A biomimetic nanocomposite, its preparation method, and its application in the preparation of products for treating atherosclerosis.

Abstract

This invention discloses a biomimetic nanocomposite, its preparation method, and its application in the preparation of products for treating atherosclerosis, belonging to the field of pharmaceutical preparation technology. The biomimetic nanocomposite is an Ir-TiO2 metal nanozyme encapsulated in an M2 macrophage membrane. This invention prepares a structurally biomimetic nanocomposite by coating Ir-TiO2 nanoparticles with an M2 macrophage membrane. After intravenous injection, the formed biomimetic nanocomposite actively targets endothelial cells in atherosclerotic lesions, releasing Ir-TiO2 antagonistically into the cytoplasm of plaque-containing endothelial cells and inflammatory macrophages. Once Ir-TiO2 reaches the lesion site, it exerts an enzymatic effect to achieve anti-inflammatory activity and promotes cholesterol excretion from inflammatory cells such as macrophages and smooth muscle cells at the lesion site, reducing intracellular lipid deposition, reducing foam cell formation, and promoting plaque growth. This nanotherapy can effectively prevent the progression of inflammation in atherosclerotic lesions and alleviate atherosclerosis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically a biomimetic nanocomposite, its preparation method, and its application in the preparation of products for treating atherosclerosis. Background Technology

[0002] Atherosclerosis is a leading cause of death from coronary artery disease, stroke, and other fatal cardiovascular diseases, and is one of the leading causes of death in humans. Chronic unresolved inflammation is a key driver of atherosclerosis, characterized by the gradual deposition of plaques in the arteries. Currently, medication is the most commonly used treatment for atherosclerosis. Although its pathogenesis is not fully understood, atherosclerosis is generally considered a chronic inflammatory disease caused by endothelial dysfunction. Vascular inflammation participates in the formation of atherosclerosis through many different molecular and cellular pathways. In other words, high levels of reactive oxygen species (ROS) are considered closely related to the atherosclerotic process. Reactive oxygen species (ROS) are produced in many physiological processes in the human body, including superoxide anions, hydroxyl radicals, and hydrogen peroxide (H2O2). It is important to note that excessive ROS under pathological conditions can easily lead to lipid peroxidation (LPO), causing DNA damage, which not only induces oxidative stress and apoptosis but also activates pro-inflammatory signaling pathways, exacerbating the inflammatory response. ROS oxidize low-density lipoprotein (LDL) into oxidized low-density lipoprotein (ox-LDL), causing macrophages to transform 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 ankylosing spondylitis (AS). Among them, natural antioxidants such as vitamin A (VA) and vitamin C (VC) are considered due to their safety advantages. Unfortunately, due to their limited ROS elimination capacity and rapid metabolism, natural antioxidant therapy has suffered from unfavorable clinical results in large-scale human trials.

[0003] In recent years, with the development of nanotechnology, researchers have discovered a variety of nanomaterials with unique ROS regulation properties to guide the spatiotemporal dynamics of ROS in the biological environment, thus giving rise to a new generation of therapeutic methods: nanomaterial-guided in vivo ROS evolution therapy. However, as with any strategy, the use of nanoparticles (NPs) also has limitations, such as low utilization rates. Therefore, how to deliver more nanomedicines to the lesion site remains a major challenge. Summary of the Invention

[0004] The purpose of this invention is to provide, for the first time, a biomimetic nanocomposite, its preparation method, and its application in the preparation of products for treating atherosclerosis. This invention involves coating the surface of Ir-TiO2 nanoparticles with M2 macrophage membranes (M2), thus preparing a structurally biomimetic nanocomposite (M2@Ir-TiO2). After intravenous injection, the formed biomimetic nanocomposite actively targets endothelial cells in atherosclerotic lesions, releasing Ir-TiO2 into the cytoplasm of endothelial cells and inflammatory macrophages within the plaque. Once at the lesion site, Ir-TiO2 exerts an enzymatic effect to achieve anti-inflammatory activity and promotes cholesterol excretion from inflammatory cells such as macrophages and smooth muscle cells, reducing intracellular lipid deposition, decreasing foam cell formation, and promoting plaque growth. This nanotherapy can effectively prevent the progression of inflammation in atherosclerotic lesions and alleviate atherosclerosis.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] The first technical solution: This invention provides a biomimetic nanocomposite, which is a metal nanozyme Ir-TiO2 encapsulated in the membrane of an M2 macrophage.

[0007] Macrophages are large and highly versatile white blood cells, essentially key cellular effector cells in inflammation and tissue repair processes. They play a crucial role in the pathogenesis of many innate and adaptive diseases, particularly those involving the infiltration and transport of immune tissue cells. Among all cell types involved in the pathogenesis of atherosclerosis, macrophages play the most important role. In atherosclerotic plaques, macrophages mainly exhibit two phenotypes: pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages. The former are polarized by TNF-α, IFN-γ, or lipopolysaccharide (LPS) and participate in foam cell formation, secretion of pro-inflammatory cytokines and chemokines, plaque instability and rupture, ultimately inducing cardiovascular events. The latter are mainly polarized by anti-inflammatory factors such as IL-4 and IL-10. The secretion of anti-inflammatory factors promotes inflammation resolution and improves plaque stability, thereby inhibiting the development of atherosclerosis.

[0008] Based on this, the present invention provides a metal nanozyme with excellent anti-inflammatory properties. It not only rapidly removes excess reactive oxygen species but also inhibits the formation of ox-LDL, lowers cholesterol levels, and suppresses foam cell formation. Particularly important, we utilize the M2 macrophage membrane (M2) as a targeted drug delivery platform to treat atherosclerosis by mimicking the inherent targeting of plaques by M2 macrophages. The advanced M2@Ir-TiO2 exhibits good biocompatibility.

[0009] Furthermore, considering the important anti-inflammatory role of M2 macrophages in inflammatory lesions, this study used M2 macrophage membranes as a modifier. In vivo, M2-coated nanoparticles effectively targeted and accumulated in atherosclerotic lesions. After a 2-month treatment regimen, M2@Ir-TiO2 was shown to significantly delay the progression of AS. In addition, M2@Ir-TiO2 exhibited good safety profiles after long-term administration.

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

[0011] S1. Preparation of TiO2;

[0012] S2. Preparation of Ir-TiO2: Ethanol and IrCl3 are added to the TiO2, and after sufficient reaction, Ir-TiO2 is obtained;

[0013] S3. Preparation of biomimetic nanocomposite: The Ir-TiO2 and cell membrane fluid containing membrane proteins are mixed and dispersed evenly under sonication. After centrifugation at low temperature, the supernatant is discarded and the mixture is resuspended in PBS to obtain the biomimetic nanocomposite, namely M2@Ir-TiO2.

[0014] Preferably, the concentrations of the metal nanozyme Ir-TiO2 and the membrane proteins in the cell membrane fluid are both 1 mg / mL, and the volume ratio of the metal nanozyme Ir-TiO2 to the cell membrane fluid is 1:1.

[0015] The third technical solution: This invention provides an application of biomimetic nanocomposites in the preparation of products for treating atherosclerosis.

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

[0017] This invention provides a biomimetic method for effectively loading Ir-TiO2 onto inflammatory atherosclerotic lesions. Considering the positive role of M2 macrophages in atherosclerotic plaques, this study uses M2 macrophage membranes to encapsulate the metal nanozyme Ir-TiO2 with good antioxidant capacity, targeting the plaque site. An M2 macrophage membrane (M2) is coated onto the surface of Ir-TiO2 nanoparticles to prepare a biomimetic nanocomposite (M2@Ir-TiO2). After intravenous injection, the formed biomimetic nanocomposite actively targets endothelial cells in atherosclerotic lesions, releasing Ir-TiO2 into the cytoplasm of endothelial cells and inflammatory macrophages within the plaque. Once at the lesion site, Ir-TiO2 exerts its enzymatic effect to achieve anti-inflammatory effects and promotes cholesterol excretion from inflammatory cells such as macrophages and smooth muscle cells, reducing intracellular lipid deposition, reducing foam cell formation, and promoting plaque growth. Therapeutic studies have shown that this nanotherapy can effectively prevent the progression of inflammation in atherosclerotic lesions and alleviate atherosclerosis.

[0018] A metal nanozyme with excellent anti-inflammatory properties was designed, which not only rapidly removes excess reactive oxygen species but also inhibits the formation of ox-LDL, lowers cholesterol levels, and suppresses foam cell formation. Crucially, we utilized the M2 macrophage membrane (M2) as a targeted drug delivery platform to treat atherosclerosis by mimicking the inherent targeting of plaques by M2 macrophages. The advanced M2@Ir-TiO2 exhibits good biocompatibility. Attached Figure Description

[0019] Figure 1 Enzyme activity and characterization of Ir-TiO2.

[0020] Figure 1 In the image, (a) is a schematic diagram of the multi-enzyme activity of Ir-TiO2; (b) shows the CAT-like enzyme activity of Ir-TiO2; (c) shows the SOD-like enzyme activity of Ir-TiO2; (d) shows the SEM image (left) and HRTEM image (right) of Ir-TiO2; (e) shows the Zeta potential of Ir-TiO2 and M2@Ir-TiO2; (f) shows the particle size of Ir-TiO2 and M2@Ir-TiO2; (g) shows the TEM image of M2@Ir-TiO2; (h) shows the fluorescence colocalization of M2@Ir-TiO2; and (i) shows the Coomassie brilliant blue staining of Ir-TiO2 and M2@Ir-TiO2.

[0021] Figure 2 M2@Ir-TiO2's targeting effect on endothelial cells in plaque sites and its ability to scavenge reactive oxygen species.

[0022] Figure 2In the figures, a. Schematic diagram of the mechanism by which macrophage membranes target endothelial cells. Confocal laser scanning microscopy images (scale bar 100 μm) of DiR-Ir-TiO2 and DiR-M2@Ir-TiO2 internalized in HUVECs (b) and quantification of cellular uptake (n = 3, mean ± SD) (c). Representative CLSM images (scale bar = 100 μm) of activated endothelial cells co-incubated with DiR-M2@Ir-TiO2 for 0, 2, 4, 8, and 12 h (d) and fluorescence statistical analysis (e). g. Flowchart of the M2@Ir-TiO2 assay for scavenging intracellular reactive oxygen species. Fluorescence statistical analysis of intracellular ROS content in LPS-induced HUVECs (h) and RAW264.7 cells (i) after different treatments (control group, LPS, LPS + TiO2, LPS + Ir-TiO2, LPS + M2@Ir-TiO2) (n = 3, mean ± SD). Flow cytometry statistical analysis of LPS-induced HUVECs (j) and RAW264.7 cells (k) after different treatments (control group, LPS, LPS + TiO2, LPS + Ir-TiO2, LPS + M2@Ir-TiO2).

[0023] Figure 3 M2@Ir-TiO2 exhibits anti-inflammatory properties, promotes cholesterol efflux in vitro, and inhibits foam cell formation.

[0024] Figure 3 a. Intracellular concentrations of TNF-α, INF-γ, and IL-1β in LPS-induced RAW264.7 and HUVEC cells under different treatments. j. Representative confocal fluorescence images of intracellular oxidized low-density lipoprotein accumulation in LPS-induced RAW264.7 cells after different treatments (control group, LPS, LPS + TiO2, LPS + Ir-TiO2, LPS + M2@Ir-TiO2), scale bar 50 μm (g), and fluorescence statistical analysis (i) and 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 M2@Ir-TiO2 targets plaques in vivo, clearing reactive oxygen species and enhancing in vivo therapeutic effects.

[0026] Figure 4 in, a. ApoE − / −Flowchart of the establishment and treatment of the male rat in vivo model. (a, b) In vivo pharmacokinetics of IR-Ir-TiO2 (10 mg / kg) and IR-M2@Ir-TiO2 (10 mg / kg) in mice after intravenous injection. (A) In vitro image and (B) Quantitative analysis of whole blood collected at different time points after intravenous injection. Control group (A) shows whole blood results of mice without any treatment. (b) In vitro image and (c) Quantitative analysis of whole blood collected at different time points after intravenous injection. Control group (A) shows whole blood results of mice without any treatment. (d) In vitro fluorescence image. ApoE − / − Mice were fed a high-fat diet for 8 weeks and then intravenously injected with IR-Ir-TiO2 (10 mg / kg) and IR-M2@Ir-TiO2 (10 mg / kg). Twelve hours after administration, the mice were euthanized, and the aorta was dissected for in vitro imaging. Representative photographs (e) and statistical analysis (f) of ORO-stained aortic tissue from mice treated with different formulations are shown. In vivo plaque ROS clearance results (g) and fluorescence intensity statistics (h) after different treatments are also presented.

[0027] Figure 5 Representative images (a) and radar images (b) of aortic root sections stained with ORO, H&E, Masson, anti-α-SMA antibody, and anti-CD68 antibody. (c) Quantitative analysis of aortic root sections stained with ORO (n = 3−5, mean ± SD). Levels of alanine aminotransferase (ALT), uric acid (U), and creatine kinase (CK) in mice treated with M2@Ir-TiO2 two months later.

[0028] Figure 6 Representative confocal fluorescence images of LPS-induced HUVECs cells after different treatments (control group, LPS, LPS + TiO2, LPS + Ir-TiO2, LPS + M2@Ir-TiO2), scale bar 100 μm.

[0029] Figure 7 Representative confocal fluorescence images of LPS-induced RAW264.7 cells after different treatments (control group, LPS, LPS + TiO2, LPS + Ir-TiO2, LPS + M2@Ir-TiO2), scale bar 100 μm.

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

[0031] Figure 9 Distribution of M2@Ir-TiO2 (10 mg / kg) in different organs at different time points after tail vein injection in C57BL / 6 mice.

[0032] Figure 10 RAW264.7 (a) and HUVEC (b) were subjected to cytotoxicity assays using CCK8 at different concentrations of M2@Ir-TiO2.

[0033] Figure 11 Hemolysis rate of M2@Ir-TiO2 at different concentrations. Detailed Implementation

[0034] Example 1

[0035] 1. Enzyme activity, preparation and characterization of Ir-TiO2 ( Figure 1 )

[0036] Preparation of Ir-TiO2: 3 g terephthalic acid, anhydrous methanol (6 mL), and 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 reactor, and reacted at 150 ℃ for 48 h. After the reaction was completed, the mixture was washed with DMF and methanol (centrifuged twice at 10000 rpm for 5 min each), and then heat-treated at 600 ℃ for 2 h in air to obtain TiO2. Subsequently, 200 mg TiO2 and 40 mL ethanol were ultrasonically dispersed, 60 mg IrCl3 was added, and the mixture was hydrothermally heated at 80 ℃ for 24 h to obtain Ir-TiO2.

[0037] Preparation of biomimetic nanocomposites: 1 mg / mL Ir-TiO2 was mixed with cell membrane fluid containing 1 mg / mL membrane protein at a volume ratio of 1:1; then dispersed by sonication for 30 min, with continuous mixing by pipette during dispersion; then centrifuged at 4 ℃ for 10 min, the supernatant was discarded, and then 1 mL PBS was added for resuspending to prepare a biomimetic nanocomposite of cell membrane-encapsulated nanomaterials, namely M2@Ir-TiO2, which was stored in a refrigerator.

[0038] (1) Nanozyme activity

[0039] In the environment of atherosclerotic plaques, high concentrations of reactive oxygen species (ROS) play a crucial role, thus reducing ROS (such as H2O2, O2) is essential. - This invention presents a metal nanozyme, Ir-TiO2, with excellent ROS scavenging capabilities, which may be an effective strategy for treating plaques. Its catalytic activity against hydrogen peroxide (CAT) and superoxide dismutase (SOD) is also demonstrated. Figure 1As shown in Figure a. To verify the excellent catalytic performance of Ir-TiO2 nanoparticles in producing oxygen from hydrogen peroxide, the scavenging rate of Ir-TiO2 for hydrogen peroxide was detected by colorimetry [319.2 mg Ti(SO4)2 and 8.33 mL H2SO4]. The O2 produced by xanthine and xanthine oxidase was detected using the WST-8 assay. - The SOD-like activity of NPs was evaluated. The results showed that, compared with TiO2, Ir-TiO2 exhibited stronger hydrogen peroxide scavenging rate and O2 scavenging activity. - The clearance rate is approximately 90%. Figure 1 b) and 77% Figure 1 c).

[0040] (2) Characterization

[0041] SEM ( Figure 1 d left) and HRTEM ( Figure 1 The image (d, right) shows that Ir-TiO2 has an approximately spherical morphology with an average diameter of about 200 nm. Its particle size, measured by a Malvern particle size analyzer, is approximately 214.6 nm, consistent with the results of SEM and HRTEM. Modifying Ir-TiO2 with macrophage membranes (M2) yielded M2@Ir-TiO2. Malvern particle size analyzer results showed a slight increase in the size of the biomimetic nanoparticles M2@Ir-TiO2, with a particle size of approximately 254.9 nm. Figure 1 e), the zeta potential shows that the potential of Ir-TiO2 after M2 modification changed from -38 to -24 (e). Figure 1 f), all of the above results indicate that the nano-coating was successfully applied to the M2@Ir-TiO2 surface.

[0042] To further confirm the successful coating of macrophage membranes on the Ir-TiO2 surface, the morphological characteristics of Ir-TiO2 and M2@Ir-TiO2 were examined using visualization TEM. The dense "shell" structure encapsulating the Ir-TiO2 surface visually demonstrates the efficient coating of macrophage membranes on the Ir-TiO2 surface. Figure 1 Next, M2 was labeled with DiI, and Ir-TiO2 was labeled with FITC. Under an upright fluorescence microscope, overlapping DIR and FITC fluorescence was observed, confirming that Ir-TiO2 was encapsulated within M2. Figure 1 h). Simultaneously, Coomassie brilliant blue staining results showed a high degree of overlap between the proteins in the M2 macrophage membrane and those present in M2@Ir-TiO2, further confirming the successful preparation of M2@Ir-TiO2. Figure 1 i).

[0043] 2. Inhibition of foam cell formation by M2@Ir-TiO2 in vitro.

[0044] (1) Targeting effect of M2@Ir-TiO2 on endothelial cells in plaque sites Figure 2 ).

[0045] Mounting evidence suggests that macrophage membrane (MM)-coated nanoparticles can target atherosclerotic plaque sites. Figure 2 a). This invention aims to investigate the targeting effect of M2@Ir-TiO2 on inflammatory HUVECs. HUVECs were stimulated with LPS to mimic the site of inflammation, and DiR-Ir-TiO2 and DiR-M2@Ir-TiO2 were added to activated or inactivated endothelial cells. Fluorescence microscopy was used to observe the uptake of DiR-Ir-TiO2 and DiR-M2@Ir-TiO2 by cells. Images showed that DiR-M2@Ir-TiO2 exhibited a higher degree of internalization in activated endothelial cells compared to DiR-Ir-TiO2, displaying a stronger red fluorescence signal. Mean fluorescence intensity analysis showed that the uptake signal of DiR-M2@Ir-TiO2 in activated endothelial cells was 2.0 times higher than that of DiR-Ir-TiO2 (…). Figure 2 b, c). Compared to HUVECs, activated HUVECs co-incubated with M2@Ir-TiO2 showed significant red fluorescence, which increased in a time-dependent manner over 12 h. Figure 2 d). Flow cytometry analysis further revealed that after 12 h of co-incubation, the foam cells achieved the highest uptake efficiency of DiR-M2@Ir-TiO2, reaching 96.4%, indicating that M2@Ir-TiO2 has excellent targeting ability for foam cells. Figure 2 e, f).

[0046] (2) The active oxygen scavenging effect of M2@Ir-TiO2.

[0047] To evaluate the ROS scavenging ability, i.e., anti-inflammatory ability, of M2@Ir-TiO2, this invention used lipopolysaccharide (LPS)-treated RAW264.7 cells and HUVECs cells as in vitro models, and used the fluorescent probe-2,7-dichlorodihydrofluorescein-diacetate (DCFH-DA) to observe ROS production in foam cells. Figure 2 g). First, CLSM images showed significant green fluorescence signals in both the LPS and TiO2 groups, indicating that macrophages produced a large amount of ROS under LPS treatment, while the intracellular reactive oxygen species content remained high after TiO2 treatment, suggesting that Ir-TiO2's ROS scavenging ability was insufficient. Conversely, the IrTiO2 group showed only weak green fluorescence, indicating its excellent reactive oxygen species scavenging ability. Furthermore, compared to Ir-TiO2, M2@Ir-TiO2 exhibited a stronger ROS removal capacity ( Figure 6 , Figure 7 and Figure 2 h, i). Flow cytometry results were consistent with the above data; the intracellular fluorescence intensity was strongest after LPS stimulation, approximately 73% or 90%, further confirming that the fluorescence signal of foam cells treated with M2@Ir-TiO2 was the weakest (only 13.9% or 23.4%). Figure 2 (j, k). Why is it that, despite using the same concentration and time for both Ir-TiO2 and M2@Ir-TiO2, M2@Ir-TiO2 exhibits stronger reactive oxygen species (ROS) scavenging ability? It is speculated that this may be due to M2's targeting ability to plaque sites, allowing more Ir-TiO2 to concentrate on clearing ROS at the lesion site, thus giving M2@Ir-TiO2 a stronger ROS scavenging ability.

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

[0049] To further evaluate the intracellular antioxidant activity of MM@Ir-TiO2, the expression of various inflammatory factors (such as IL-4, IL-1β, TNF-α, and γ-INF) and anti-inflammatory factors (such as IL-10) in macrophages and endothelial cells was detected using an ELISA kit. The results showed that LPS-stimulated inflammatory cells exhibited the pro-inflammatory factor TNF-α (…). Figure 3 a, b), INF-γ Figure 3 c, d), IL-1β Figure 3 High expression of e, f), and anti-inflammatory factor IL-10 ( Figure 8 Low expression of a) and b). Surprisingly, compared with TiO2, M2@Ir-TiO2 treatment significantly reduced the levels of TNF-α, IL-6, and INF-γ, while increasing the level of IL-10. These data confirm that M2@Ir-TiO2 has significant antioxidant effects, effectively alleviating cellular inflammatory responses and improving the inflammatory environment within plaques.

[0050] (4) The in vitro cholesterol efflux-promoting effect of M2@Ir-TiO2 ( Figure 3 ).

[0051] Reactive oxygen species (ROS) can oxidize low-density lipoprotein (LDL) to oxidized low-density lipoprotein (ox-LDL), causing macrophages to transform into foam cells and inducing an inflammatory response, ultimately leading to plaque rupture. The above results demonstrate that M2@Ir-TiO2 has excellent reactive oxygen species scavenging capabilities. This invention further investigates whether M2@Ir-TiO2 treatment can reduce macrophage uptake of oxidized low-density lipoprotein (ox-LDL) and thus reduce foam cell formation. In this invention, RAW264.7 cells were pretreated with various formulations and then incubated with DiI-labeled ox-LDL (DiI-ox-LDL) for 4 hours. Fluorescence microscopy and flow cytometry analysis showed that RAW264.7 cells treated with LPS exhibited the strongest red fluorescence, while the M2@Ir-TiO2 group showed the weakest red fluorescence.

[0052] The above results indicate that LPS stimulation of macrophages to generate reactive oxygen species can increase the accumulation of intracellular ox-LDL. After 4 hours of co-incubation with M2@Ir-TiO2, the red fluorescence (DiI-ox-LDL) in macrophages was significantly reduced, indicating that it significantly inhibited the accumulation of DiI-ox-LDL in RAW264.7 cells. Figure 3 g, h, i). Simultaneously, the promoting effect of Ir-TiO2 on the cholesterol transport pathway was detected using a cholesterol content assay kit. The results showed that the cholesterol content in the supernatant of the Ir-TiO2 group and the M2@Ir-TiO2 group was significantly increased (g, h, i). Figure 3 The above results indicate that M2@Ir-TiO2 helps reduce intracellular lipid retention and inhibit foam cell formation. Similarly, Oil Red O2 (ORO) staining confirmed a reduction in foaming, meaning that the number of foam cells in the M2@Ir-TiO2 group was significantly reduced compared to the LPS group. Figure 3 k).

[0053] 3. In vivo therapeutic effects of M2@Ir-TiO2.

[0054] Among them, M2@Ir-TiO2 targets plaques in vivo and clears reactive oxygen species (ROS). Figure 4 Representative images and radar images of aortic root sections stained with ORO, H&E, Masson, anti-α-SMA antibody, and anti-CD68 antibody ( ); Figure 5 ).

[0055] Specifically as follows:

[0056] Using 6-8 weeks of apolipoprotein E deficiency (ApoE) − / −An atherosclerosis model was established in male mice, and the pharmacokinetic characteristics of M2@Ir-TiO2 in C57BL / 6 mice were studied. Following intravenous injection of IR-labeled M2@Ir-TiO2, fluorescence imaging showed that M2@Ir-TiO2 was almost completely cleared from the blood after 24 hours. Figure 4 (b, c). Then, in ApoE carrying atherosclerotic plaques − / − The in vivo atherosclerosis-targeting ability of IR-labeled M2@Ir-TiO2 injected via tail vein was verified in mice. Twelve hours after intravenous injection, IR-labeled M2@Ir-TiO2 accumulated in an isolated aorta. Mice were sacrificed, and their major organs and aorta were harvested for in vitro processing. In vitro imaging clearly showed the accumulation of IR-M2@Ir-TiO2 in atherosclerotic plaques. Strong fluorescence was observed in the arcuate region of the aorta, a region prone to atherosclerosis development. In contrast, the fluorescence in the atherosclerotic plaque region of the IR-Ir-TiO2 group was weaker, significantly lower than that of the IR-M2@Ir-TiO2 group. This result indicates that M2 functionalization can enhance the accumulation of IR-M2@Ir-TiO2 in plaque regions in vivo. Figure 4 d). 12 h post-injection, the fluorescence signal was mainly distributed in the liver and kidneys. 24 h later, the fluorescence in the liver significantly decreased, while the fluorescence in the kidneys remained strong, suggesting that the nanoparticles in mice may be mainly excreted through the kidneys. Figure 9 ).

[0057] Twenty mice were randomly divided into four groups (n=5): control group, model group, Ir-TiO2 group, and M2@Ir-TiO2 group. They were treated twice weekly via tail vein injection at a dose of approximately 10 mg / kg each time. After two months of treatment, the aorta was harvested for ORO staining; the red areas after staining indicated plaque areas. Figure 4 a). ORO staining clearly showed atherosclerotic plaque areas in the model group. Ir-TiO2 treatment reduced plaque area by up to 20%. Compared with Ir-TiO2, M2@Ir-TiO2 treatment showed a slight improvement, possibly due to M2 functionalization enhancing the aggregation ability of IR-TiO2 in plaques. M2@Ir-TiO2 treatment significantly improved the treatment effect, with the plaque rate reduced to 6.77%, indicating that M2@Ir-TiO2 effectively inhibited the progression of atherosclerosis. Figure 4 e, f). Intracellular ROS production after M2@Ir-TiO2 treatment was detected using a DHE probe. The control group had the lowest ROS levels among all groups. When plaques form, the plaques contain a high concentration of ROS. Ir-TiO2 significantly reduced ROS levels within the plaques, with the M2@Ir-TiO2 group showing a more pronounced effect, as M2 can cause more Ir-TiO2 to accumulate at the plaque site, thus clearing ROS. Figure 4g, h). Simultaneously, ORO-stained frozen sections of the aorta yielded similar results, demonstrating the effective anti-atherosclerotic activity of M2@Ir-TiO2 ( Figure 5 These results indicate that M2@Ir-TiO2 is effective in slowing the progression of atherosclerosis.

[0058] Immunohistochemical analysis of CD68 (macrophage marker) Figure 5 a) M2@Ir-TiO2 was found to significantly reduce the number of macrophages in aortic plaques. Notably, increased expression of necrotic cores and macrophage infiltration may promote the progression of atherosclerosis. In contrast, increased proliferation of vascular smooth muscle cells (VSMCs) and increased collagen concentration are considered beneficial at all stages of atherosclerosis. Furthermore, anti-α-smooth muscle actin (α-SMA) antibody staining showed a significant increase in the number of SMCs in the lesion area after M2@Ir-TiO2 treatment. Figure 5 a). Consistent with these results, Masson staining also showed a significant upregulation of collagen concentration. These findings suggest that M2@Ir-TiO2 can stabilize further plaque development and inhibit the occurrence of atherosclerosis.

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

[0060] To investigate whether nanomedicines would cause unnecessary damage to healthy cells, the biosafety of M2@Ir-TiO2 at different concentration gradients was tested at the cellular level using a CCK8 assay kit. RAW264.7 cells were treated with M2@Ir-TiO2 at concentrations of 5 µg / ml, 10 µg / ml, 20 µg / ml, 40 µg / ml, and 80 µg / ml. Figure 10 a) and HUVECs cells ( Figure 10 b) The results showed that when the concentration of M2@Ir-TiO2 was below 40 μg / ml, the cell viability still reached 75%, indicating that M2@Ir-TiO2 has high safety. Figure 10 The hemolysis test showed that although the concentration of M2@Ir-TiO2 was 200 μg / mL, no obvious hemolysis occurred. Figure 11 ).

Claims

1. A biomimetic nanocomposite, characterized in that, It is a metal nanoscale enzyme Ir-TiO2 wrapped by M2 macrophage membrane.

2. The method for preparing a biomimetic nanocomposite according to claim 1, characterized in that, The method comprises the following steps: S1. Preparing TiO2; S2. Preparing Ir-TiO2: adding ethanol and IrCl3 into the TiO2, and obtaining Ir-TiO2 after sufficient reaction; S3. Preparing the biomimetic nanocomposite: mixing the Ir-TiO2 and cell membrane liquid containing membrane protein, uniformly dispersing under ultrasonic, then low-temperature centrifugation, discarding the supernatant, adding PBS for resuspension, and obtaining the biomimetic nanocomposite.

3. The method for preparing a biomimetic nanocomposite according to claim 2, characterized in that, The concentration of the metal nanoscale enzyme Ir-TiO2 and the membrane protein in the cell membrane liquid is 1 mg / mL, and the volume ratio of the metal nanoscale enzyme Ir-TiO2 and the cell membrane liquid is 1:

1.

4. Application of the biomimetic nanocomposite according to claim 1 in the preparation of a product for treating atherosclerosis.

Images