozen and thawed 5 times, and ground 20 times with a glass homogenizer to fully break the cells. The ground cell suspension was centrifuged at 1000 rpm for 10 min. The supernatant was centrifuged at 14000 rpm for 20 min to obtain a macrophage membrane (MM) precipitate. The protein content in the obtained MM was measured using a BCA protein concentration assay kit.

[0040] (3) Preparation of MPR@PBNPs

[0041] The PR@PBNPs solution prepared above was mixed with macrophage membranes (MM) at a mass ratio of 9:1. The mixture was vortexed three times every 10 minutes (to coat the macrophage membranes on the nanoparticles through external vibration). The mixture was then sonicated in an ultrasonic bath for 3 minutes (ultrasound parameters set at 42 kHz and 100 W power) to obtain MM-coated MPR@PBNPs. DiD-labeled MPR@PBNPs (DiD-MPR@PBNPs) were prepared using the same method as above.

[0042] (4) Characterization of nanomedicines

[0043] The particle size, potential and morphology of PR@PBNPs and MPR@PBNPs were detected using DLS, Zeta potential instrument (90Plus PALS, Brookhaven, USA) and TEM (Tecnai G212, FEI, USA).

[0044] (5) Cytotoxicity test

[0045] RAW264.7 cells or HUVECs were plated at 1 × 104 Cells were seeded in 96-well plates. After 12 hours of culture, different concentrations of PR@PBNPs and MPR@PBNPs (25, 50, 75, 100, 150, and 200 μg / mL) were used to incubate the cells for 24 hours. Subsequently, a culture medium solution containing CCK-8 was added (CCK-8 solution: culture medium = 1:9). After being placed in a cell incubator for 30 minutes, the absorbance was measured at 450 nm using a microplate reader (51119180ET, Thermo Fisher, China). The cell viability was calculated as follows:

[0046] Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%

[0047] in:

[0048] As: absorbance of the experimental group (culture medium containing cells, CCK-8, and test drug)

[0049] Ac: absorbance of the control group (culture medium containing cells, CCK-8, and no test drug)

[0050] Ab: absorbance of blank group (culture medium without cells and test drugs, CCK-8)

[0051] (6) Hemolysis test

[0052] Mix 1 mL of C57 mouse anticoagulated blood with 1.25 mL of 1× PBS to obtain diluted anticoagulated blood. Add 1 mL of 1 mg / mL PR@PBNPs or MPR@PBNPs solution to each tube in the experimental group. Add 1 mL of 1× PBS to each tube in the negative control group. Add 1 mL of pure water to each tube in the positive control group. Add 20 μL of diluted anticoagulated blood to each sample. Incubate at 37°C for 1 hour, then centrifuge at 2000 rpm for 5 minutes. Measure the absorbance of the supernatant at 545 nm using a microplate reader. Calculate the hemolysis rate using the following formula.

[0053] The hemolysis rate formula is as follows:

[0054] HR (%) = (AB) / (CB) × 100%

[0055] Where: HR: sample hemolysis rate

[0056] A: Absorbance of the sample

[0057] B: absorbance of negative control group

[0058] C: Absorbance of positive control group

[0059] (7) MPR@PBNPs protein characterization

[0060] Raw264.7 cells, MM, MPR@PBNPs, and PR@PBNPs were lysed using RIPA lysis buffer containing PMSF, and the protein lysates were collected. A 10% SDS-PAGE gel was prepared using a rapid gel kit. After the samples were loaded (15 μL per sample), the gel was run at 60 V for 25 minutes. When the protein sample reached the boundary between the stacking gel and the separation gel, the electrophoresis was switched to 110 V and continued for 80 minutes. The gel was stained with SimplyBlue and photographed using a gel imaging device.

[0061] Western blotting (WB) was used to verify the expression of CD47, CD68, integrin α4, and integrin β1 in Raw264.7 cells, MM, MPR@PBNPs, and PR@PBNPs. A 10% SDS-PAGE gel was prepared. After electrophoresis, the gel was electrotransferred on ice. The primary antibodies (CD47, CD68, integrin α4, and integrin β1) were then incubated overnight. After incubation with the secondary antibody for 1 hour, the gel was immediately visualized using a ChemiDoc MP (Bio-Rad, USA) instrument.

[0062] (8) Uptake of nanomedicines by macrophages and endothelial cells

[0063] Raw264.7 cells were collected at 3 × 10 5 Cells were seeded into each well of a confocal dish and cultured overnight for 12 hours. The cells were treated with LPS (treatment concentration of 1 μg / mL) for 24 hours. DiD-PR@PBNPs and DiD-MPR@PBNPs (treatment concentration of 100 μg / mL) were added to each group and treated with cells for 2 hours. The culture medium was removed, and the cell nuclei were then stained with Hoechst. Finally, photos were taken using a confocal microscope (SP8, Leica, Germany). For flow cytometry analysis, after incubation with nanodrugs, a Raw264.7 cell suspension was prepared. The ability of macrophages to take up nanodrugs was detected using a flow cytometer (CytoFLEX, Beckman Coulter, USA).

[0064] HUVECs were collected at 3 × 10 5 Cells were seeded into each well of a confocal dish and cultured overnight for 12 hours. The cells were treated with TNF-α (treatment concentration 10 ng / mL) for 12 hours. Subsequently, DiD-PR@PBNPs and DiD-MPR@PBNPs (treatment concentration 100 μg / mL) were added and incubated with the cells for 30 minutes. The culture medium was removed, and the cell nuclei were stained with Hoechst. Finally, photos were taken using a confocal microscope. For flow cytometry analysis, HUVECs suspension was prepared after nanodrug incubation. The ability of endothelial cells to take up nanodrugs was detected using flow cytometry.

[0065] (9) transwell

[0066] HUVECs were seeded in the upper chamber of a transwell chamber (14152, Corning, America), and Raw264.7 cells were seeded in the lower chamber where the slides were placed. HUVECs were stimulated with TNF-α (treatment concentration 10 ng / mL) for 12 h. DiD-PR@PBNPs and DiD-MPR@PBNPs (treatment concentration 200 μg / mL) were added to the upper chamber and treated with the cells for 24 h. The cell slides in the lower chamber were removed and fixed with 4% paraformaldehyde at room temperature for 5 min. The cell nuclei were stained with DAPI. After adding an anti-fluorescence quencher, the slides were photographed using a confocal microscope. For flow cytometry analysis, the Raw264.7 cell suspension in the lower chamber was collected for flow cytometry to detect fluorescence intensity.

[0067] (10) MPR@PBNPs' ability to scavenge ROS in inflammatory macrophages and endothelial cells

[0068] Raw264.7 cells were plated at 4 × 10 5 The cells were seeded in a 24-well plate at a density of 100 μg / mL. After incubation for 12 hours until the cells adhered, LPS (treatment concentration 1 μg / mL) was added to stimulate the cells for 24 hours. Subsequently, naked drug PR-PB, PR@PBNPs and MPR@PBNPs (treatment concentration of 50 μg / mL) were added to each group to treat the cells for 9 hours. The cells were stained with DCFH-DA, and the cell nuclei were stained with Hoechst. Finally, the pictures were taken with a microscope (DM3000, Leica, Germany). For flow cytometry analysis, the cells in different groups were stained with DCFH-DA, the cell suspensions were collected, and the ROS levels in Raw264.7 cells were detected by flow cytometry.

[0069] HUVECs were plated at 4 × 10 per well. 5 Cells were seeded at a density of 100 μg / mL in 24-well plates and stimulated with LPS (1 μg / mL) for 4 hours. The cells were then treated with bare PR-PB, PR@PBNPs, and MPR@PBNPs (at a treatment concentration of 50 μg / mL) for 6 hours. The cells were stained with DCFH-DA, and the nuclei were stained with Hoechst. Finally, the cells were photographed using a microscope. For flow cytometry analysis, cells from different groups were stained with DCFH-DA, and the cell suspensions were collected. ROS levels in HUVECs were measured using flow cytometry.

[0070] (11) Ability of MPR@PBNPs to inhibit foam cell formation

[0071] Raw264.7 cells or HUVECs were seeded in 96-well plates at a density of 5000 cells / well. The cells were pretreated with bare PR-PB, PR@PBNPs, and MPR@PBNPs (at a treatment concentration of 50 μg / mL) for 12 hours. Subsequently, the cells were treated with ox-LDL (at a treatment concentration of 50 μg / mL) for 12 hours. The cells were then stained with Oil Red O according to the kit protocol and photographed using a microscope.

[0072] (12) Anti-apoptosis ability of MPR@PBNPs

[0073] 4×10 per well 5 Raw264.7 cells or HUVECs were seeded in 24-well plates at a density of 100 μg / mL and cultured overnight. Cells were pretreated with naked PR-PB, PR@PBNPs, and MPR@PBNPs (treatment concentration of 50 μg / mL) for 12 hours. TBHP (treatment concentration of 5 mM / L) was then added for co-incubation. After incubation for 2 hours, the cells were stained with AM and PI. Finally, photos were taken using a microscope. For flow cytometry analysis, cells were stained with AM and PI, the cell suspension was collected, and cell apoptosis was detected using a flow cytometer.

[0074] (13)ApoE - / - Mouse modeling and treatment

[0075] Male ApoE - / - Mice were fed a high-fat diet for 7 weeks to establish an AS model. The AS model mice were divided into four groups. Each group received PR+PB (naive drug group), PR@PBNPs, or MPR@PBNPs via tail vein injection every three days for 45 days (injection dose: PR 6 mg / kg, PB 2 mg / kg). A high-fat diet was maintained throughout treatment.

[0076] (14) In vivo targeting ability

[0077] To verify the targeting effect of the nanomedicine on AS lesions, we injected DiD-PR@PBNPs and DiD-MPR@PBNPs into AS model mice via the tail vein. Twenty-four hours later, the mice were anesthetized and sacrificed, and the aorta and major organs (heart, liver, spleen, lungs, and kidneys) were removed. Images were taken using an in vivo animal imager (NightOWL II LB 983, Berthold, Germany) at an excitation wavelength of 640 nm, and the mean fluorescence intensity was analyzed using Image software.

[0078] (15) Effect of MPR@PBNPs on AS in vivo

[0079] After treatment, the mice were anesthetized and sacrificed. The entire aorta was isolated and fixed with 4% paraformaldehyde, stained with ORO, and photographed. Root sections of the aorta were prepared and stained with Oil Red O, TB, and Masson's stains. Immunohistochemistry for α-SMA and CD68 was also performed. The stained sections were photographed using a stereomicroscope.

[0080] (16) In vivo animal experiments to investigate the safety of MPR@PBNPs in AS mice

[0081] After the treatment, the blood of the mice was obtained. The blood routine levels of the mice were tested, and the levels of cholesterol (TC), triglycerides (TG), glutathione transaminase (ALT), aspartate transaminase (AST), urea nitrogen (BUN) and creatinine (CREA) in the blood were tested. Finally, the heart, liver, spleen, lungs and kidneys of the mice were obtained and fixed with 4% paraformaldehyde. After preparing their paraffin sections, hematoxylin-eosin (H&E) staining was performed. The H&E-stained sections were photographed using a stereomicroscope.

[0082] 3. Results

[0083] (1) Preparation and characterization of MPR@PBNPs

[0084] First, PR@PBNPs were prepared by nanoprecipitation. Dynamic light scattering (DLS) analysis showed that the hydrodynamic diameter of PR@PBNPs was 98.51±0.80nm ( Figure 1 A), and its polydispersity index (PDI) value was 0.14±0.01( Figure 1 C), and the zeta potential was -24.28±2.98mV( Figure 1 B). Transmission electron microscopy (TEM) showed that PR@PBNPs were uniformly spherical ( Figure 1 D). The hydrodynamic diameter of the extracted macrophage membrane (MM) was 272.50±6.15nm ( Figure 1 A), PDI was 0.26±0.003( Figure 1 C), and the zeta potential was -38.45±0.44mV( Figure 1 B). TEM shows that MM has obvious cell membrane structure ( Figure 1 E). The above results indicate that we have successfully prepared PR@PBNPs and obtained MM. MM was coated on the surface of PR@PBNPs by ultrasound to obtain MPR@PBNPs. The hydrodynamic diameter of MPR@PBNPs was 123.32±3.64nm( Figure 1 A), PDI was 0.19±0.03( Figure 1C), the hydrodynamic diameter of the uncoated PR@PBNPs increased by about 24.81 nm. The Zeta potential of MPR@PBNPs was -38.22±0.20 mV ( Figure 1 B), showing a Zeta potential similar to that of MM. The above results indirectly indicate that MM is coated on the surface of PR@PBNPs. The morphology of MPR@PBNP was observed by TEM to further confirm whether MM is coated on the surface of PR@PBNPs. TEM images showed that the surface of MPR@PBNPs had a clear cell membrane structure ( Figure 1 F).

[0085] The retention of proteins on MPR@PBNPs plays an important role in the biomimetic function of MPR@PBNPs. The protein composition on macrophages, MM, MPR@PBNPs and PR@PBNPs was detected by gel electrophoresis (SDS-PAGE). The results showed that MPR@PBNPs better retained the protein composition on MM ( Figure 1 G) Studies have shown that the CD47 protein on the surface of macrophages can recognize and bind to the SIRP-α receptor, thereby exerting a "don't eat me" signal. In addition, the surface of MMs contains integrins α4 / β1 that can specifically recognize VCAM-1, which is highly expressed on inflammatory endothelial cells. We used Western blot technology to detect these characteristic proteins on the surfaces of macrophages, MMs, and MPR@PBNPs. Figure 1 As shown in Figure H, we detected macrophage marker proteins CD68, CD47, and integrin α4 / β1 on the surfaces of macrophages, MMs, and MPR@PBNPs. These results indicate that MPR@PBNPs retain the protein composition and related characteristic proteins on MMs, providing a guarantee for their subsequent biological functions.

[0086] To preliminarily evaluate the biosafety of PR@PBNPs and MPR@PBNP, we tested their cytotoxicity and blood compatibility. At concentrations below 200 μg / ml, PR@PBNPs and MPR@PBNP had no significant effect on the viability of macrophages and HUVECs, and had a certain ability to promote cell proliferation. This may be due to the biological activity of puerarin (PR) in promoting cell proliferation. In vitro hemolysis experiments showed that PR@PBNPs and MPR@PBNPs did not induce hemolysis under high concentration conditions (1.5 mg / mL). The above results indicate that PR@PBNPs and MPR@PBNPs have good cell and blood compatibility.

[0087] (2) MPR@PBNPs promoted endothelial cell uptake and effectively crossed the endothelial layer

[0088] To reach the AS plaque, nanomedicines need to be effectively taken up by the endothelium of the AS lesion site and pass through the endothelial layer. To evaluate this, HUVEC cells activated by tumor necrosis factor-α (TNF-α) were co-incubated with DiD-labeled PR@PBNPs and MPR@PBNPs. Confocal microscopy (CLSM) and flow cytometry (FACS) were used to detect the uptake of nanomedicines by HUVEC cells ( Figure 2 AC). Compared with DiD-PR@PBNPs, DiD-MPR@PBNPs showed a stronger red fluorescence signal in activated HUVEC cells. These results indicate that MPR@PBNPs promoted the uptake of inflammatory endothelial cells. To further study the ability of nanomedicines to penetrate the vascular endothelium, we established an in vitro transweel model. DiD-PR@PBNPs and DiD-MPR@PBNPs were added to the upper chamber. After 24 hours, the uptake of nanomedicines by Raw264.7 cells in the lower chamber was observed by CLSM and FACS ( Figure 2 DF). Compared to DiD-PR@PBNPs, macrophages in the lower chamber took up significantly more DiD-MPR@PBNPs. Macrophage uptake of DiD-MPR@PBNPs was 1.2 times greater than that of DiD-PR@PBNPs. These results demonstrate that MPR@PBNPs are effectively taken up by inflammatory endothelial cells and cross the vascular endothelium, contributing to their accumulation in AS plaques.

[0089] (3) MPR@PBNPs inhibit ROS production, apoptosis, and lipid uptake in endothelial cells

[0090] During the development of AS, endothelial cells produce excessive reactive oxygen species (ROS) under various adverse factors, causing oxidative stress, thereby promoting the progression of AS. Therefore, anti-oxidative stress in endothelial cells can inhibit the development and deterioration of AS. HUVEC cells were treated with lipopolysaccharide (LPS) to produce ROS. Fluorescence microscopy and FACS analysis showed that naked drug PR-PB, PR@PBNPs and MPR@PBNPs significantly reduced ROS production in HUVEC cells after treatment ( Figure 3 AC). In addition, we evaluated the effect of nanomedicine on HUVEC cell apoptosis ( Figure 3 DF). The apoptosis rate of HUVEC cells was significantly reduced after treatment with PR@PBNPs and MPR@PBNPs. Studies have shown that endothelial cells in plaques take up lipids and transport them to the intima, thereby promoting the development of AS. Therefore, we tested the ability of nanomedicines to inhibit lipid uptake by endothelial cells. Both PR@PBNPs and MPR@PBNPs can effectively inhibit the uptake of lipids by endothelial cells ( Figure 3 G and Figure 3H). The above results indicate that MPR@PBNPs can effectively protect against endothelial cell oxidative stress, reduce endothelial cell apoptosis, and inhibit endothelial cell lipid uptake.

[0091] (4) MPR@PBNPs inhibit macrophage ROS production, apoptosis, and foam cell formation

[0092] Macrophages are important cells involved in the development of AS. Therefore, we evaluated the effects of nanomedicines on macrophage function. First, we investigated the uptake of PR@PBNPs by macrophages. Compared with DiD-PR@PBNPs, inflammatory macrophages have a stronger uptake capacity for DiD-MPR@PBNPs ( Figure 4 AC). Further studies found that MPR@PBNPs were able to enter the cytoplasm as the uptake time increased. The above results indicate that MPR@PBNPs can be effectively taken up by inflammatory Raw264.7 cells and enter the cytoplasm, which is conducive to the therapeutic effect of MPR@PBNPs on macrophages. In order to evaluate the ability of MPR@PBNPs to eliminate ROS in macrophages. We used LPS to stimulate Raw264.7 cells to produce ROS. The ROS level of Raw264.7 cells in the model group increased significantly ( Figure 4 D). After treatment with bare drug PR-PB, PR@PBNPs and MPR@PBNPs, the content of intracellular ROS was significantly reduced, among which MPR@PBNPs had the best effect ( Figure 4 E and Figure 4 F). In addition, MPR@PBNPs can effectively inhibit macrophage apoptosis ( Figure 4 GI). After macrophages take up a large amount of lipids, they form foam cells, which aggravates inflammation and plaque development. ORO staining was used to evaluate the inhibitory effect of MPR@PBNPs on foam cell formation. Compared with PR-PB, PR@PBNPs and MPR@PBNPs can significantly inhibit the formation of foam cells, and MPR@PBNPs has the best inhibitory effect ( Figure 4 JK).

[0093] (5) MPR@PBNPs targeting AS lesions in vivo and its anti-AS efficacy

[0094] First, the ability of MPR@PBNPs to target AS lesions in vivo was studied. DiD-labeled PR@PBNPs and MPR@PBNPs were injected into AS model mice via the tail vein. 24 hours later, the aorta and major organs were removed for fluorescence imaging. Compared with PR@PBNPs, MPR@PBNPs were more abundant and more strongly distributed in the aorta, indicating that MPR@PBNPs can efficiently target and aggregate at AS lesions ( Figure 5 A and Figure 5C). In addition, both PR@PBNPs and MPR@PBNPs were mainly enriched in the liver, indicating that they were mainly metabolized by the liver ( Figure 5 B and Figure 5 D) To investigate the effect of MPR@PBNPs on AS in vivo, ApoE - / - Mice were divided into four groups and treated with 5% glucose, bare PR+PB, PR@PBNPs, and MPR@PBNPs, respectively. Aortic oil red O staining showed that the plaque area in the MPR@PBNPs treatment group was the smallest compared with the other groups ( Figure 5 EF). Further analysis of aortic root plaques was performed. Compared with the treatment group, MPR@PBNPs effectively reduced lipid deposition, necrotic core formation, and collagen production within the plaques. In addition, MPR@PBNPs effectively reduced the content of smooth muscle cells and macrophages in the plaques ( Figure 5 GL). The above results show that MPR@PBNPs can effectively inhibit the formation of AS plaques.

[0095] (6) Safety of MPR@PBNPs in vivo

[0096] The biosafety of nanomedicines is crucial. We performed HE staining on sections of major organs to observe pathological changes. Compared with the PBS-injected control group, no significant damage to major organs was found after treatment with bare PR-PB, PR@PBNPs, and MPR@PBNPs ( Figure 6 A). Routine blood tests showed that after treatment, there were no significant differences in the white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB) and platelet (PLT) indicators in each group ( Figure 6 B). Blood lipid level examination revealed no significant difference in HDL, LDL, TG and CHO levels among the treatment groups ( Figure 6 C). More importantly, the main biochemical indicators of each group after treatment were normal, indicating that there was no damage to liver and kidney function ( Figure 6 D). In general, MPR@PBNPs have no obvious toxic side effects and have good biosafety.

Claims

1. A method for preparing a macrophage membrane-coated biomimetic carrier-free nanomedicine, characterized in that: The steps include: S1. Preparation of PR@PBNPs Puerarin PR and probucol PB were prepared into carrier-free self-assembled nanoparticles PR@PBNPs by nanoprecipitation method; S2. Preparation of MPR@PBNPs PR@PBNPs are mixed with macrophage membranes, and the macrophage membranes are coated on the surface of PR@PBNPs to prepare the biomimetic carrier-free nanomedicine MPR@PBNPs.

2. The preparation method according to claim 1, wherein: S1. Preparation of PR@PBNPs: Puerarin, probucol, and DSPE-PEG-COOH were dissolved in an organic solvent and mixed evenly. The resulting mixed solution was dropped into pure water and then transferred to a dialysis bag for dialysis. After dialysis, the liquid in the dialysis bag was collected to obtain a PR@PBNPs solution; the molecular weight of PEG in the DSPE-PEG-COOH was 1000-3000.

3. The preparation method according to claim 2, wherein: The amounts of puerarin, probucol, DSPE-PEG-COOH, organic solvent and pure water are in the ratio of 4-6 mg:8-12 mg:1 mg:0.8-1.2 mL:3-5 mL; the organic solvent is selected from DMF or DMSO.

4. The preparation method according to claim 2, wherein: The dialysis time is 5 to 14 hours, and the molecular weight cut-off (MWCO) of the dialysis bag is 3000 to 4000 Da.

5. The preparation method according to claim 2, wherein: S2. Preparation of MPR@PBNPs: Mixing the PR@PBNPs solution and the macrophage membrane in a mass ratio of 7 to 11:1, and then ultrasonicating in an ultrasonic water bath to coat the macrophage membrane on the PR@PBNPs to obtain macrophage membrane-coated bionic carrier-free nanodrugs MPR@PBNPs; the mass ratio of the raw materials puerarin and probucol to the macrophage membrane in step S1 is 4 to 6:8 to 12:

1.

6. The preparation method according to claim 5, characterized in that: After the PR@PBNPs and macrophage membranes are mixed, they are vortexed every 7 to 12 minutes, and vortexed 2 to 3 times in total to ensure that the two are fully mixed; the ultrasonic time is 2.5 to 4 minutes, and the ultrasonic conditions are 40 to 44 kHz and 90 to 110 W.

7. The preparation method according to claim 1, wherein: The method for obtaining the macrophage membrane comprises: culturing macrophages until the cell confluence reaches 80%-90%, collecting the cells and centrifuging to obtain a cell pellet, adding a buffer to resuspend the cells, repeatedly freezing and thawing the cell suspension in liquid nitrogen 4-5 times, and grinding to fully break the cells, centrifuging the ground cell suspension in a centrifuge at low temperature and low speed for 7-12 minutes, and then taking the supernatant and centrifuging it at high speed for 15-20 minutes to obtain the macrophage membrane pellet.

8. A macrophage membrane-coated biomimetic carrier-free nanomedicine, characterized by: The product is prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the macrophage membrane-coated biomimetic carrier-free nanomedicine according to claim 8 in the preparation of a drug for treating atherosclerosis.

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