Bionic nano-composite, preparation method thereof and application of bionic nano-composite in preparation of products for treating atherosclerosis

Abstract

The invention discloses a bionic nano-composite, a preparation method thereof and application of the bionic nano-composite in preparation of a product for treating atherosclerosis, and belongs to the technical field of medicine preparation. The bionic nano compound is a metal nano enzyme Ir-TiO2 coated by an M2 macrophage cell membrane. According to the invention, the surfaces of Ir-TiO2 nanoparticles are coated with M2 macrophage membranes, so that the nanocomposite with a bionic structure is prepared. After intravenous injection, the formed bionic nanocomposite can actively target endothelial cells in atherosclerotic lesions, and release Ir-TiO2 antagonism to endothelial cells in plaques and cytoplasm of inflammatory macrophages. After the Ir-TiO2 reaches the diseased region, the Ir-TiO2 can play an enzyme role to achieve an anti-inflammatory effect, and can promote inflammatory cells such as macrophages and smooth muscle cells at the diseased region to discharge cholesterol, reduce lipid deposition in the cells, reduce the generation of foam cells and promote the growth of plaques. The nanotherapy can effectively prevent the progress of atherosclerosis lesion inflammation and relieve atherosclerosis.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a biomimetic nanocomposite, a preparation method thereof, and an application thereof in the preparation of products for treating atherosclerosis. Background Art

[0002] Atherosclerosis is the main cause of lethal cardiovascular diseases such as coronary artery disease and stroke, and is one of the main causes of human death. Chronic unresolved inflammation is a key driver of atherosclerosis, which is characterized by the gradual deposition of arterial plaques. Currently, drugs are the most commonly used method for treating atherosclerosis. Although its pathogenesis is still unclear, atherosclerosis is generally considered a chronic inflammatory disease caused by endothelial dysfunction. Vascular inflammation is involved in the formation of atherosclerosis through many different molecular and cellular pathways. In other words, high levels of reactive oxygen species (ROS) are considered to be closely related to the AS (atherosclerosis) process. The human body produces reactive oxygen species (ROS) in many physiological processes, including superoxide anions, hydroxyl radicals, hydrogen peroxide (H 2 O 2 ) and other free radicals. It should be noted that excessive ROS under pathological conditions is prone to cause lipid peroxidation (LPO) and DNA damage, which will not only induce oxidative stress and apoptosis, but also activate pro-inflammatory signaling pathways and exacerbate the inflammatory response. ROS oxidizes low-density lipoprotein (LDL) to oxidized low-density lipoprotein (ox-LDL), turning macrophages into foam cells and inducing an inflammatory response, ultimately leading to plaque rupture. Based on the oxidation theory, various antioxidants have been explored for the treatment of AS. Among them, natural antioxidants such as vitamin A (VA), vitamin C (VC), etc. Because of their advantages in terms of safety. Unfortunately, due to their limited ROS scavenging ability and rapid metabolism, natural antioxidant therapy has suffered poor clinical outcomes in large-scale human trials.

[0003] In recent years, with the development of nanotechnology, various nanomaterials with unique ROS regulation properties have been explored to guide the spatio-temporal dynamics of ROS in the biological environment, thus giving rise to a new generation of treatment methods, namely, nanomaterial-guided in vivo ROS evolution therapy. However, like any strategy, the use of nanoparticles (NPs) also has limitations, such as low utilization rate. Therefore, how to deliver more nanodrugs to the lesion site is a major problem. Summary of the Invention

[0004] The purpose of the present invention is to provide for the first time a biomimetic nanocomposite, a preparation method thereof, and an application thereof in the preparation of products for treating atherosclerosis. The present invention is in Ir-TiO 2The surface of the nanoparticles is coated with M2 macrophage membranes (M2) to prepare a structurally biomimetic nanocomposite (M 2 @Ir-TiO 2 ). After intravenous injection, the formed biomimetic nanocomposite can actively target endothelial cells in atherosclerotic lesions and release Ir-TiO into the cytoplasm of endothelial cells and inflammatory macrophages in the plaque 2 to exert its function. After Ir-TiO 2 reaches the lesion site, it can act as an enzyme to achieve an anti-inflammatory effect, and can promote inflammatory cells such as macrophages and smooth muscle cells in the lesion site to excrete cholesterol, reduce intracellular lipid deposition, reduce the formation of foam cells, and promote plaque growth. This nanotherapy can effectively prevent the progression of atherosclerotic lesion inflammation and relieve atherosclerosis.

[0005] The object of the present invention is achieved through the following technical solutions:

[0006] The first technical solution: The present invention provides a biomimetic nanocomposite, which is a metal nanozyme Ir-TiO encapsulated by M2 macrophage membranes 2 .

[0007] Macrophages are a type of large and highly versatile white blood cells and are essentially the main cellular effectors in the processes of inflammation and tissue repair. They play a key role in the pathogenesis of many congenital and adaptive diseases, especially those involving the infiltration and transportation of immune histiocytes. Among all cell types involved in the pathogenesis of atherosclerosis, macrophages play the most important role. In atherosclerotic plaques, macrophages mainly have two phenotypes, namely pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages. The former is polarized by TNF-α, IFN-γ or lipopolysaccharide (LPS) and is involved in the formation of foam cells, the secretion of pro-inflammatory cytokines and chemokines, plaque instability and rupture, and ultimately induces cardiovascular events. The latter is mainly polarized by anti-inflammatory factors such as IL-4 and IL-10, and the secretion of anti-inflammatory factors is conducive to the regression of inflammation and the improvement of plaque stability, thereby preventing the development of atherosclerosis.

[0008] Based on this, the present invention provides a metal nanozyme with excellent anti-inflammatory performance, which can not only quickly remove excess reactive oxygen species, but also inhibit the formation of ox-LDL, reduce cholesterol levels, and inhibit the formation of foam cells. Particularly importantly, we use M2 macrophage membranes (M2) as a targeted drug delivery platform to treat atherosclerosis by mimicking the inherent targeting of M2 macrophages to plaques. The advanced M 2 @Ir-TiO 2 has good biocompatibility.

[0009] In addition, considering the important anti-inflammatory role of M2 macrophages in inflammatory lesions, M2 macrophage membranes were used as modifiers in this study. In vivo, M2-coated nanoparticles effectively targeted and accumulated in atherosclerotic lesions. After a 2-month treatment regimen, M 2 @Ir-TiO 2 was shown to significantly delay the progression of AS. In addition, M 2 @Ir-TiO 2 exhibited good safety performance after long-term administration.

[0010] The second technical solution: The present invention provides a method for preparing a biomimetic nanocomposite, comprising the following steps:

[0011] S1. Prepare TiO 2 ;

[0012] S2. Prepare Ir-TiO 2 : Add ethanol and IrCl 2 to the TiO 3 , and obtain Ir-TiO 2 after sufficient reaction;

[0013] S3. Prepare the biomimetic nanocomposite: Mix the Ir-TiO 2 and membrane proteins, disperse them evenly under ultrasound, then centrifuge at low temperature, discard the supernatant, and resuspend with PBS to obtain the biomimetic nanocomposite, namely M 2 @Ir-TiO 2 .

[0014] Preferably, the concentrations of both the metal nanozyme Ir-TiO 2 and the membrane proteins are 1 mg / mL, and the volume ratio of the metal nanozyme Ir-TiO 2 to the membrane proteins is 1:1.

[0015] The third technical solution: The present invention provides an application of a biomimetic nanocomposite in the preparation of products for treating atherosclerotic diseases.

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] The present invention provides a biomimetic method to deliver the payload Ir-TiO 2 to inflammatory atherosclerotic lesions. Considering the positive role of M2 macrophages in atherosclerotic plaques, M2 macrophage membranes were used to coat the metal nanozyme Ir-TiO 2 with good antioxidant capacity to target the plaque site. Coating the surface of Ir-TiO 2 nanoparticles with M2 macrophage membranes (M2) to prepare a structurally biomimetic nanocomposite (M2 @Ir-TiO 2 ) After intravenous injection, the formed biomimetic nanocomposite can actively target endothelial cells in atherosclerotic lesions and release Ir-TiO into the cytoplasm of endothelial cells and inflammatory macrophages in the plaque 2 to exert its function. After Ir-TiO 2 reaches the lesion site, it can act as an enzyme to achieve an anti-inflammatory effect, and can promote inflammatory cells such as macrophages and smooth muscle cells in the lesion site to excrete cholesterol, reduce intracellular lipid deposition, reduce the formation of foam cells, and promote plaque growth. Therapeutic studies have shown that this nanotherapy can effectively prevent the progression of atherosclerotic lesion inflammation and relieve atherosclerosis.

[0018] A metal nanozyme with excellent anti-inflammatory properties was designed. It can not only rapidly remove excess reactive oxygen species, but also inhibit the formation of ox-LDL, reduce cholesterol levels, and inhibit the formation of foam cells. Particularly importantly, we used M2 macrophage membrane (M2) as a targeted drug delivery platform to treat atherosclerosis by mimicking the inherent targeting of M2 macrophages to plaques. Advanced M 2 @Ir-TiO 2 has good biocompatibility. Brief Description of the Drawings

[0019] Figure 1 : Enzyme activity and characterization of Ir-TiO 2 .

[0020] Figure 1 Among them, (a) Schematic diagram of the multi-enzyme activity of Ir-TiO 2 ; (b) Catalase-like enzyme activity ability of Ir-TiO 2 ; (c) Superoxide dismutase-like enzyme activity ability of Ir-TiO 2 ; (d) SEM image (left) and HRTEM image (right) of Ir-TiO 2 ; (e) Zeta potential of Ir-TiO 2 and M 2 @Ir-TiO 2 ; (f) Particle size of Ir-TiO 2 and M 2 @Ir-TiO 2 ; (g) TEM of M 2 @Ir-TiO 2 ; (h) Fluorescence co-localization of M 2 @Ir-TiO 2 ; (i) Coomassie brilliant blue staining map of Ir-TiO 2 and M 2 @Ir-TiO 2 .

[0021] Figure 2 : M 2 @Ir-TiO 2 Targeting effect on endothelial cells at the plaque site and reactive oxygen species scavenging ability.

[0022] Figure 2 In a, schematic diagram of the mechanism of macrophage membrane targeting endothelial cells. Confocal laser scanning microscopy images of DiR-Ir-TiO internalized by HUVECs cells 2 and DiR-M 2 @Ir-TiO 2 (scale bar 100 μm) (b) and quantitative cell uptake (n = 3, mean ± SD) (c). Representative CLSM images (scale bar = 100 μm) (d) and fluorescence statistical analysis (e) of activated endothelial cells co-incubated with DiR-M 2 @Ir-TiO 2 for 0, 2, 4, 8, and 12 h. g. M 2 @Ir-TiO 2 Flow chart of the experiment for scavenging intracellular reactive oxygen species. Fluorescence statistical analysis of the ROS content in LPS-induced HUVECs cells (h) and RAW264.7 cells (i) after different treatments (control group, LPS, LPS + TiO 2 , LPS + Ir-TiO 2 , LPS + M 2 @Ir-TiO 2 ) (n = 3, mean ± SD). Flow cytometry fluorescence statistical analysis of LPS-induced HUVECs cells (j) and RAW264.7 cells (k) after different treatments (control group, LPS, LPS + TiO 2 , LPS + Ir-TiO 2 , LPS + M 2 @Ir-TiO 2 ).

[0023] Figure 3 : M 2 @Ir-TiO 2 Anti-inflammatory ability, promoting cholesterol efflux in vitro and inhibiting foam cell formation.

[0024] Figure 3 In a - f, concentrations of TNF-α, INF-γ, and IL-1β in LPS-induced RAW264.7 and HUVEC cells under different treatments. LPS-induced RAW264.7 cells under different treatments (control group, LPS, LPS + TiO 2 , LPS + Ir-TiO 2 , LPS + M2 @Ir-TiO 2 ) Representative confocal fluorescence images of the accumulation of oxidized low-density lipoprotein in cells after treatment, scale bar: 50 μm (g), and their fluorescence statistical analysis (i), flow cytometry fluorescence intensity statistics (h). j. Cholesterol content in macrophage supernatant (n = 3). k. ORO staining of RAW264.7 cells pretreated with various protocols.

[0025] Figure 4 : M 2 @Ir-TiO 2 In vivo targeted therapy for plaques, scavenging of reactive oxygen species and in vivo therapeutic effects.

[0026] Figure 4 In, a. ApoE - / - Flowchart of the establishment and treatment of male mouse in vivo models. (a, b) IR-Ir-TiO 2 (10 mg / kg) and IR-M 2 @Ir-TiO 2 (10 mg / kg) in vivo pharmacokinetics after intravenous injection in mice. (A) Ex vivo images and (B) quantitative analysis of whole blood collected at different time points after intravenous injection. The control group (A) shows the results of mouse whole blood without any treatment. (b) Ex vivo images and (c) quantitative analysis of whole blood collected at different time points after intravenous injection. The control group (A) shows the results of mouse whole blood without any treatment. (d) Ex vivo fluorescence images. ApoE - / - Male mice were fed a high-fat diet for 8 weeks and then intravenously injected with IR-Ir-TiO 2 (10 mg / kg) and IR-M 2 @Ir-TiO 2 (10 mg / kg). Twelve hours after administration, the mice were euthanized and the aortas were isolated for ex vivo imaging. Representative photographs (e) and statistical analysis (f) of ORO-stained aortic tissues from mice treated with different formulations. Results of ROS scavenging (g) and fluorescence intensity statistics (h) in vivo plaques after different treatments.

[0027] Figure 5 : Representative images (a) and radar charts (b) of ORO, H&E, Masson, anti-α-SMA antibody, and anti-CD68 antibody staining of aortic root sections. (c) Quantitative analysis of ORO in aortic root sections (n = 3 - 5, mean ± SD). M2@Ir-TiO 2 Levels of alanine aminotransferase (d), uric acid (e), and creatine kinase (f) in mice two months after treatment.

[0028] Figure 6: Representative confocal fluorescence images of LPS-induced HUVECs cells after different treatments (control group, LPS, LPS+TiO 2 , LPS+Ir-TiO 2 , LPS+M 2 @Ir-TiO 2 ), scale bar 100 μm.

[0029] Figure 7 : Representative confocal fluorescence images of LPS-induced RAW264.7 cells after different treatments (control group, LPS, LPS+TiO 2 , LPS+Ir-TiO 2 , LPS+M 2 @Ir-TiO 2 ), scale bar 100 μm.

[0030] Figure 8 : Intracellular IL-10 concentration in LPS-induced RAW264.7 (a) and HUVEC (b) under different treatments.

[0031] Figure 9 : Distribution of M 2 @Ir-TiO 2 (10 mg / kg) in different organs at different time points after tail vein injection in C57BL / 6 mice.

[0032] Figure 10 : Cytotoxicity of RAW264.7 (a) and HUVEC (b) treated with different concentrations of M 2 @Ir-TiO 2 detected by CCK8.

[0033] Figure 11 : Hemolysis rate of M 2 @Ir-TiO 2 at different concentrations. Detailed implementation manners

[0034] Example 1

[0035] 1. Enzyme activity, preparation and characterization of Ir-TiO 2 ( Figure 1 )

[0036] Preparation of Ir-TiO 2: 3g of terephthalic acid, anhydrous methanol (6 mL), anhydrous DMF (54 mL) were slowly added to tetrabutyl titanate (1.5 mL) under continuous stirring. The mixture was stirred for 15 min, transferred to a 100 mL hydrothermal autoclave, and reacted at 150 °C for 48 h. After the reaction, it was washed with DMF and methanol (centrifuged twice at 10000 rpm for 5 min each), and then heat-treated at 600 °C for 2 h in an air atmosphere to obtain TiO 2 ; Subsequently, 200 mg of TiO 2 and 40 mL of ethanol were ultrasonically dispersed, and 60 mg of IrCl 3 was added. Hydrothermal reaction was carried out at 80 °C for 24 h to obtain Ir-TiO 2 .

[0037] Preparation of biomimetic nanocomposites: 1 mg / mL of Ir-TiO 2 was mixed with 1 mg / mL of membrane protein in a volume ratio of 1:1; Subsequently, it was dispersed by an ultrasonic instrument for 30 min, and continuously mixed with a pipette during dispersion; Subsequently, it was centrifuged at 4 °C for 10 min, and the supernatant was discarded. Then, 1 mL of PBS was added to resuspend it to prepare a biomimetic nanocompound of cell membrane-coated nanomaterials, namely M 2 @Ir-TiO 2 , and stored in the refrigerator.

[0038] (1) Nanozyme activity

[0039] In the environment of atherosclerotic plaques, high concentrations of reactive oxygen species play an important role. Therefore, reducing ROS (such as H 2 O 2 , O 2 - ) may be an effective strategy for treating plaques. In this invention, metal nanozyme Ir-TiO 2 with excellent ROS scavenging ability was prepared, and its catalytic effects on catalase (CAT) and superoxide dismutase (SOD) are shown in Figure 1 a. To verify that the Ir-TiO 2 nanoparticles have good performance in catalyzing the production of oxygen from hydrogen peroxide, the colorimetric method [319.2 mg of Ti(SO 4 ) 2 and 8.33 mL of H 2 SO 4 was used to detect the scavenging rate of Ir-TiO 2 for hydrogen peroxide. The WST-8 method was used to detect the O 2 - generated by xanthine and xanthine oxidase to evaluate the SOD-like activity of NPs. The results showed that compared with TiO 2 , Ir-TiO 2 had a stronger scavenging rate for hydrogen peroxide and O2 - Clearance rate, the clearance rate is about 90%( Figure 1 b) and 77%( Figure 1 c).

[0040] (2) Characterization

[0041] SEM( Figure 1 d left) and HRTEM( Figure 1 d right) images show that Ir-TiO 2 has an approximately spherical morphology with an average diameter of about 200 nm. The particle size is measured by a Malvern particle size analyzer, about 214.6 nm, which is consistent with the results of SEM and HRTEM. By modifying Ir-TiO 2 with macrophage cell membrane (M2), M 2 @Ir-TiO 2 is obtained. The results of the Malvern particle size analyzer show that the size of the biomimetic nanoparticles M 2 @Ir-TiO 2 increases slightly, and its particle size is about 254.9 nm( Figure 1 e). The zeta potential shows that the potential of Ir-TiO 2 changes from -38 to -24 after M2 modification( Figure 1 f). The above results all indicate that the nano-coating is successfully coated on the surface of M 2 @Ir-TiO 2 .

[0042] To further confirm that the macrophage cell membrane is successfully coated on the surface of Ir-TiO 2 , the morphological characteristics of Ir-TiO 2 and M 2 @Ir-TiO 2 are detected by visual TEM. The dense "shell layer" structure wrapped on the surface of Ir-TiO 2 intuitively proves that the macrophage cell membrane is efficiently coated on the surface of Ir-TiO 2 ( Figure 1 g). Next, M2 is labeled with DiI and Ir-TiO 2 is labeled with FITC. The overlap of DIR and FITC fluorescence can be seen under an upright fluorescence microscope, confirming that Ir-TiO 2 is wrapped inside M2( Figure 1 h). At the same time, the Coomassie brilliant blue staining results show that the proteins of the M2 macrophage cell membrane highly coincide with the proteins present in M 2 @Ir-TiO 2 , also confirming the successful preparation of M 2 @Ir-TiO 2 ( Figure 1 i).

[0043] 2.M 2 @Ir-TiO 2 Inhibition of foam cell formation in vitro.

[0044] (1)M 2 @Ir-TiO 2 Targeting effect on endothelial cells at the plaque site ( Figure 2 ).

[0045] Increasing evidence indicates that nanoparticles coated with macrophage membranes (MM) can target atherosclerotic plaque sites ( Figure 2 a). This invention attempts to study M 2 @Ir-TiO 2 's targeting effect on inflammatory HUVECs. HUVECs cells were stimulated with LPS to simulate the inflammatory site, and DiR-Ir-TiO 2 and DiR-M 2 @Ir-TiO 2 were added to activated or non-activated endothelial cells. The uptake of DiR-Ir-TiO 2 and DiR-M 2 @Ir-TiO 2 by cells was observed under a fluorescence microscope. The images showed that DiR-M 2 @Ir-TiO 2 showed a higher internalization degree in activated endothelial cells compared with DiR-Ir-TiO 2 , showing a stronger red fluorescence signal. Average fluorescence intensity analysis showed that the absorption signal of DiR-M 2 @Ir-TiO 2 by activated endothelial cells was 2.0 times higher than that of DiR-Ir-TiO 2 ( Figure 2 b, c). Compared with HUVECs, obvious red fluorescence could be observed in activated HUVECs cells co-incubated with M 2 @Ir-TiO 2 , and it increased in a time-dependent manner within 12 h ( Figure 2 d). Flow cytometry analysis further found that after co-incubation for 12 h, the uptake efficiency of foam cells for DiR-M 2 @Ir-TiO 2 reached up to 96.4%, indicating that M 2 @Ir-TiO 2 has excellent targeting ability for foam cells ( Figure 2 e, f).

[0046] (2)M 2 @Ir-TiO2 Reactive oxygen species scavenging effect.

[0047] To evaluate the ROS scavenging ability of M 2 @Ir-TiO 2 , namely the anti-inflammatory ability, the present invention uses lipopolysaccharide (LPS)-treated RAW264.7 cells and HUVECs cells as in vitro models, and uses the fluorescent probe - 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) to observe the production of ROS in foam cells ( Figure 2 g). First, CLSM images showed that obvious green fluorescence signals were observed in both the LPS and TiO 2 groups, indicating that a large amount of ROS was produced by macrophages under the action of LPS. However, after treatment with TiO 2 , the intracellular reactive oxygen species content was still very high, indicating that Ir-TiO 2 had insufficient scavenging ability for ROS. On the contrary, the IrTiO 2 group only showed weak green fluorescence, indicating its excellent reactive oxygen species scavenging ability. At the same time, compared with Ir-TiO 2 , M 2 @Ir-TiO 2 had a stronger ability to remove ROS ( Figure 6 , Figure 7 and Figure 2 h, i). The results of flow cytometry were consistent with the above data. The intracellular fluorescence intensity was the strongest after LPS stimulation, about 73% or 90%, further confirming that the fluorescence signal of foam cells treated with M 2 @Ir-TiO 2 was the weakest (only 13.9% or 23.4%) ( Figure 2 j, k). Why, despite the same use concentration and time of Ir-TiO 2 and M 2 @Ir-TiO 2 , M 2 @Ir-TiO 2 had a stronger reactive oxygen species scavenging ability. It was conjectured that this might be due to the targeting ability of M2 to the plaque site, enabling more Ir-TiO 2 to concentrate at the lesion site to scavenge ROS. Therefore, M 2 @Ir-TiO 2 had a stronger reactive oxygen species scavenging ability.

[0048] (3) In vitro anti-inflammatory effect of M2@Ir-TiO2 ( Figure 3 , Figure 8 and Figure 9 ).

[0049] To further evaluate the intracellular antioxidant effect of MM@Ir-TiO2, an ELISA kit was used to detect the expression of various inflammatory factors (such as IL-4, IL-1β, TNF-α, γ-INF) and anti-inflammatory factors (such as IL-10) in macrophages and endothelial cells. The results showed that LPS-stimulated inflammatory cells exhibited high expression of pro-inflammatory factors TNF-α( Figure 3 a, b), INF-γ( Figure 3 c, d), IL-1β( Figure 3 e, f), and low expression of the anti-inflammatory factor IL-10( Figure 8 a, b). Surprisingly, compared with TiO 2 , M 2 @Ir-TiO 2 treatment significantly reduced the levels of TNF-α, IL-6, and INF-γ, while increasing the level of IL-10. These data confirmed that M 2 @Ir-TiO 2 has a significant antioxidant effect and can effectively reduce the cellular inflammatory response and improve the inflammatory environment within the plaque.

[0050] (4) The in vitro cholesterol efflux-promoting effect of M 2 @Ir-TiO 2 ( Figure 3 ).

[0051] ROS can oxidize low-density lipoprotein (LDL) to oxidized low-density lipoprotein (ox-LDL), transform macrophages into foam cells, and induce an inflammatory response, ultimately leading to plaque rupture. The above results demonstrated that M 2 @Ir-TiO 2 has good reactive oxygen species scavenging ability. Next, the present invention explored whether M 2 @Ir-TiO 2 treatment could reduce the uptake of oxidized low-density lipoprotein (ox-LDL) by macrophages and decrease foam cell formation. The present invention pretreated RAW264.7 with various formulations and then incubated the cells with DiI-labeled ox-LDL (DiI-ox-LDL) for 4 h. Fluorescence microscopy and flow cytometry analysis showed that the red fluorescence of RAW264.7 cells was the strongest after LPS treatment, while the red fluorescence of the M2@Ir-TiO 2 group was the weakest.

[0052] The above results indicated that after LPS-stimulated macrophages generated reactive oxygen species, the intracellular accumulation of ox-LDL could be increased, while the addition of M 2 @Ir-TiO 2After co-incubation for 4 hours, the red fluorescence (DiI-ox-LDL) in macrophages was significantly weakened, indicating that it significantly inhibited the accumulation of DiI-ox-LDL in RAW264.7 cells ( Figure 3 g, h, i). At the same time, the promoting effect on the cholesterol transport pathway was detected by a cholesterol content detection kit for Ir-TiO 2 . The results showed that the cholesterol content in the supernatant of the Ir-TiO 2 group and the M 2 @Ir-TiO 2 group increased significantly ( Figure 3 j). The above results indicate that M 2 @Ir-TiO 2 is beneficial to reducing intracellular lipid retention and inhibiting foam cell formation. Similarly, Oil Red O (ORO) staining confirmed a reduction in the foaming process, that is, the number of foam cells in the M 2 @Ir-TiO 2 group was significantly reduced compared with the LPS group ( Figure 3 k).

[0053] 3. In vivo therapeutic effect of M 2 @Ir-TiO 2 .

[0054] Among them, M 2 @Ir-TiO 2 targets plaques in vivo and scavenges reactive oxygen species ( Figure 4 ); Representative images and radar charts of ORO, H&E, Masson, anti-α-SMA antibody, and anti-CD68 antibody staining of aortic root sections ( Figure 5 ).

[0055] Specifically as follows:

[0056] Atherosclerosis models were established using 6-8-week-old apolipoprotein e-deficient (ApoE - / - ) male mice, and the pharmacokinetic characteristics of M 2 @Ir-TiO 2 in C57BL / 6 mice were studied. After intravenous injection of IR-labeled M 2 @Ir-TiO 2 , fluorescence imaging showed that M 2 @Ir-TiO 2 was almost completely cleared from the blood after 24 hours ( Figure 4 b, c). Then, the in vivo atherosclerotic targeting ability of tail vein injection of IR-labeled M - / - @Ir-TiO 2 was verified in mice carrying atherosclerotic plaques ApoE 2 . 12 h after intravenous injection, IR-labeled M2 @Ir-TiO 2 Accumulate in the isolated aorta, sacrifice the mice, and take their main organs and the isolated aorta for treatment. In vitro imaging can clearly observe the accumulation of IR-M in atherosclerotic plaques 2 @Ir-TiO 2 . Strong fluorescence was found in the aortic arch region, which is an area prone to developing atherosclerosis. In contrast, the fluorescence in the atherosclerotic plaque area of the IR-Ir-TiO 2 group was weak, much lower than that of IR-M 2 @Ir-TiO 2 group. This result indicates that M2 functionalization can enhance the accumulation of IR-M in the plaque area in vivo 2 @Ir-TiO 2 ( Figure 4 d). At 12 h after injection, the fluorescence signal was mainly distributed in the liver and kidneys. After 24 h, the fluorescence in the liver was significantly weakened, while the fluorescence in the kidneys was still strong, suggesting that the nanoparticles in the mice may be mainly excreted through the kidneys Figure 9 ).

[0057] Twenty mice were evenly divided into 4 groups (n = 5): control group, model group, Ir-TiO 2 group, and M 2 @Ir-TiO 2 group. Treat twice a week via the tail vein injection, with each treatment dose about 10 mg / kg. After two months of treatment, take the aorta for ORO staining, and the red area after staining is the plaque area Figure 4 a). The atherosclerotic plaque area was clearly shown by ORO staining in the model group. Treatment with Ir-TiO 2 could reduce the plaque area to 20%. Compared with Ir-TiO 2 , the treatment effect of M2@Ir-TiO 2 was slightly improved, probably because M2 functionalization enhanced the aggregation ability of IR-TiO 2 in the plaque. Treatment with M 2 @Ir-TiO 2 significantly improved the treatment effect, and the plaque rate was reduced to 6.77%, indicating that M 2 @Ir-TiO 2 effectively inhibited the progression of atherosclerosis Figure 4 e, f). Detect the production of intracellular ROS after treatment with M 2 @Ir-TiO 2 using the DHE probe. The ROS in the control group was the least among all groups. When plaques form, the inside of the plaque is in a state of high concentration of ROS, and Ir-TiO 2 can significantly reduce the ROS in the plaque, and M2 @Ir-TiO 2 The group effect is even more significant because M2 can cause more of the @Ir-TiO 2 to aggregate at the plaque, enabling it to scavenge ROS ( Figure 4 g, h). Meanwhile, similar results were obtained from ORO-stained frozen aortic sections, indicating the effective anti-atherosclerotic activity of @Ir-TiO 2 @Ir-TiO 2 ( Figure 5 a-c). These results suggest that @Ir-TiO 2 @Ir-TiO 2 is effective in slowing the progression of atherosclerosis.

[0058] Immunohistochemical analysis of CD68 (a macrophage marker) ( Figure 5 a) revealed that @Ir-TiO 2 @Ir-TiO 2 could significantly reduce the macrophage content in aortic plaques. Notably, the increased expression of necrotic cores and macrophage infiltration may promote the progression of atherosclerosis. In contrast, the proliferation of vascular smooth muscle cells (VSMCs) and the increase in collagen concentration are considered beneficial at all stages of atherosclerosis. Additionally, anti-α-smooth muscle actin (α-SMA) antibody staining showed that the number of SMCs in the lesion area increased significantly after @Ir-TiO 2 @Ir-TiO 2 treatment ( Figure 5 a). Consistent with this result, Masson staining showed that the collagen concentration was also significantly upregulated. These findings indicate that @Ir-TiO 2 @Ir-TiO 2 can stabilize the further development of plaques and inhibit the occurrence of atherosclerosis.

[0059] 4. In vivo and in vitro biosafety.

[0060] To investigate whether the nanodrug would cause unnecessary damage to healthy cells, at the cellular level, the biosafety of @Ir-TiO 2 @Ir-TiO 2 at different concentration gradients was tested using a CCK8 kit. RAW264.7 ( 2 @Ir-TiO 2 treated with concentrations of 5 μg / ml, 10 μg / ml, 20 μg / ml, 40 μg / ml, and 80 μg / ml) and HUVECs cells ( Figure 10 a) and ( Figure 10 b), and the results showed that @Ir-TiO 2 @Ir-TiO 2When the concentration is within 40 μg / ml, the cell survival rate still reaches 75%, indicating that M 2 @Ir-TiO 2 has high safety( Figure 10 ). The hemolysis test shows that although the concentration of M 2 @Ir-TiO 2 is 200 μg / mL, no obvious hemolysis occurs( Figure 11 ).

Claims

1. A bionic nanocomposite, characterized in that: It is a metal nanozyme Ir-TiO2 wrapped in the membrane of M2 macrophages.

2. The method for preparing a biomimetic nanocomposite according to claim 1, characterized in that: The steps include: S1. Preparation of TiO2; S2. Preparation of Ir-TiO2: adding ethanol and IrCl3 to the TiO2, and reacting sufficiently to obtain Ir-TiO2; S3. Preparation of biomimetic nanocomposite: The Ir-TiO2 and membrane protein are mixed and dispersed evenly under ultrasound, followed by low-temperature centrifugation, the supernatant is discarded, and PBS is added for re-suspending to obtain the biomimetic nanocomposite.

3. The method for preparing a biomimetic nanocomposite according to claim 2, characterized in that: The concentrations of the metal nanozyme Ir-TiO2 and the membrane protein are both 1 mg / mL, and the volume ratio of the metal nanozyme Ir-TiO2 and the membrane protein is 1:

1.

4. Use of the bionic nanocomposite according to claim 1 in preparing a product for treating atherosclerosis.

Images