Macrophage membrane functionalized manganese dioxide nano-drug as well as preparation method and application thereof in treatment of atherosclerosis

Abstract

The invention relates to the technical field of atherosclerosis related drugs, in particular to a macrophage membrane functionalized manganese dioxide nano-drug as well as a preparation method and application thereof in treatment of atherosclerosis. The invention relates to a macrophage membrane functionalized manganese dioxide nano-drug, which comprises an HMnO2 carrier, colchicine is loaded on the HMnO2 carrier, and a macrophage membrane is coated outside the HMnO2 carrier. The preparation can be applied to practical operation of preparing medicines for treating atherosclerosis. According to the technical scheme, the technical problem of limited treatment effect caused by single action mechanism of medicines for treating atherosclerosis in the prior art can be solved. The nano-drug in the scheme can remove ROS to play an anti-inflammatory effect, reprogram macrophages and promote cholesterol excretion, and is used for synergistically treating atherosclerosis. The macrophage membrane can target an inflammation part and escape from an immune system. And the side effect of the nano-drug is relatively small, so that the nano-drug has ideal popularization and application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of atherosclerosis-related drugs, and in particular to macrophage membrane functionalized manganese dioxide nanomedicine, a preparation method thereof, and application thereof in treating atherosclerosis. Background Art

[0002] Atherosclerosis (AS) is a common disease that seriously affects cardiovascular health. With the accumulation of lipids and the formation of plaques, the load on the circulatory system increases, leading to an increased risk of cardiovascular disease and increased susceptibility to plaques. Once vulnerable plaques are formed, they may lead to a series of irreversible complications, including myocardial infarction and heart failure, seriously endangering the patient's life and health. Therefore, in clinical practice, comprehensive management of AS has always been a key issue.

[0003] Studies have shown that macrophages play a crucial role in the development of AS because of their polarization, production of inflammatory factors, and lipid accumulation. Monocytes are recruited to the site of atherosclerosis and transformed into proinflammatory M1 macrophages, which produce reactive oxygen species (ROS). Excessive ROS production at the site of inflammation triggers a series of chain reactions of inflammation through lipid peroxidation. Therefore, some studies aim to alleviate AS by reducing ROS levels and thus inhibiting the production of inflammatory factors. In addition, some internalized lipids, such as ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1), are usually removed from cells by transporters; however, the hypoxic microenvironment at the AS site leads to upregulation of hypoxia-inducible factor 1α (HIF-1α), reducing the expression of ABCA1 or ABCG1 proteins, inhibiting cholesterol transport, and thus increasing lipid accumulation at the AS site. In this case, macrophages with abnormal lipid load are more likely to transform into foam cells, thereby exacerbating the formation of AS. Therefore, some studies have also attempted to treat AS by regulating cholesterol efflux. However, in recent years, the treatment of plaques has rarely increased the targeting of nanotherapy, while regulating the inflammatory microenvironment of plaques to synergistically promote cholesterol efflux to achieve a dual therapeutic effect. In addition, AS patients have individual differences, and plaques of different development stages can often be found in a single patient.

[0004] The traditional therapeutic drug colchicine (Col) is widely used as an anti-inflammatory alkaloid drug. Recent large-scale clinical trials have reported that colchicine can improve the survival rate of patients with cardiovascular disease (CVD) by about 26-29% by reducing the risk of ischemic stroke and myocardial infarction. However, the narrow therapeutic range and lack of targeting of Col have hindered its clinical application. Therefore, we need to explore a drug carrier that can be loaded with Col to synergistically enhance the therapeutic effect of AS while achieving drug targeting. Summary of the invention

[0005] The present invention is intended to provide a macrophage membrane functionalized manganese dioxide nanomedicine to solve the technical problem that the drugs for treating atherosclerosis in the prior art have limited therapeutic effects due to a single mechanism of action.

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

[0007] A macrophage membrane functionalized manganese dioxide nanomedicine comprising HMnO 2 Support, HMnO 2 The carrier is loaded with colchicine, HMnO 2 The carrier is coated with a macrophage membrane.

[0008] Using the above scheme, nanomedicine (Col@HMnO 2 -MM), which can remove ROS, exert anti-inflammatory effects, reprogram macrophages, and promote cholesterol efflux, and can be used for synergistic treatment of AS. Col@HMnO 2 -MM uses hollow mesoporous manganese dioxide (HMnO 2 ) nanozymes as carriers, which have a hollow mesoporous structure to deliver the anti-inflammatory drug Col and are coated with MM ( Figure 1 a) Due to the targeting and immune escape ability of MM, nanomedicines can quickly accumulate at AS sites and release drugs in the acidic inflammatory microenvironment. In addition, Col@HMnO 2 -MM has high antioxidant enzyme activity, can effectively eliminate ROS, inhibit the generation of inflammatory storms, promote macrophage reprogramming from M1 to M2, relieve hypoxic microenvironment, and promote cholesterol efflux ( Figure 1 b) Therefore, Col@HMnO 2 -MM has the potential to be developed into a targeted drug to synergistically treat AS by alleviating the inflammatory storm and promoting cholesterol efflux, and may provide a new therapeutic means for further exploration of nanomedicines for the treatment of various inflammatory cardiovascular diseases.

[0009] Furthermore, the HMnO 2 The support was prepared by the following method: 2 Surface construction of mesoporous MnO 2 layer, forming SiO 2 @MnO 2 The SiO 2 @MnO 2 SiO in the composite 2 , forming HMnO 2 Basic support; in HMnO 2 The base carrier is sequentially covered with polyacrylamine hydrochloride and polyacrylic acid to obtain HMnO 2 carrier.

[0010] Furthermore, HMnO 2 The surface area of ​​the carrier is 86.3m 2 / g, and the average pore size is about 11.7nm.

[0011] Furthermore, the macrophage membrane functionalized manganese dioxide nanomedicine had a particle size of 211.63±8.71nm.

[0012] The technical solution also provides a method for preparing macrophage membrane functionalized manganese dioxide nanomedicine, comprising the following steps performed in sequence:

[0013] S1: Synthesis of HMnO 2 Carrier;

[0014] S2: Colchicine and HMnO 2 The carriers were stirred and mixed to obtain Col@HMnO 2 ;

[0015] S3: Col@HMnO 2 The liposome is mixed with macrophage membrane and then extruded through a liposome extruder to obtain the liposome.

[0016] Furthermore, in S2, HMnO 2 The dosage ratio of the carrier to colchicine is 1:1-3:1.

[0017] Furthermore, in S3, Col@HMnO 2 The dosage ratio of the liposome to the macrophage membrane is 2:1-4:1; the number of extrusions is 15-25 times; and the pore size of the polycarbonate membrane of the liposome extruder is 400-800 nm.

[0018] Furthermore, in S1, SiO 2 After surface modification, KMnO 4 The solution was stirred and reacted, MnO 2 In SiO 2 The mesoporous layer is grown on the hard template to obtain SiO 2 @MnO 2 Complex;

[0019] Use alkali to etch the SiO 2 @MnO 2 SiO in the composite 2 , forming HMnO 2 Basic support; in HMnO 2 The base carrier is sequentially covered with polyacrylamine hydrochloride and polyacrylic acid to obtain HMnO 2 Carrier;

[0020] For SiO2 The surface modification method is: SiO 2 Mix with polyvinyl pyrrolidone to obtain SiO 2 @PVP; Then, in SiO 2 Water, meta-diphenol and formaldehyde are added to PVP, and then ammonia is added to the mixture to obtain SiO 2 @RF, Complete SiO 2 surface modification.

[0021] The technical solution also provides an application of a macrophage membrane functionalized manganese dioxide nanomedicine in the preparation of a drug for treating atherosclerosis.

[0022] Furthermore, drugs for treating atherosclerosis are used to eliminate reactive oxygen species, promote the transformation of macrophages from M1 to M2, inhibit verification, alleviate the hypoxic microenvironment at the site of atherosclerosis and promote cholesterol efflux.

[0023] In summary, the principle and beneficial effects of this technical solution are:

[0024] Manganese-based nanozymes have the function of simulating the activity of catalase (CAT) and superoxide dismutase (SOD). The hollow structure can be loaded with a series of drugs, providing a higher surface area to volume ratio, and having more reaction sites than the corresponding solid structure, thereby producing greater enzyme-like activity. The hollow mesoporous manganese-based nanocarrier can not only load Col, but also enhance the synergistic therapeutic effect of the drug due to its CAT effect. Nanoparticles wrapped with macrophage membranes (MM) can naturally target inflammatory sites, escape phagocytosis by the immune system, and achieve effective circulation in the body. Col has a narrow therapeutic range and lacks targeting. The use of the carrier of this scheme can completely overcome the above defects. Therefore, using manganese-based nanozymes as carriers to load Col and wrap MM can prevent it from being phagocytosed by phagocytes, so that the drug has a longer circulation time while enhancing the targeting of the drug, thereby improving its clinical application.

[0025] This technical solution is the first to load Col into HMnO 2 -MM, the two played a synergistic role. In particular, in terms of promoting the transformation of macrophages from M1 to M2 and reducing the area of ​​aortic lesions, the two produced a synergistic effect. For details, see Example 6 (for example: Figure 6 b), Example 9 (for example: Figure 8 f).

[0026] This technical scheme designed and prepared the multifunctional nanodrug Col@HMnO 2-MM to achieve macrophage-targeted AS treatment. Nanomedicines can evade immune system engulfment to achieve long-term circulation and can target AS inflammatory sites to achieve drug release. Col@HMnO 2 -MM has the expected ROS scavenging ability and can reprogram macrophages to inhibit the inflammatory storm of AS. In addition, nanomedicine can downregulate the expression of HIF-1α through its own oxygen production, improve the hypoxic microenvironment of AS, and thus promote cholesterol efflux. Compared with a single treatment approach, the synergistic combination of the two treatment approaches can improve the efficacy of Col@HMnO 2 -The efficacy of MM on AS. Col@HMnO 2 -MM has been used for APOE - / - Mice, and studies have shown that it has enhanced pharmacokinetic properties and active targeting properties, with the expected anti-AS efficacy. 2 -MM provides a more comprehensive and targeted therapeutic strategy for the management of AS. However, there are many ways that can lead to the development of AS. In addition, manganese-based nanozymes can also be used for magnetic resonance imaging, and the dynamic monitoring of their therapeutic effects may be a hot topic for future research.

[0027] In addition, compared with a single treatment strategy, the synergistic treatment strategy can achieve the expected AS treatment effect. To combat chronic AS disease, long-term chronic treatment is usually required. The nanoparticles provided in this scheme are plaque-targeted therapeutic drugs with fewer side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Col@HMnO 2 -Schematic diagram of MM nanomedicine-mediated synergistic therapy for AS.

[0029] Figure 2 Col@HMnO of Example 1 2 -MM characterization and Col release experimental results (ad: SiO 2 、SiO 2 @MnO 2 、HMnO 2 and Col@HMnO 2 - TEM image of MM; eg: SiO 2 、SiO 2 @MnO 2 and HMnO 2 SEM image; h: Western blot analysis of Col@HMnO 2 , MM and Col@HMnO 2 -MM;i:Col@HMnO 2 -HRTEM image of MM; j: SiO2 、SiO 2 @MnO 2 and HMnO 2 XRD analysis; k: XPS spectrum; l: HMnO 2 Mn 2p XPS spectrum; m: Col@HMnO 2 -Elemental mapping of MM; n: MM, Col@HMnO 2 and Col@HMnO 2 -MM particle size (n=3); o: SiO 2 、SiO 2 @MnO 2 、HMnO 2 , MM and Col@HMnO 2 -Zeta potential measurement of MM (n=3); p: Col, HMnO 2 and Col@HMnO 2 UV-visible spectra of Col@HMnO at pH 5.5 or 7.4 2 and Col@HMnO 2 - Release rate of Col in MM; **p<0.01).

[0030] Figure 3 In vitro evaluation of Col@HMnO for Examples 2 and 3 2 -MM's active oxygen scavenging properties (a: Col@HMnO 2 -Schematic diagram of MM's mechanism of clearing ROS; b: dissolved oxygen meter detects O 2 Concentration; ch: Col@HMnO 2 -MM's H 2 O 2 , ·OH, ·O 2 - , DPPH, ·R, ·OR clearance; i: Col@HMnO 2 -MM inhibitory effect on LPO with increasing concentration; j: Col@HMnO 2 - CCK-8 analysis of MM; k: Col@HMnO 2 -MM hemolysis assay).

[0031] Figure 4 The in vitro Col@HMnO 2 -MM on the cell internalization and antioxidant capacity of experimental results (ab: CLSM detection of LPS-stimulated or not stimulated RAW264.7 cells DiI-Col@HMnO 2 and DiI-Col@HMnO 2-MM uptake; c: activated macrophages and Col@HMnO 2 -MM co-incubation for 0.5h and 4h; d: CCK-8 method to evaluate cell survival; e: flow cytometry to detect apoptosis of RAW264.7 cells after different treatments; f: fluorescence image of stained macrophages is consistent with the results in e; g: immunofluorescence image of JC-1 stained macrophages under different treatments; h: quantitative fluorescence intensity analysis of the red-green ratio of JC-1 detection in g; **p<0.01, **p<0.001, ****p<0.0001; scale bar: 50μm).

[0032] Figure 5 The macrophages Col@HMnO of Example 6 2 -MM's ROS scavenging ability and anti-inflammatory properties. (a: Total ROS, ·O 2 - and H 2 O 2 CLSM images, scale bar: 50 μm; bd: total ROS, ·O under different treatments 2 - and H 2 O 2 Fluorescence quantitative intensity analysis; e: ROS levels of RAW264.7 cells in different pathways; Effects of different treatments on the content of activated macrophage inflammatory factors (f) IL-1β, (g) IL-6, (h) TNF-α; i: CLSM images of macrophages stained with phalloidin. Scale bar: 25μm; j: Representative CLSM images of RAW264.7 cell surface biomarkers (M1: CD86, M2: CD206), scale bar: 50μm; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0033] Figure 6 These are the results of the macrophage M1 to M2 polarization experiment in Example 6 (relative expression levels of M1 and M2 related genes of RAW 264.7 after different treatments; data format: mean ± standard deviation, n = 3; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0034] Figure 7 Col@HMnO of Example 7 2 -MM on macrophage ox-LDL internalization, cholesterol transport and foam cell formation (a: Col@HMnO 2-Schematic diagram of the cholesterol transport pathway regulated by MM; b: CLSM images showing the cellular uptake of DiI-oxLDL by LPS-treated macrophages; c: CLSM staining of HIF-1α in macrophages under different treatments; d: Relative expression of HIF-1α mRNA after different treatments; e: Western blot detection of HIF-1α protein expression; f: CLSM staining of ABCG1 in macrophages under different treatments; g: Fluorescence quantitative intensity analysis of f; h: Relative expression level of ABCA1 mRNA under different treatments; i: CLSM images showing the remaining total cholesterol in RAW264.7 cells; j: Cholesterol efflux from RAW264.7 cells under different treatments; k: Optical microscopy images of ox-LDL internalization in activated macrophages; l: ORO-positive area in quantification (h); **p<0.01, **p<0.001, ****p<0.0001; Scale bar: 50μm).

[0035] Figure 8 Col@HMnO of Examples 8 and 9 2 -MM in vivo targeting ability and therapeutic effect (a: intravenous administration of DiR labeled Col@HMnO 2 -MM nanomedicine after the aorta was collected at different time points; b: DiR labeled Col@HMnO in CLSM imaging 2 -MM (red) and CD68-labeled cells (green) colocalization; c: Schematic diagram of AS treatment regimen; d: ORO staining of whole aorta of mice in each group; e: H&E stained aortic root sections; f: quantitative evaluation of ORO-positive areas in aorta; g: quantitative analysis of plaque size based on H&E staining images; scale bar: 100 μm; *p < 0.05, ***p < 0.001, ****p < 0.0001).

[0036] Fig. 9 APOE of Example 9 - / - Results of the study on the mechanism and effect of AS treatment in mice (a: Typical CLSM images of aortic sections stained with CD68 antibody, MMP-9 antibody, α-SMA antibody and Masson's trichrome staining; b: Quantitative analysis of macrophages, MMP-9, plaque collagen area and VSMCs in a, scale bar: 100 μm; fg: APOE after various treatments - / -Fluorescence images and quantitative analysis of DHE-stained sections of mouse aorta, scale bar: 200 μm; h: iNOS immunofluorescence staining of aortic segments of mice treated with different methods, scale bar: 100 μm; i-1: ELISA kits to detect serum CRP, IL-6, IL-1β, and TNF-α levels; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0037] Fig.10 The SiO 2 @RF's electron microscope image.

[0038] Fig.11 The HMnO of Comparative Example 1 2 Electron microscope image of . DETAILED DESCRIPTION

[0039] The present invention is further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial sources. More specifically, the information of materials and reagents is as follows:

[0040] Tetraethyl orthosilicate (TEOS), polyvinyl pyrrolidone (PVP, Mw = 10000), polyacrylamine hydrochloride (PAH, Mw ≈ 15000), polyacrylic acid (PAA, Mw ≈ 1800), resorcinol (AR, 99%), formaldehyde solution, sodium carbonate (NaOH), potassium permanganate (KMnO 4), ammonia solution, and hexadecyltrimethoxysilane (HDTMS, GC ≥ 85%) were purchased from Chinese Medicine Chemical Reagent Co., Ltd. Colchicine was purchased from Chengdu Must Biotechnology Co., Ltd. (Chengdu, China). RAW 264.7 cells were purchased from the Chinese Academy of Sciences (Shanghai, China). Fetal bovine serum (FBS) was purchased from Inner Mongolia Opcell Biotechnology Co., Ltd. (Inner Mongolia, China). Calcein-AM, propidium iodide (PI), DiI, DiR, DHE, 4,6-diamino-2-phenylindole (DAPI), paraformaldehyde, DCFH-DA, DAF-FM DA, and actin tracker red rhodamine were purchased from Shanghai Biyuntian Biotechnology Co., Ltd. Cholesterol was from Avanti Polar Lipids, Inc (USA). Cell counting kit-8 (CCK-8) was purchased from Beitian Biotechnology Institute (Shanghai, China). CD86, CD206, HIF-1α, and ABCG1 antibodies were purchased from Shanghai Biyuntian Biotechnology Co., Ltd. Integrin α, integrin β, and CD47 antibodies were from Hunan Aifang Biological Co., Ltd. ELISA kits for IL-6, IL-1β, TNF-α, IL-18, and CRP were purchased from Jiangsu Sumei Biotechnology Co., Ltd.

[0041] Example 1: Col@HMnO 2 -MMM Synthesis

[0042] (1) Silica nanospheres (SiO 2 )

[0043] 98 mL of ethanol and 10 mL of water were mixed, and then 1.25 mL of ammonia (28%) and 5 mL of TEOS were added. After stirring at room temperature (25°C) for 20 h, the SiO 2 , rinse with ethanol and water several times.

[0044] (2)SiO 2 @MnO 2 Synthesis

[0045] First, 50 mg SiO 2 The SiO2 was prepared by stirring 0.5 g of PVP (with a particle size of 60-80 nm) at room temperature overnight (25 ± 5 ° C, 12 h-16 h; stirring was performed by conventional 800-1200 rpm magnetic stirring, or conventional 400-600 rpm shaking, both of which can achieve the purpose of this scheme; the stirring operation or overnight at room temperature mentioned below all adopts the above conditions and will not be repeated), and then washed by centrifugation at 8000 r / min of deionized water for 3 times, each time for 10 min, to obtain SiO2. 2@PVP product. Then, 25.5 mL of water, 20 mg of m-diphenol and 28 μL of formaldehyde were added to the obtained product. After thorough mixing, 100 μL of (2.8%) ammonia water was added, the temperature was raised to 60°C, stirred for 2 h, the temperature was raised to 100°C, stirred for 2 h, washed by centrifugation with water and cooled 3 times, the reaction was terminated, and SiO 2 @RF product. Add 5 mL of water to 10 mg of SiO 2 @RF, mix thoroughly, then add 3mL KMnO 4 The mixture was stirred at room temperature for 4 hours and then washed with water three times by centrifugation to obtain the desired SiO 2 @MnO 2 Through the above process, it is possible to 2 Hard template surface covered with mesoporous MnO 2 layer, in order to prepare the hollow MnO 2 Create conditions.

[0046] (3)HMnO 2 Synthesis

[0047] To achieve a hollow structure, 10 mL of 1 M NaOH and 50 mg of SiO 2 @MnO 2 The mixture was stirred at room temperature overnight. The mixture was then washed with water and centrifuged three times to obtain HMnO 2 (Hollow mesoporous manganese dioxide). 2 Dispersed in 5 ml H 2 O, add 5 ml of cationic polymer polyacrylamine hydrochloride solution (PAH, 5 mg / mL), perform conventional ultrasonic treatment for 3 min (optional time range 3-5 min), stir for 3 h, and collect the solution. Rinse the sample with deionized water until a colorless and transparent supernatant is obtained. 2 -PAH was slowly added into 5 mL (5 mg / mL) of anionic polymer polyacrylic acid (PAA) solution, conventionally sonicated for 3 min, stirred for 3 h, and washed and collected for later use.

[0048] (4)MM preparation

[0049] RAW264.7 cells were inoculated in culture dishes and incubated for 24 h. The culture medium was collected and centrifuged to separate cells and debris. The supernatant was concentrated in a 10 kDa molecular weight spin column (Millipore, USA) for 8 min (2500×g, 4°C). After the collected liquid was centrifuged at 4°C for 40 min, the resulting precipitate was resuspended in sterile phosphate buffered saline (PBS) and stored at -80°C for subsequent experiments.

[0050] (5)Col@HMnO2 -MM's preparation

[0051] Due to the modification of PAA / PAH, HMnO 2 Stir with Col at room temperature for 24 h (HMnO 2 The mass ratio of HMnO to Col is 1:2. In actual operation, HMnO 2 :Col=1:2-3:1), and obtain Col@HMnO 2 Preparation of MM coated with Col@HMnO 2 -MM, Col@HMnO 2 The mixture with MM was extruded by a liposome extruder (Col@HMnO 2 The mass ratio of HMnO to MM is 3:1. In actual operation, Col@HMnO 2 :MM=2:1-4:1), the liposome extruder uses a polycarbonate membrane with a pore size of 400nm (pore size of 400-800nm ​​can be used), and the extrusion process is repeated 18 times (15-25 times of extrusion can be used). Then, centrifugation at 13000rpm for 10min was performed to obtain Col@HMnO 2 -MM nanomedicine.

[0052] (6)Col@HMnO 2 -MM characterization experiments

[0053] SiO was observed by TEM (FEI Tecnai G2F30, USA). 2 、SiO 2 @RF、SiO 2 @MnO 2 、HMnO 2 and Col@HMnO 2 -MM morphology. TEM was used to analyze the Col@HMnO 2 XRD (BRUKER D8ADVANCE, Germany), XPS (Thermo Scientific K-Alpha) and FTIR (Thermo Scientific Nicolet iS5) were used to study the HMnO 2 The structure of HMnO was determined by BET surface area adsorption instrument (ASAP 2460, USA). 2 The specific surface area and corresponding pore size distribution of Col@HMnO were determined by BCA protein assay kit. 2 There is no Col in the supernatant, so the drug loading and encapsulation efficiency are calculated: Encapsulation efficiency = (W Total added Col -WUnloaded Col / W Total added Col )×100%;Drug loading efficiency=(W Total added Col -W Unloaded Col ) / (W Loaded Col +W Total added HMnO2 )×100%. Dynamic light scattering (DLS) laser particle size analyzer (Zetasizer Nano ZS90, UK) was used to analyze the MM, Col@HMnO 2 and Col@HMnO 2 -MM particle size and Zeta potential, and observed Col@HMnO 2 -MM particle size stability at 1, 3, 5 and 7 days. Western blot analysis was used to verify the HMnO 2 , MM and Col@HMnO 2 -Protein profile of MM.

[0054] (7) Col release characteristics experiment

[0055] To evaluate the drug release pattern of Col, 1 mL of Col@HMnO 2 -MM (1 mg / mL) or Col@HMnO 2 (2 mg / mL) was placed in dialysis bags with a molecular weight of 3500 Da. These dialysis bags were immersed in 50 mL of PBS at pH 7.4 or 5.5 and maintained at 37°C. At specific time intervals, samples of the release medium (500 μL) were obtained. The cold concentration was determined by UV-visible spectroscopy.

[0056] (8) Experimental results

[0057] SiO 2 The initial core structure is covered with MnO 2 , and then use NaOH to etch the inner SiO 2 ( Figure 1 a) to construct hollow mesoporous HMnO 2 Nanozyme. Transmission electron microscopy (TEM) results show that SiO 2 ( Figure 2 a) SiO 2 @RF、SiO 2 @MnO 2 ( Figure 2 b) and HMnO 2 ( Figure 2 c) Successful synthesis, especially HMnO 2 The hollow mesoporous structure of SiO 2 、SiO 2 @MnO 2 and HMnO2 The structure of Figure 2 e-2g). for SiO 2 、SiO 2 @MnO 2 and HMnO 2 X-ray diffraction (XRD) analysis showed that HMnO 2 Made of birnite-type manganese oxide (δ-MnO 2 ,JCPDS card number 18-0802)( Figure 2 j). X-ray photoelectron spectroscopy (XPS) shows a strong Mn signal ( Figure 2 k,2l), showing two characteristic peaks at 642.4eV and 654.2eV. The spin-orbit peaks of Mn(IV)2p2 / 2 and Mn(IV)2p2 / 3 further strengthen its formation. Then, we further analyzed the valence state of manganese and found that the synthesized hollow mesoporous manganese dioxide has 2-valence and 4-valence. According to the pore size analyzer (BET) measurement, HMnO 2 The surface area is 86.3m 2 / g, and the average pore size is about 11.7nm. These results are for HMnO 2 The mesoporous structure of Col provided strong evidence and suggested its potential for drug loading. 2 In the nanozyme carrier, Col@HMnO 2 ( Figure 2 p). According to the standard curve generated by the BCA kit, the HMnO 2 The percentage of Col in the HMnO@Col@H ... 2 Coating macrophage membrane MM to obtain Col@HMnO 2 -MM. Figure 2 TEM images of d and Figure 2 High-resolution transmission electron microscopy (HRTEM) confirmed that Col@HMnO 2 The successful preparation of -MM visually proves the existence of MM coating. In addition, elemental mapping analysis ( Figure 2 m) and energy dispersive X-ray (EDX) spectroscopy confirmed the Col@HMnO 2 -The element ratio of Mn, O and P in MM, with special attention paid to the phosphorus (P) content in MM. Dynamic light scattering (DLS) shows that Col@HMnO 2 The average diameter of the particles was approximately 181.10 ± 4.36 nm, which increased to 211.63 ± 8.71 nm after adding the MM coating (307.47 ± 8.15 nm). Figure 2 n) SiO 2 、SiO2 @MnO 2 、HMnO 2 , MM and Col@HMnO 2 The Zeta potentials of -MM were -37.83±0.42mV, -35.40±0.61mV, -34.60±1.14mV, -16.17±0.55mV and -22.87±0.61mV respectively. Figure 2 O). The above results indicate that the MM coating was successfully synthesized. 2 、SiO 2 @RF、SiO 2 @MnO 2 and Col@HMnO 2 -MM particle size and dispersibility remained stable within one week. Integrin α, CD47 and integrin β were widely expressed on the surface of MM and played an important role in the targeting and activation of macrophages in plaques. Western blot analysis showed that the above characteristic protein markers were expressed in MM and Col@HMnO 2 -MM, confirming that Col@HMnO 2 -MM can replicate the corresponding role of macrophages in plaques ( Figure 2 h). In vitro drug release experiments were performed at pH 7.4 or 5.5, demonstrating that Col can be slowly released into the inflammatory environment ( Figure 2 q). At pH 5.5, after 4 hours, Col@HMnO 2 -MM or Col@HMnO 2 Almost 80% of Col was released from the solution at pH 7.4, while Col@HMnO 2 Only 40% of Col in HMnO-MM was released within 4 hours. 2 -MM nanomedicine has been successfully synthesized and can be degraded in the inflammatory acid environment of atherosclerosis, which is expected to achieve a therapeutic effect.

[0058] Example 2: In vitro Col@HMnO 2 -MM's performance in removing ROS

[0059] (1) ·O 2 - Clearing performance

[0060] The superoxide anion (·O) is produced by the reaction of xanthine oxidase with xanthine 2 - ). Then add Col@HMnO 2 -MM, reacted for 5 min and 10 min, and the residual ·O was measured by electron paramagnetic resonance (EPR)2 - .

[0061] (2) OH scavenging performance

[0062] Using TiO 2 / UV system was used to prepare hydroxyl radicals (·OH) under 340nm ultraviolet light. Then 30μg / mL Col@HMnO 2 -MM, react for 5min and 10min, and determine the remaining ·OH by EPR method.

[0063] (3) Clear H 2 O 2 Performance

[0064] H 2 O 2 (10 mM) was used as the control group, and the sample Col@HMnO 2 -MM was added to the reaction mixture for 5 min and 10 min, respectively, and H 2 O 2 Clearance rate.

[0065] (4)O 2 Concentration monitoring

[0066] Col@HMnO 2 -MM (30 μg / mL) was added to 10 ml H 2 O 2 (10mM) solution. Col@HMnO alone 2 -MM group and individual H 2 O 2 The solution group served as the control group. Dissolved oxygen was used to measure the O 2 Horizontal, lasts 10 minutes.

[0067] (5) DPPH scavenging performance

[0068] DPPH solution (0.04 mg / mL) was prepared in anhydrous ethanol. Then Col@HMnO 2 -MM, react for 5min and 10min, and determine the DPPH residue by EPR method.

[0069] (6) Eliminate alkyl and alkoxy free radicals

[0070] The PAA / UV system was used to prepare alkyl radicals and alkyl radicals. Then Col@HMnO 2 -MM, reaction time 5min and 10min, the residual alkyl radicals and alkoxy radicals were determined by EPR method.

[0071] (7)Col@HMnO 2 -MM LPO inhibition experiment

[0072] 400 μL of different Col@HMnO 2 -MM solutions (10, 20, 30, 40 and 50 μg / mL) were mixed with the prepared MDA working solution. Next, the samples were heated in boiling water at 95°C for 50 minutes. After cooling, the test solution was centrifuged at 15,000 rpm for 20 minutes, and the absorbance of the supernatant was measured at 535 nm using a UV-visible spectrophotometer.

[0073] (8) Experimental results

[0074] As a highly reactive molecule, reactive oxygen species plays a unique role in maintaining vascular homeostasis. However, excessive production of ROS can lead to irreparable damage to blood vessels, ultimately triggering a series of inflammatory responses and exacerbating the progression of AS. Therefore, it is urgent to study new nanomedicines with the ability to scavenge reactive oxygen species. We tested Col@HMnO 2 -MM's active oxygen scavenging properties and its overall antioxidant properties Figure 3 As shown in a. Among various ROS, H 2 O 2 , ·OH and ·O 2 - are the main types of free radicals. We first examine these three free radicals, such as Figure 3 As shown in c, 3d and 3e, in Col@HMnO 2 After 5 and 10 minutes of treatment with -MM, all three free radicals were significantly reduced. In addition, analysis of dissolved oxygen levels showed that Col@HMnO 2 -MM and H 2 O 2 After the reaction, O 2 The concentration increased significantly, indicating that Col@HMnO 2 -MM promotes H 2 O 2 To O 2 The conversion of Figure 3 b). Inspired by the above results, we used DPPH (a common free radical compound) to further explore the ability of the nanomedicine to remove reactive oxygen and nitrogen species (RONS). 2 -MM also showed strong nitrogen scavenging activity ( Figure 3 f). Alkyl radicals (·R) and alkoxy radicals (·OR) are specific free radicals for lipid peroxidation. Therefore, we also confirmed that Col@HMnO 2 -MM's ability to remove lipid peroxides. Figure 3As shown in Figures 3g and 3h, the levels of ·R and ·OR decreased significantly after 5min and 10min of treatment. Similarly, lipid peroxidation (LPO) is also closely involved in the entire oxidative damage process, and various excessive ROS-related free radicals can damage vascular cells by inducing LPO. Therefore, we evaluated the ability of nanomedicines to scavenge lipid peroxides. Figure 3 As shown in i, the nanodrug Col@HMnO 2 -MM reduced the level of lipid peroxides in a concentration-dependent manner, thereby hindering LPO to a certain extent. The above in vitro experimental results prove that the nanodrug has a strong ROS scavenging ability and plays an important role in regulating vascular homeostasis.

[0075] Example 3: Col@HMnO 2 - Biosafety of MM

[0076] (1) Hemolysis analysis

[0077] Fresh blood samples of SD rats were centrifuged (3000 r / min) and washed three times to obtain red blood cells. 2 -MM, Col or Col@HMnO 2 -MM incubated for 4 hours, then centrifuged at 13500rpm for 5 minutes. The absorbance of the supernatant at 540nm was evaluated using an ELISA instrument. Red blood cells were placed in water as a positive control and in PBS as a negative control. The hemolysis rate was calculated as follows: Hemolysis rate % = (absorbance of the experimental group - absorbance of the negative group) / (absorbance of the positive group - absorbance of the negative group) × 100%.

[0078] (2) Cytotoxicity

[0079] RAW264.7 cells were incubated in 96-well plates for 24 h, and different concentrations (10, 20, 30, 40, and 50 μg / mL) of Col@HMnO 2 -MM was incubated for another 24 h, and its cytotoxicity was determined by CCK-8.

[0080] (3) In vivo biosafety

[0081] Healthy C57BL / 6J mice injected with HMnO 2 -MM, Col and Col@HMnO 2 -MM. After 3 days, we tested the body weight of mice in each group. Fresh blood was collected for biochemical and blood routine tests, and then organs and tissues were collected for H&E staining.

[0082] (4) Experimental results

[0083] We tested whether this synthesized nanomedicine can cause hemolysis in biological systems. The hemolysis rate of each experimental group was significantly lower than that of the positive control group (water), and far lower than 5%, which verified the good blood compatibility of the nanomedicine ( Figure 3 j). CCK-8 method was used to evaluate the activity of Col@HMnO at different concentrations. 2 -MM (10, 20, 30, 40 and 50 μg / mL) was toxic to RAW264.7 cells, and no obvious cell abnormalities were observed within 24 hours ( Figure 3 k). Subsequently, we evaluated the damage of different treatments to macrophages. Live / dead fluorescence staining showed no obvious abnormalities in RAW264.7 cells treated with nanomedicine. Body weight measurement, hematological analysis, blood biochemical analysis, and H&E staining histological analysis of major organs were used for in vivo biosafety evaluation. 2 -MM biosafety. After intravenous injection, the four groups (normal saline, HMnO 2 -MM, free Col and Col@HMnO 2 -MM) mice. In addition, clinical biochemical analysis showed that there was no difference in the levels of ALT, AST, CREA and urea among the four groups, indicating that systemic administration did not affect the biological functions of the liver and kidneys. In addition, hematological analysis showed that there was no significant difference between the treated group and the untreated normal group of mice. H&E staining histology confirmed that there were no significant changes in the main organs (including heart, liver, spleen, lung, and kidney) of the four groups of mice. In summary, the above results confirm that Col@HMnO 2 -MM has good biocompatibility and safety both in vivo and in vitro.

[0084] Example 4: Cellular Uptake

[0085] (1) Uptake of nanomedicine by unactivated macrophages

[0086] RAW 264.7 cells were obtained from Chongqing Key Laboratory of Ultrasound Molecular Imaging. RAW 264.7 cells (5×10 4 ) were inoculated in a confocal dish and incubated for 24 h. DiI-labeled Col@HMnO 2 and Col@HMnO 2 -MM (30 μg / mL), cultured for 4 h. After paraformaldehyde fixation, DAPI was used to stain the cell nucleus for 15 min. Finally, the cells were observed using CLSM.

[0087] (2) Activated macrophages for nanodrug uptake

[0088] RAW264.7 cells (5×10 4) were cultured in a confocal dish for 24 h and then treated with LPS (1 μg / mL). 2 and Col@HMnO 2 -MM, cells were cultured for 4 h, washed with PBS, fixed with paraformaldehyde and stained with DAPI for 15 min. Then the cells were visualized using CLSM. In addition, biological transmission electron microscopy was used to observe the activated macrophages Col@HMnO after 0.5 h and 4 h. 2 -MM's situation.

[0089] (3) Experimental results

[0090] To explore whether MMs confer immune escape to nanomedicines and enhance targeting under inflammatory conditions, we labeled Col@HMnO with the probe DiI. 2 -MM, the probe DiI emits red fluorescence. In this study, we used confocal laser microscopy (CLSM) to investigate the effect of macrophages on DiI-Col@HMnO under lipopolysaccharide (LPS)-stimulated and non-stimulated conditions. 2 and DiI-Col@HMnO 2 -MM uptake and internalization. CLSM shows that macrophages take DiI-Col@HMnO 2 - Efficient uptake and internalization of MM, especially by LPS-stimulated macrophages ( Figure 4 a, 4b). and DiI-Col@HMnO 2 In contrast, LPS-activated macrophages showed a strong affinity for DiI-Col@HMnO 2 -MM uptake and internalization were significantly increased. 2 -MM uptake and internalization were significantly reduced. This shows that MM coating promoted the activation of macrophages to Col@HMnO 2 -MM uptake and internalization, reducing the possibility of phagocytosis by phagocytes. To explore the immune escape effect of nanomedicines, we used LysoTracker Green to specifically label lysosomes and DiI to label Col@HMnO 2 -MM. Col@HMnO 2 After being taken up and internalized by LPS-activated macrophages, Col@HMnO was highly colocalized with lysosomes in the early stage. The fluorescence signal intensity of intracellular lysosomes decreased over time, indicating that lysosomes and nanomedicines were gradually separated. Bio-TEM was used to more intuitively observe the Col@HMnO 2 -The location of MM in LPS-activated macrophages. Col@HMnO 2 -MM is mainly distributed in the cytoplasm over time ( Figure 4 c). Taken together, these results suggest that nanomedicines have the ability to evade immune cells and target sites of inflammation.

[0091] Example 5: In vitro antioxidant protection

[0092] (1) CCK8 assay for cell viability

[0093] RAW 264.7 macrophages were plated in 96-well plates and incubated with HMnO 2 -MM (30 μg / mL), free Col (10 μg / mL) or Col@HMnO 2 -MM (30 μg / mL) for 2 hours. Using the above concentrations, free Col and Col@HMnO 2 -MM, the amount of Col remained basically the same. The cells in the control group were cultured in the culture medium without any treatment. 2 O 2 The cells were treated for 24 h. As a control, the cells were treated with fresh culture medium for comparison. On the second day, CCK-8 was used to measure the cell survival rate.

[0094] (2) JC-1 detection of mitochondrial membrane potential changes

[0095] RAW264.7 cells (5×10 4 ) were inoculated in a confocal culture dish and HMnO 2 -MM, free Col or Col@HMnO 2 -MM for 2 hours (drug dosage is the same as above). Then, the cells were exposed to H 2 O 2 (400 μM) for 24 hours. The changes of mitochondrial membrane potential were detected by JC-1 detection kit.

[0096] (3) Flow cytometry detection of cell apoptosis

[0097] RAW 264.7 cells were plated at 1 × 10 6 The cells were plated at a density of 10 cells / mL in a six-well plate and incubated overnight to allow adhesion. HMnO 2 -MM, Col and Col@HMnO 2 -MM pretreatment for 2 h (drug dosage as above). 2 O 2 The cells were stimulated for 24 h, and macrophage apoptosis was detected by flow cytometry.

[0098] (4) Experimental results

[0099] Compared with normal tissues, AS lesions produce excessive ROS, causing irreversible damage to intracellular lipids, carbohydrates, proteins, and DNA. Based on the above analysis, we studied Col@HMnO 2 -MM cell protection against hydrogen peroxide-induced oxidative stress. We used the CCK-8 method to evaluate the effect of hydrogen peroxide on macrophages by measuring changes in cell viability. Figure 4 As shown in d, H 2 O 2 After 24 h, the cell viability decreased significantly, indicating that H 2 O 2 Can cause macrophage damage. 2 O 2 ) compared to HMnO 2 -MM, free Col or Col@HMnO 2 -MM pretreatment increased the survival rate of macrophages, indicating that Col@HMnO 2 -MM can eliminate H 2 O 2 Similarly, flow cytometry was used to detect the damage caused by Col@HMnO 2 -MM's ability to protect macrophages from oxidative stress-induced apoptosis. Figure 4 As shown in e, with HMnO 2 Compared with the free Col group, the Col@HMnO 2 -MM pretreatment of RAW264.7 cells significantly reduced the apoptosis and necrosis ratios. The same results were obtained by live / dead staining, where live cells were stained green with fluorescein (AM) and dead cells were stained red with propidium iodide (PI). Figure 4 f). Changes in mitochondrial membrane potential (MMP) during apoptosis are widely considered to be a hallmark of cell death. 2 -MM's effect on MMP, using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazole-carbonyl carbonyl cyanide iodide (JC-1) to stain macrophages. JC-1 aggregates in the mitochondria of cells with high MMP to form J aggregates, producing strong red fluorescence. 2 O 2 After treatment, the red fluorescence intensity decreased and the green fluorescence intensity increased, indicating changes in MMP. 2 -MM pretreatment resulted in a significant decrease in green fluorescence, indicating that Col@HMnO 2 -MM can prevent the change of MMP and thus inhibit cell apoptosis ( Figure 4 g). Figure 4 h shows that untreated macrophages have a larger ratio of red to green fluorescence intensity, while H2 O 2 Stimulation reduced this ratio. Notably, after pretreatment with nanodrugs, the ratio increased again. Overall, Col@HMnO 2 -MM protects macrophages from damage induced by hydrogen peroxide-triggered oxidative stress.

[0100] Example 6: Intracellular ROS scavenging activity, in vitro anti-inflammatory effect, and polarization of macrophages M1 to M2

[0101] (1) Intracellular ROS scavenging activity

[0102] RAW 264.7 macrophages were cultured in confocal culture dishes and exposed to HMnO 2 -MM, free Col or Col@HMnO 2 -MM treatment for 2 hours (drug dosage is the same as in Example 5). The control group cells were not treated in the culture medium. Then the cells were treated with 1 μg / mL LPS for 24 hours, and the negative control was treated with fresh culture medium again. After PBS washing, they were incubated with 10 μM DCFH-DA in serum-free culture medium for 30 minutes. The cells were then visualized using CLSM.

[0103] (2) Anti-inflammatory effect in vitro

[0104] After receiving the same treatment as above, the cells were stimulated with LPS, and various inflammatory factors in the culture supernatant were detected by ELISA kits, and the total protein level was detected by BCA kits.

[0105] (3) Polarization of macrophages from M1 to M2

[0106] In the RT-PCR experiment, RAW 264.7 macrophages were seeded into 6-well plates and activated with LPS (1 μg / mL) to induce M1 polarization. Then, HMnO 2 -MM, free Col and Col@HMnO 2 -MM was incubated for 24h (drug dosage is the same as in Example 5). After collecting the cells, total RNA was extracted using TRIzol reagent. RT-PCR was used to detect the level of macrophage mRNA. Then, we used CLSM to observe the expression of CD86 and CD206. The cells were treated in the same way and fixed with 4% paraformaldehyde for immunofluorescence staining. They were exposed to the primary antibody and incubated overnight at 4°C. Fluorescently labeled secondary antibodies were added and incubated for 1h, the cell nuclei were co-stained with DAPI, and the images were observed using CLSM.

[0107] (4) Experimental results

[0108] We studied Col@HMnO 2-MM inhibits the generation of ROS in cells. After 12 hours of stimulation, the ROS level in the model group cells increased significantly. On the other hand, activated macrophages and HMnO 2 -MM, free Col or Col@HMnO 2 After 4 h of co-incubation with HMnO-MM, the green fluorescence intensity of (DCFH-DA) in the cells decreased significantly, especially for Col@HMnO 2 -MM group. This indicates that the total intracellular ROS level decreased ( Figure 5 a, 5b). H 2 O 2 (green) DHE (red) and DAF-FM DA (green) staining results are consistent with the above results, indicating that Col@HMnO 2 -MM makes H 2 O 2 , ·O 2 - and NO levels were reduced to a minimum ( Figure 5 In addition, flow cytometry was used to quantitatively analyze the total intracellular ROS level to confirm the Col@HMnO 2 -MM treatment can effectively inhibit the production of ROS in RAW264.7 cells ( Figure 5 e) These results confirm that Col@HMnO 2 -MM can protect macrophages from damage by clearing intracellular ROS. Figure 5 In b, the control group, model group, and HMnO 2 -MM group, free Col group and Col@HMnO 2 The results of -MM group were: 0.013±0.006; 36.717±0.343; 24.182±1.933; 12.748±1.045; 0.233±0.193; Figure 5 c, control group, model group, HMnO 2 -MM group, free Col group and Col@HMnO 2 The results of -MM group were: 0.694±0.598; 30.492±2.573; 15.755±0.989; 15.932±2.471; 4.627±0.757; Figure 5 d, control group, model group, HMnO 2 -MM group, free Col group and Col@HMnO 2 The results of the -MM group were: 0.306±0.277; 54.177±9.777; 24.284±1.951; 23.757±5.075; 2.742±0.872. It can be seen from the experimental data that Col@HMnO 2-MM group loads Col into HMnO 2 -MM, the two factors have a synergistic effect, Col@HMnO 2 The -MM group produced a significant improvement in inhibiting ROS generation in cells.

[0109] It is known that excessive production of intracellular ROS can activate a series of signal transduction cascades, thereby inducing inflammation and aggravating the formation of AS. 2 -MM has good ROS scavenging activity. Next, we investigated whether this scavenging activity could inhibit inflammation. Figure 5 f) IL-6 ( Figure 5 g), TNF-α( Figure 5 h) and other inflammatory factors were significantly upregulated, indicating that macrophages were polarized toward the pro-inflammatory M1 phenotype. 2 Pre-incubation with -MM or Col partially inhibited the expression of these cytokines, but it is worth noting that pre-incubation with Col@HMnO 2 -MM treatment significantly suppressed these cytokines, indicating that the nanomedicine has effective anti-inflammatory properties. 2 Compared with the -MM group, Col@HMnO 2 -MM effectively reduces inflammation in macrophages. Figure 5 f, control group, model group, HMnO 2 -MM group, free Col group and Col@HMnO 2 -The results of the MM group were: 76.52±1.05; 100.96±1.67; 91.66±0.89; 91.17±2.82; 82.65±0.44 (n=3); Figure 5 g, control group, model group, HMnO 2 -MM group, free Col group and Col@HMnO 2 The results of -MM group were: 128.52±0.84; 184.42±1.03; 153.82±5.24; 152.08±1.15; 142.59±2.26 (n=3); Figure 5 h, control group, model group, HMnO 2 -MM group, free Col group and Col@HMnO 2 The results of the -MM group were: 808.60±11.24; 1285.02±44.42; 1040.17±17.60; 1041.35±17.28; 954.37±1.14 (n=3). 2-MM group loads Col into HMnO 2 -MM, the two factors have a synergistic effect, Col@HMnO 2 -MM group produced a significant improvement in anti-inflammatory effect.

[0110] During the development of AS, two main types of macrophages are observed: M1 pro-inflammatory and M2 anti-inflammatory macrophages. In atherosclerotic lesions, macrophages are mainly polarized toward the M1 type. A potential therapeutic approach to manage AS is to reprogram M1 macrophages into M2 macrophages. As ROS and cytokines play a crucial role in this process, an increasing number of antioxidant and anti-inflammatory nanomedicines are designed to promote the conversion of M1 to M2. 2 -MM's reactive oxygen species scavenging and anti-inflammatory abilities, we explored its ability to reprogram macrophages. To this end, we first analyzed the mRNA levels of M1 and M2 macrophage-related markers. After LPS treatment, the expression of M1 macrophage markers (such as iNOS) was significantly upregulated, while the expression of M2 macrophage markers (such as CD206 and Arg-1) was relatively reduced, indicating that we successfully induced M1 macrophage polarization. 2 After pre-incubation with -MM or free Col, the expression of M1 markers decreased and the expression of M2 markers increased. 2 Compared with the MM group and the Col group, Col@HMnO 2 -MM group has better therapeutic effect on reprogramming of M1 to M2 macrophages. Figure 6 ,exist Figure 6 In a, the control group, model group, HMnO 2 -MM group, free Col group and Col@HMnO 2 The results of -MM group were: 1.03±0.04; 1.77±0.15; 1.46±0.19; 0.97±0.10; 0.62±0.06 (n=3); Figure 6 In b, the control group, model group, and HMnO 2 -MM group, free Col group and Col@HMnO 2 -The results of the MM group were: 1.03±0.03; 1.03±0.09; 1.54±0.54; 2.32±0.19; 4.16±0.02 (n=3); Figure 6 c, control group, model group, HMnO 2 -MM group, free Col group and Col@HMnO 2The results of the -MM group were: 0.98±0.02; 0.78±0.07; 1.29±0.04; 1.50±0.11; 2.08±0.59 (n=3). 2 -MM group loads Col into HMnO 2 -MM, the two factors have a synergistic effect, Col@HMnO 2 -MM group was significantly more effective in promoting the transformation of M1 to M2 than free Col group or Col@HMnO 2 -The sum of the effects of the MM group. Figure 6 b is used as an example to illustrate that the effect of increasing the markers of M2 macrophages due to the application of free Col is (2.32-1.03)=1.29; 2 -MM application brought about the effect of increasing M2 macrophage markers (1.54-1.03) = 0.51; due to Col@HMnO 2 -MM application brought about the effect of increasing the M2 macrophage markers: (4.16-1.03)=3.13>1.29+0.51. 2 The phenomenon of synergistic enhancement of -MM does exist, and in promoting the transformation of macrophages from M1 to M2, a 1+1>2 effect is produced. Macrophages are innate immune cells that use different activation states to respond to their microenvironment. 2 -MM can be used in combination to synergistically improve the microenvironment of atherosclerotic lesions and enhance the therapeutic effect.

[0111] Based on the above results, we used CLSM to observe the morphological changes of macrophages. 2 -After MM nanomedicine treatment, the multi-antennary structure unique to M1 macrophages disappeared ( Figure 5 i) To verify this transformation, immunofluorescence analysis of CD86 and CD206 proteins (red) was performed using CLSM to analyze the cell phenotype. We observed that CD86 2 -MM, free Col or Col@HMnO 2 -MM treatment significantly decreased the fluorescence intensity. In contrast, the fluorescence intensity of CD206 increased significantly, especially Col@HMnO 2 -MM Group( Figure 5 j,5k). In conclusion, Col@HMnO 2 -MM can effectively induce the reprogramming of M1 to M2 macrophages for the treatment of AS.

[0112] Example 7: Col@HMnO 2 Effects of -MM on ox-LDL internalization, cholesterol transport and foam cell formation in macrophages

[0113] (1) Cellular uptake of ox-LDL

[0114] Exploring Col@HMnO 2 -MM nanomedicine inhibits oxidized low-density lipoprotein (ox-LDL) uptake. RAW264.7 cells were initially exposed to 1 μg / mL LPS for 24 h and then treated with Col@HMnO 2 -MM was incubated for 2 hours (experimental grouping and drug dosage were the same as in Example 5). Then, DiI-oxLDL at a concentration of 40 μg / mL was used for 4 hours, and after Hoechst 33342 staining for 30 minutes, the uptake of ox-LDL by the cells was observed by CLSM.

[0115] (2) Regulation of HIF-1α, ABCA1, and ABCG1 protein expression

[0116] To induce upregulation of HIF-1α protein expression, cells were treated with CoCl 2 (100 μM) was incubated in serum-free medium for 4 h, and then the cells were subjected to various treatments for 24 h (experimental grouping and drug dosage were the same as in Example 5), and protein expression was detected by CLSM, RT-PCR and Western blot. In order to detect the expression of ABCA1, we subjected the cells to different treatments. After total RNA was extracted with TRIzol reagent, the relative expression of ABCA1mRNA was detected by RT-PCR. In order to detect the expression of ABCG1, we subjected the cells to different treatments. The results were visualized using CLSM.

[0117] (3) Cholesterol efflux assay

[0118] We used fluorescent NBD-cholesterol to study cholesterol efflux from macrophages. After the cells were treated in different ways for 12 hours (experimental grouping and drug dosage were the same as in Example 5), 1 μg / mL NBD-cholesterol was added to the cell culture dish. The cells were then washed with PBS and cultured for another 6 hours. The culture supernatant was collected and centrifuged (14000rpm, 6min) to remove debris. At the same time, the cells were lysed with sodium hydroxide (1mL, 0.05M). The NBD-cholesterol concentration was detected at Ex=500nm and Em=535nm by a fluorescent enzyme spectrometer to calculate the cholesterol efflux rate.

[0119] (4) Oil Red O staining of foam cell formation

[0120] After the cells were treated with 30 μg / mL ox-LDL, the prepared nanomedicine was added to different groups and incubated for 24 h (experimental grouping and drug dosage were the same as in Example 5), fixed with 4% paraformaldehyde for 25 min, stained with Oil Red O for 15 min, then washed once with isopropanol, washed twice with water, and stained with hematoxylin for 1 min. After washing with water, the intracellular lipid droplets were observed under a light microscope.

[0121] (5) Experimental results

[0122] The ability of macrophages to take up ox-LDL and the formation of foam cells due to dysregulated cholesterol transport ultimately lead to plaque formation. Therefore, we investigated whether nanodrugs released under acidic conditions could effectively promote cholesterol efflux pathways ( Figure 7 a). First, we study Col@HMnO 2 -How does MM affect the uptake of ox-LDL by macrophages? After incubation with DiI-labeled ox-LDL (DiI-ox-LDL) and cells for 4 hours, Figure 7 b shows that untreated activated macrophages (model group) exhibited more prominent red fluorescence signals, while HMnO 2 -MM group, Col group and Col@HMnO 2 The fluorescence intensity of the -MM group was significantly reduced, especially Col@HMnO 2 -MM group. These results indicate that the nanomedicine can effectively reduce the cellular uptake of ox-LDL.

[0123] Under normal circumstances, lipids are internalized by macrophages and hydrolyzed in lysosomes to generate free cholesterol. After part of the cholesterol is converted into cholesterol esters, the remaining cholesterol binds to transport proteins such as ABCA1 and is excreted from the cell, directing lipid metabolism. However, due to the extreme hypoxia at the site of atherosclerosis, more lipids accumulate in abnormally aggregated macrophages than in normal blood vessels. This is mainly due to the significant upregulation of HIF-1α. The abnormal upregulation of HIF-1α can offset the effects of cholesterol on macrophages by inhibiting cholesterol transporters ABCA1 and ABCG1, thereby enhancing the formation of foam cells. Carrier HMnO 2 Promote H 2 O 2 Decomposition to generate O 2 , thereby alleviating the hypoxia in the atherosclerotic area. To verify the above point of view, we used CoCl 2 Treatment of macrophages to induce hypoxia modulated the protein expression level of HIF-1α in cells. As expected, in the presence of HMnO 2 and Col@HMnO 2 -MM pretreatment group was observed to have an effective downregulation of HIF-1α expression, suggesting that HMnO2 Group and Col@HMnO 2 -MM group can improve the hypoxic microenvironment of the plaque by producing oxygen ( Figure 7 e). Immunofluorescence staining ( Figure 7 c) and qPCR results ( Figure 7 d) shows the same trend. After verifying the inhibition of HIF-1α protein expression, we further studied the regulatory effect of nanomedicine on transporters ABCA1 and ABCG1. 2 Group and Col@HMnO 2 -MM groups showed a significant increase in the expression of transport proteins ABCG1 and ABCA1. This effect was mainly due to the inhibitory effect of nanomedicine on HIF-1α protein expression, suggesting that nanomedicine enhances cholesterol transport ( Figure 7 f, 7g, 7h). Thus, we validated the roles of ABCA1 and ABCG1, which are important cholesterol efflux pumps that can effectively reduce the excessive accumulation of lipids in cells.

[0124] Since ATP transporters ABCA1 and ABCG1 were upregulated, we next investigated the cholesterol transport pathway by examining high-density lipoprotein (HDL)-mediated cholesterol efflux. We used CLSM to visually inspect the intracellular residual cholesterol. CLSM results showed that Col@HMnO 2 -MM group showed the lowest intracellular cholesterol retention (green). We also observed a relative decrease in the amount of residual cholesterol in the free Col group, which may be due to its anti-inflammatory effect leading to reduced lipid influx ( Figure 7 i) The fluorescence enzyme spectrometer obtained the same result. Figure 7 j As shown in Figure 2, compared with the untreated group, HMnO 2 The HDL-mediated cholesterol efflux in the -MM group and free Col group was slightly larger, while Col@HMnO 2 Due to the synergistic effect, the HDL-mediated cholesterol efflux in the MM-treated group was significantly greater than that in the untreated group. 2 -MM can reduce lipid accumulation in macrophages.

[0125] We then studied Col@HMnO 2 -MM can inhibit foam cells by reducing LDL uptake and increasing cholesterol efflux. Macrophages were pretreated with different nanomedicine groups, incubated with ox-LDL for 24 h, and then stained with Oil Red O (ORO). Abundant lipid droplets were observed in the cells of the model group, and foam cell formation was observed ( Figure 7 k). In contrast, Col@HMnO 2-MM group had the most significant inhibitory effect on foam cell formation. Quantitative analysis of ORO intracellular deposition results ( Figure 7 The microscopic observation results shown in l) provide support. In summary, the above results show that Col@HMnO 2 -MM may enhance the inhibition of foam cell formation by synergistically reducing LDL uptake and increasing cholesterol efflux.

[0126] Example 8: In vivo distribution and pharmacokinetic studies

[0127] Feeding APOE with a high-fat diet - / - The atherosclerosis model was established in mice at 13 weeks. DiR-labeled Col@HMnO 2 (30 μg / mL) and Col@HMnO 2 -MM (30 μg / mL) was injected into the tail vein of AS model mice (n=3). After different cycle times (2, 4, and 6 h), the aorta was removed and fluorescence imaging was performed in vivo to evaluate the targeting of atherosclerotic plaque sites in mice. Fluorescence imaging of the heart, liver, spleen, lung, and kidney was performed at 6 and 48 h after administration to evaluate the metabolism of nanomedicines in organs. In the pharmacokinetic study, DiR-labeled Col@HMnO was injected into the tail vein. 2 Blood samples (100 μL) were collected from the eye of MM mice at specific time points (2, 4, 6, 8, 10, 12, 24, and 48 h) and collected by centrifugation (3000 r × 10 min) for fluorescence imaging to evaluate the nanodrug Col@HMnO 2 - Half-life of MM.

[0128] Fluorescence imaging (FLI) can be used to visualize Col@HMnO 2 -MM's main accumulation site and metabolism in vivo. We first demonstrated in vitro that nanomedicine can be used for FLI. Col@HMnO 2 The fluorescence (FL) signal intensity of -MM (labeled with DiR) increased with the increase of concentration and finally reached equilibrium. Fluorescence quantitative analysis confirmed the above results. 2 -MM-specific targeting of apolipoprotein E deficiency (APOE - / - ) mouse plaque sites. DiR-labeled Col@HMnO 2 -MM can be seen accumulating in the aorta 2 hours after tail vein injection, reaching a higher concentration 6 hours after injection ( Figure 8 a). The fluorescence brightness of the target group is higher than that of the non-target group. These results indicate that Col@HMnO 2-MM has the expected plaque targeting properties, which may be related to MM modification. We also observed that at 6 h, the nanodrugs mainly accumulated in the liver and spleen, and Col@HMnO 2 -MM group and Col@HMnO 2 There was no significant difference in the fluorescence signal intensity between different organs of the group. In addition, the nanodrug was almost eliminated after 48 hours, indicating that the nanodrug can be metabolized by the liver and spleen and has a certain biosafety. Finally, we detected the in vivo pharmacokinetic parameters of the nanodrug. The drug was administered through the tail vein of mice. We collected blood samples from mice at different time points and found that the fluorescence intensity of the blood samples gradually decreased over time. Nanodrug Col@HMnO 2 -MM has a half-life of 14.997 h, which prolongs the half-life of Col in the blood. This may be attributed to the ability of nanomedicine to escape phagocytic cells. 2 -MM has the potential to improve the therapeutic effect of Col. We also used immunofluorescence staining to visualize the ability of the synthesized nanomedicine to target the AS site. Figure 8 b shows DiR-labeled Col@HMnO 2 -MM (red) co-localized with the macrophage-specific protein CD68 (green). In contrast, in the non-targeted group, the DiR-labeled Col@HMnO 2 A weak red fluorescence was observed.

[0129] Example 9: In vivo evaluation of therapeutic effects and study of therapeutic mechanisms

[0130] In in vivo animal experiments, APOE - / - The AS model was established by feeding mice with a high-fat diet, and the mice in the control group were fed with a normal diet. At the end of the fifth week, the mice in the model groups were randomly divided into a normal saline group, a HMnO 2 -MM group (30 μg / mL), Col group (30 μg / mL), Col@HMnO 2-MM group (30μg / mL), with different treatment methods, the nanodrug was injected into the tail vein twice a week, 0.125mL each time. After 8 weeks of treatment, the aorta was removed and the mice were killed. The aorta was washed with 10% paraformaldehyde for 30 minutes and followed up for evaluation. The whole aorta was stained with Oil Red O, and the plaque area was analyzed using Image-Pro Plus 6.0 software. The aortic sections were fixed with 10% paraformaldehyde, embedded in 3.5μm thick paraffin for H&E staining and histological examination. The paraffin sections were then identified with Masson staining and CD68, MMP-9 and α-SMA antibodies. The aortic sections were frozen and the production of ROS in the plaque site was evaluated with DHE fluorescent dye. To study the polarization of macrophages, we incubated the aortic sections with anti-ARG-1 and anti-iNOS antibodies. Serum samples were collected, and serum TNF-α, IL-6, CRP, IL-1β, and IL-18 levels were detected using ELISA kits to evaluate the serum inflammation level. We collected eye blood from mice and measured their serum cholesterol levels. During the treatment period, major organs were collected for H&E staining and biosafety assessment.

[0131] Based on the above encouraging results, we studied Col@HMnO 2 -MM's effect on plaques in vivo. APOE - / - The mice were fed a Western diet (high-fat diet) for 5 weeks and randomly divided into groups at week 6. Each group was given different drug treatments: control group (C57BL / 6 mice), model group (APOE - / - Mouse), HMnO 2 -MM, free Col group and Col@HMnO 2 -MM. Then inject twice a week for 8 weeks ( Figure 8 c). In this way, we were able to clearly compare each treatment to the control and model treatments to explore the effects of the drugs on plaques. At the end of week 13, the entire aorta was removed from each mouse and stained with ORO ( Figure 8 d,8f). Figure 8 As shown in d and 8f, the ORO staining area in the model group was the largest, indicating that the AS model was successfully constructed. 2 -MM and Col groups had smaller ORO positive areas, while Col@HMnO 2 The ORO-positive area in the -MM group was the smallest. The same results were found in the ORO-stained aortic sections of different groups. Based on macroscopic observation and ORO staining results, H&E-stained sections of the aortic root also confirmed that the nanodrug Col@HMnO 2 -MM is most effective in treating plaques, such as Figure 8 As shown in e and 8h. Figure 8As shown in e, a large number of fat-rich necrotic cores were observed in the model group. 2 -MM group and Col group had a decrease in plaque area, indicating that monotherapy can alleviate the progression of AS to a certain extent. 2 Compared with the -MM group, Col@HMnO 2 -The plaque area in the MM group was significantly reduced, indicating that the synergistic effect was significantly stronger than that of single-drug treatment.

[0132] exist Figure 8 f, control group, model group, HMnO 2 -MM group, free Col group and Col@HMnO 2 The results of -MM group were: 0.27±0.21; 41.02±0.68; 27.97±1.44; 21.13±1.42; 7.24±1.29 (n=3); Figure 8 g, control group, model group, HMnO 2 -MM group, free Col group and Col@HMnO 2 The results of the -MM group were: 0.05±0.05; 38.31±8.11; 23.86±6.52; 14.49±0.42; 0.81±0.39 (n=3). 2 -MM group loads Col into HMnO 2 -MM, the two factors have a synergistic effect, Col@HMnO 2 -MM group was significantly more effective in treating aortic lesions than free Col group or Col@HMnO 2 -The sum of the effects of the MM group. Figure 8 f as an example, the effect of reducing aortic lesions due to the application of free Col is (41.02%-21.13%)=19.89%; due to HMnO 2 -MM application brought about the effect of reducing aortic lesions (41.02%-27.97%) = 13.05%; due to Col@HMnO 2 -MM application brought about the effect of reducing aortic lesions: (41.02%-7.24%) = 33.78% > 19.89% + 13.05%. 2 The dosage of Col in the -MM group was basically the same, which shows that the specific data showed that Col and HMnO 2 The synergistic effect of -MM does exist, and it has produced a 1+1>2 effect in the treatment of aortic lesions. Similarly, for the reduction of plaque area, Col@HMnO 2-MM also showed outstanding results.

[0133] We performed further histochemical evaluation of mouse aorta sections to better understand the mechanism of synergistic plaque treatment. Antibodies against CD68, matrix metalloproteinase-9 (MMP-9), and α-smooth muscle actin (α-SMA), and Masson staining were used to evaluate the composition of plaques after different treatments ( Fig. 9 a). Anti-CD68 antibody shows the synthesized nanodrug Col@HMnO 2 -MM minimized macrophage infiltration in the plaque site, while a significant decrease in the concentration of MMP-9 antibody indicated a decrease in plaque vulnerability. Similarly, α-SMA antibody staining and Masson trichrome staining showed that Col@HMnO 2 -MM treatment leads to a significant reduction in the number of VSMCs, thereby preventing excessive accumulation of collagen and facilitating the stabilization of AS plaques ( Fig. 9 b-9e).

[0134] exist Fig. 9 In b, the control group, model group, and HMnO 2 -MM group, free Col group and Col@HMnO 2 -The results of the MM group were: 0.05±0.05; 33.97±5.99; 22.57±3.34; 15.57±3.58; 2.41±0.79 (n=3); Fig. 9 c, control group, model group, HMnO 2 -MM group, free Col group and Col@HMnO 2 -The results of the MM group were: 0.07±0.05; 8.23±0.24; 4.24±0.43; 3.89±0.31; 0.59±0.27 (n=3); Fig. 9 d, control group, model group, HMnO 2 -MM group, free Col group and Col@HMnO 2 The results of -MM group were: 0.01±0.01; 13.90±0.45; 9.21±0.30; 5.49±0.45; 1.37±0.25 (n=3); Fig. 9 e, control group, model group, HMnO 2 -MM group, free Col group and Col@HMnO 2 The results of -MM group were: 0.05±0.05; 10.41±0.43; 4.54±0.85; 3.61±0.72; 0.18±0.27 (n=3); Fig. 9 g, control group, model group, HMnO 2-MM group, free Col group and Col@HMnO 2 The results of the -MM group were: 0.67±0.58; 35.24±1.97; 19.85±3.88; 15.59±2.15; 4.84±2.44 (n=3). 2 -MM group loads Col into HMnO 2 -MM, the two factors have a synergistic effect, Col@HMnO 2 -MM group had a significantly higher plaque treatment effect than free Col group or Col@HMnO 2 -MM group.

[0135] Considering our recently developed nanomedicine with exceptional ROS scavenging and anti-inflammatory properties, we performed DHE experiments to evaluate the performance of Col@HMnO 2 -MM's ROS scavenging ability in vivo. Compared with the model group, Col@HMnO 2 The red fluorescence signal of the -MM group was stronger, and the red signal intensity was the lowest, indicating that Col@HMnO 2 -MM may inhibit plaque formation by removing ROS ( Fig. 9 f, 9g). Encouraged by the above findings, we investigated whether the synthesized nanomedicine affects the reprogramming of macrophages in the plaque site by immunofluorescence staining of iNOS (a marker of M1 macrophages) and Arg-1 (a marker of M2 macrophages). Fig. 9 h, Strong iNOS (M1) fluorescence signal and weak Arg-1 (M2) fluorescence signal were observed in the AS site of mice in the model group, which was considered to indicate that the polarization of macrophages in the pathological AS site was unfavorable. 2 The fluorescence signal intensities observed in the -MM treatment group were opposite, indicating that the reprogramming from M1 to M2 was achieved. In addition, we also tested the anti-inflammatory activity of the nanomedicine in vivo. Fig. 9 The data of i-9l and HMnO 2 Compared with the Col group, the levels of various representative pro-inflammatory factors (including CRP, IL-6, IL-1β, TNF-α, and IL-18) decreased more significantly in the MM group, suggesting that the nanomedicine has a synergistic anti-inflammatory effect in vivo. These results demonstrate the effectiveness of nanomedicine in treating plaques from an anti-inflammatory perspective.

[0136] People with cardiovascular diseases often have difficulty following a healthy lifestyle, especially a healthy diet. Therefore, we also investigated the nanomedicine Col@HMnO 2 - Whether MM can delay the progression of AS by reducing lipid accumulation in mice continuously fed a Western diet.2 -MM group mice showed effective control of body weight. In addition, we explored the plasma cholesterol levels among the groups and found that Col@HMnO 2 The plasma cholesterol level of mice in the -MM group was the lowest.

[0137] Finally, we continue to observe the nanodrug Col@HMnO 2 -MM can restore the plaque area after 14 days of treatment, and evaluate the biocompatibility of the nanomedicine during the entire treatment period. H&E staining results showed that no obvious pathological changes were observed in the main organs during the 2-month treatment period, indicating that Col@HMnO 2 -It is safe during MM treatment.

[0138] Comparative Example 1: About Col@HMnO 2 -Exploration of MM synthesis method

[0139] In Example 1, "(2)SiO 2 @MnO 2 In the "synthesis" step, SiO 2 @PVP to SiO 2 The synthesis steps of @RF are very critical. SiO prepared in the manner of Example 1 2 @RF's electron microscope image shows Fig.10 (TEM and SEM images).

[0140] Prior to this, the inventors tried to 2 The synthesis method of @RF is as follows: during the synthesis process, 1.6g potassium permanganate (KMnO 4 ) solid was dissolved in 30 mL of deionized water, and then 36 g / L of (NH 4 ) 2 C 2 O 4 ·H 2 O solution was added dropwise to potassium permanganate solution and stirred for 2 h. The mixture was transferred to a polytetrafluoroethylene flask and placed in a stainless steel autoclave for reaction at 180 °C for 24 h. After cooling to room temperature, the powder product was repeatedly washed and dried. HMnO was synthesized through the above steps. 2 (Hollow Mesoporous Manganese Dioxide).

[0141] The above method omits SiO 2 @RF synthesis process, synthesized HMnO 2 (Hollow Mesoporous Manganese Dioxide) Fig.11 As shown in the figure, it is obvious that the synthesis of hollow mesoporous 2@RF synthesis), it is difficult to form spherical nanoparticles. 2 compared to( Figure 2 c), have significant differences in structure.

[0142] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A macrophage membrane functionalized manganese dioxide nanomedicine, characterized in that: The method comprises a HMnO2 carrier, on which colchicine is loaded, and the HMnO2 carrier is coated with a macrophage membrane.

2. The macrophage membrane functionalized manganese dioxide nanomedicine according to claim 1, characterized in that: The HMnO2 carrier is prepared by the following method: constructing a mesoporous MnO2 layer on the surface of SiO2 to form a SiO2@MnO2 complex, and then using alkali to etch the SiO2 in the SiO2@MnO2 complex to form a HMnO2 basic carrier; and sequentially covering the HMnO2 basic carrier with polyacrylamine hydrochloride and polyacrylic acid to obtain a HMnO2 carrier.

3. The macrophage membrane functionalized manganese dioxide nanomedicine according to claim 2, characterized in that: The surface area of ​​the HMnO2 carrier is 86.3 m 2 / g, and the average pore size is about 11.7nm.

4. The macrophage membrane functionalized manganese dioxide nanomedicine according to claim 3, characterized in that: Its particle size is 211.63±8.71nm.

5. A method for preparing a macrophage membrane functionalized manganese dioxide nanomedicine according to any one of claims 1 to 4, characterized in that: The method includes the following steps in sequence: S1: Synthesis of HMnO2 carrier; S2: Stir and mix colchicine and HMnO2 carrier to obtain Col@HMnO2; S3: Col@HMnO2 is mixed with macrophage membrane, and then extruded through a liposome extruder to obtain.

6. The method for preparing a macrophage membrane functionalized manganese dioxide nanomedicine according to claim 5, characterized in that: In S2, the dosage ratio of HMnO2 carrier and colchicine is 1:2-3:

1.

7. The method for preparing a macrophage membrane functionalized manganese dioxide nanomedicine according to claim 5, characterized in that: In S3, the dosage ratio of Col@HMnO2 to macrophage membrane is 2:1-4:1; the number of extrusions is 15-25 times; and the pore size of the polycarbonate membrane of the liposome extruder is 400-800 nm.

8. The method for preparing a macrophage membrane functionalized manganese dioxide nanomedicine according to claim 5, characterized in that: In S1, after SiO2 is surface modified, KMnO4 solution is added, and after stirring reaction, MnO2 grows on the SiO2 hard template to form a mesoporous layer, and a SiO2@MnO2 composite is obtained; Using alkali to etch SiO2 in the SiO2@MnO2 composite to form a HMnO2 base carrier; covering the HMnO2 base carrier with polyacrylamine hydrochloride and polyacrylic acid in sequence to obtain a HMnO2 carrier; The method for surface modification of SiO2 is: SiO2 and polyvinyl pyrrolidone are mixed and reacted to obtain SiO2@PVP; then, water, m-diphenol and formaldehyde are added to SiO2@PVP, and ammonia water is added after mixing, and SiO2@RF is obtained after reaction, thereby completing the surface modification of SiO2.

9. Use of a macrophage membrane functionalized manganese dioxide nanomedicine according to any one of claims 1 to 4 in the preparation of a drug for treating atherosclerosis.

10. The use of a macrophage membrane functionalized manganese dioxide nanomedicine according to claim 9 in the preparation of a drug for treating atherosclerosis, characterized in that: Drugs for treating atherosclerosis are used to eliminate reactive oxygen species, promote the transformation of macrophages from M1 to M2, inhibit proliferation, alleviate the hypoxic microenvironment at the site of atherosclerosis, and promote cholesterol efflux.

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