M2 macrophage membrane wrapped ROS response type bionic nano delivery system as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to an M2 macrophage membrane wrapped ROS response type bionic nano delivery system as well as a preparation method and application thereof. The ROS response type bionic nano delivery system wrapped by the M2 macrophage membrane is composed of ROS response nano particles carrying therapeutic drugs and the M2 macrophage membrane wrapping the ROS response nano particles. The ROS response type bionic nano delivery system wrapped by the M2 macrophage membrane provided by the invention is excellent in biocompatibility, has the functions of immune escape, precise targeting, removal of ROS in cells, inhibition of inflammatory injury and cell apoptosis and the like, and provides a bionic nano delivery system with targeting delivery, intelligent drug release and multi-dimensional regulation and control for atherosclerosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a ROS-responsive biomimetic nanodelivery system encapsulated in M2 macrophage membranes, its preparation method, and its application. Background Technology

[0002] Atherosclerosis, as the core pathological basis of cardiovascular and cerebrovascular events, involves endothelial cell damage, oxidized low-density lipoprotein (oxLDL) deposition, and a macrophage-driven inflammatory cascade. In early lesions, the accumulation of oxLDL under the intima triggers an increase in reactive oxygen species (ROS), promoting macrophage polarization towards pro-inflammatory M1 macrophages, forming a vicious cycle of inflammation, leading to plaque formation and arterial damage. Existing therapies, such as statins and PCSK9 inhibitors, can partially delay disease progression by lowering cholesterol, but their efficacy is significantly limited, and long-term use may increase the risk of new-onset diabetes and infection. Traditional nanomedicine delivery systems, while improving drug targeting, still face two major bottlenecks: 1. Immune clearance issues: Nanoparticles are easily recognized and cleared by the reticuloendothelial system (RES), resulting in low delivery efficiency to target tissues; 2. Insufficient response to the lesion microenvironment: Existing carriers cannot accurately respond to the high-ROS microenvironment within plaques, leading to uncontrollable drug release and an inability to simultaneously achieve anti-inflammatory and plaque stabilization.

[0003] In recent years, biomimetic nanotechnology has offered new solutions to these problems. For example, nanoparticles encapsulated in red blood cell membranes can extend blood circulation time by more than three times by mimicking natural cell membrane surface proteins, significantly improving drug accumulation at plaque sites. Although progress has been made in biomimetic nanotechnology, existing solutions are still limited to single-function optimization and cannot simultaneously achieve ROS clearance, macrophage phenotypic conversion, and anti-inflammatory-antioxidant synergistic regulation, severely limiting the effectiveness of targeted therapy for atherosclerosis. Therefore, there is an urgent need for a novel biomimetic nanodelivery system that combines intelligent response with multi-dimensional therapeutic functions. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a ROS-responsive biomimetic nanodelivery system encapsulated in M2 macrophage membranes, along with its preparation method and applications.

[0005] The present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a ROS-responsive biomimetic nanodelivery system encapsulated in an M2 macrophage membrane, the ROS-responsive biomimetic nanodelivery system comprising: ROS-responsive nanoparticles and an M2 macrophage membrane encapsulating the ROS-responsive nanoparticles.

[0007] Furthermore, the thickness of the M2 macrophage membrane ranges from 5 to 20 nm.

[0008] M2 macrophage membranes carry anti-inflammatory factors and specific receptors. By actively targeting inflammatory sites, neutralizing pro-inflammatory factors, inhibiting M1 macrophage polarization within plaques, promoting macrophage transformation to the M2 phenotype, and accelerating tissue repair and plaque stabilization, they can prevent ordinary macrophages from polarizing into the pro-inflammatory M1 type in the disease microenvironment, thus avoiding the exacerbation of lipid accumulation and plaque rupture.

[0009] Furthermore, the ROS-responsive biomimetic nanoparticles are particles containing phenylboronic acid ester bonds.

[0010] Secondly, the present invention provides a method for preparing a ROS-responsive biomimetic nanodelivery system encapsulated in an M2 macrophage membrane, comprising:

[0011] Therapeutic drugs were loaded onto ROS-responsive nanoparticles to obtain drug-loaded ROS-responsive nanoparticles.

[0012] The drug-carrying ROS-responsive nanoparticles were encapsulated using an M2 macrophage membrane to obtain the M2 macrophage membrane-encapsulated ROS-responsive biomimetic nanodelivery system.

[0013] Furthermore, the method for synthesizing the ROS-responsive biomimetic nanoparticles includes: dissolving 4-hydroxyphenylboronic acid pinacol ester and carbonyl diimidazole in dichloromethane at a molar ratio of 1:4-6, reacting at 20-30°C for 3-6 hours to obtain the activated product of borate ester-imidazole.

[0014] Using chitosan oligosaccharide and borate ester-imidazolium activated products as raw materials, 4-dimethylaminopyridine as catalyst, and dimethyl sulfoxide as solvent, the reaction was carried out at 20-30℃ for 8-16 h, and then purified by dialysis to obtain ROS-responsive biomimetic nanoparticles.

[0015] The molar ratio of chitosan oligosaccharide to borate ester-imidazolium activated product is 1:1.5-2.5.

[0016] Furthermore, the molar ratio of 4-hydroxyphenylboronic acid pinacol ester to carbonyl diimidazole was 1:5, the reaction temperature was 25℃, and the reaction time was 4 h. The activated product of the borate ester-imidazole was washed with water and saturated NaCl solution and dried with MgSO4.

[0017] Furthermore, the molar ratio of chitosan oligosaccharide to the activated product of borate ester-imidazole was 1:2, the reaction temperature was 25℃, and the reaction time was 12 h.

[0018] Furthermore, the synthesized ROS-responsive nanoparticles and the therapeutic drug are dissolved together in an organic solvent, and drug-loaded ROS-responsive nanoparticles are synthesized through self-assembly. Preferably, the organic solvent is dimethyl sulfoxide. Further, the mass ratio of the ROS-responsive nanoparticles to the therapeutic drug is (85-95):(5-15). Preferably, the mass ratio of the ROS-responsive nanoparticles to the therapeutic drug is 90:10.

[0019] Furthermore, the preparation method of the ROS-responsive biomimetic nanodelivery system includes: fusing the M2 macrophage membrane with the drug-carrying ROS-responsive nanoparticles using a co-extrusion method, so that the M2 macrophage membrane uniformly coats the surface of the drug-carrying ROS-responsive nanoparticles, thereby obtaining the ROS-responsive biomimetic nanodelivery system coated by the M2 macrophage membrane.

[0020] Furthermore, in the preparation method of the ROS-responsive biomimetic nanodelivery system, the therapeutic drug includes: a drug for treating atherosclerosis.

[0021] Thirdly, the present invention provides the application of an M2 macrophage membrane-encapsulated ROS-responsive biomimetic nanodelivery system in the preparation of drugs for treating atherosclerosis.

[0022] Furthermore, the present invention provides a medicament for treating atherosclerosis, the medicament comprising: an active ingredient of the medicament and pharmaceutically acceptable excipients.

[0023] Furthermore, the active ingredient of the drug includes: the ROS-responsive biomimetic nanodelivery system encapsulated in the M2 macrophage membrane obtained by the above preparation method.

[0024] Furthermore, the active ingredient of the drug also includes: a drug for treating atherosclerosis.

[0025] Furthermore, the drugs used to treat atherosclerosis include tanshinone IIA.

[0026] The present invention has the following beneficial effects: The ROS-responsive biomimetic nanodelivery system encapsulated by M2 macrophage membranes provided by the present invention has the following advantages: First, the M2 macrophage membrane endows the ROS-responsive biomimetic nanoparticles with immune escape capabilities, significantly reducing non-specific phagocytosis by macrophages and prolonging the retention time of drugs in the bloodstream; simultaneously, the specific receptors on the surface of the M2 macrophage membrane actively target the lesion site, enhancing the accumulation efficiency of drugs within atherosclerotic plaques. Second, the ROS-responsive nanoparticles achieve precise drug release at the lesion site. In a high reactive oxygen species (ROS) environment, the drug is rapidly released locally on the plaque, while in a physiological environment, the release is slow, significantly reducing the risk of systemic exposure and improving treatment safety. The system simultaneously exerts a synergistic effect of anti-inflammatory, antioxidant, and plaque stabilization. By clearing excess ROS, it reduces oxidative stress damage, inhibits the release of pro-inflammatory factors, and blocks the inflammatory cascade reaction; at the same time, it inhibits the internalization of oxidized low-density lipoprotein, reduces foam cell formation and lipid deposition, and enhances plaque structural stability and reduces the risk of rupture by increasing collagen synthesis and promoting vascular smooth muscle cell proliferation. In animal models, the system significantly slowed the progression of atherosclerosis, improved vascular endothelial function, and showed no hematologic toxicity or liver and kidney damage, demonstrating excellent biocompatibility. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the synthesis process of TIIA-NPs and MM@TIIA-NPs.

[0028] Figure 2 Characterization diagram of Oxi-COS and its intermediates; where, Figure 2 'a' in CDI-PBAP 1 H NMR spectrum; Figure 2 In this context, 'b' represents Oxi-COS. 1 H NMR spectrum; Figure 2 In the image, 'c' represents the Fourier transform infrared spectrum.

[0029] Figure 3 These are transmission electron microscope (TEM) images; among them, Figure 3 In the image, 'a' represents the TEM image of TIIA-NPs. Figure 3 In the image, b represents the TEM image of Oxi-COS; Figure 3 In this context, 'c' represents the TEM image of MM; Figure 3 In the image, d represents the TEM image of MM@TIIA-NPs.

[0030] Figure 4 The plot shows the particle size distribution and zeta potential; where, Figure 4 In this context, 'a' represents the particle size distribution analyzed by dynamic light scattering. Figure 4In the diagram, b represents the Zeta potential plots of TIIA-NPs, MM, and MM@TIIA-NPs.

[0031] Figure 5 The results of Western blotting assessment of the biological function of M2 macrophage membranes and ROS responsiveness are shown in the figures; among them, Figure 5 In the diagram, 'a' represents the WB protein band pattern. Figure 5 In the figure, b is a quantitative analysis diagram of the TLR4 strip intensity; Figure 5 In the figure, c represents the quantitative analysis of the band intensity of integrin β1; Figure 5 In the figure, d represents the quantitative analysis diagram of the CCR2 band intensity; Figure 5 In the figure, 'e' represents the quantitative analysis diagram of SRA strip intensity; Figure 5 f in the figure represents the particle size change of TIIA-NPs in different solutions over 48 hours; Figure 5 In the figure, g represents the drug release curves of TIIA-NPs in physiological and simulated inflammatory environments.

[0032] Figure 6 A graph showing the in vitro cytotoxicity assessment of MM@TIIA-NPs in different cell types.

[0033] Figure 7 This is a diagram showing cellular uptake in RAW264.7 macrophages; among them, Figure 7 In the image, 'a' represents a confocal microscope image of the uptake of MM@TIIA-NPs and TIIA-NPs under PBS conditions (scale bar = 50 μm). Figure 7 b in the figure is a confocal microscopy image of the uptake of MM@TIIA-NPs and TIIA-NPs under inflammatory stimulation (scale bar = 50 μm). Figure 7 In the figure, c represents the time-dependent uptake profile of Cy5.5-labeled nanoparticles as assessed by flow cytometry under PBS conditions. Figure 7 In the figure, d represents the time-dependent uptake profile of Cy5.5-labeled nanoparticles as assessed by flow cytometry under inflammatory conditions. Figure 7 In the figure, 'e' represents the quantitative average fluorescence intensity under PBS conditions. Figure 7 f in the figure represents the quantitative average fluorescence intensity under inflammatory stimulation.

[0034] Figure 8 A graph showing cellular uptake in HUVEC and VSMC cells; Figure 8 In the figure, 'a' represents the time-dependent uptake profile of Cy5.5-labeled nanoparticles by HUVEC cells in PBS as assessed by flow cytometry. Figure 8 In the figure, b represents the time-dependent uptake profile of Cy5.5-labeled nanoparticles by HUVEC cells under inflammatory stimulation, as assessed by flow cytometry. Figure 8c in the figure represents the quantitative average fluorescence intensity of HUVEC cells in the PBS environment; Figure 8 In the figure, d represents the quantitative average fluorescence intensity of HUVEC cells under inflammatory stimulation. Figure 8 In the figure, 'e' represents the time-dependent uptake profile of Cy5.5-labeled nanoparticles by VSMC cells in PBS as assessed by flow cytometry. Figure 8 f in the figure represents the time-dependent uptake profile of Cy5.5-labeled nanoparticles by VSMC cells under inflammatory stimulation, as assessed by flow cytometry. Figure 8 In the figure, g represents the quantitative average fluorescence intensity of VSMC cells in the PBS environment; Figure 8 In the figure, h represents the quantitative average fluorescence intensity of VSMC cells under inflammatory stimulation.

[0035] Figure 9 This diagram illustrates the regulatory role of MM@TIIA-NPs in the production of reactive oxygen species in macrophages; among them, Figure 9 In the image, 'a' represents a confocal microscopy image of reactive oxygen species generation in macrophages of each group. Figure 9 In the figure, b represents a quantitative analysis of the level of reactive oxygen species (ROS) generation within macrophages.

[0036] Figure 10 This is a diagram illustrating the inhibitory effect of MM@TIIA-NPs on reactive oxygen species (ROS) production in macrophages; where, Figure 10 In this context, 'a' represents the reactive oxygen species level in macrophages of each group as detected by flow cytometry. Figure 10 b in the figure represents the quantitative analysis of the average fluorescence intensity of reactive oxygen species (ROS) in macrophages.

[0037] Figure 11 This diagram illustrates the inhibitory effects of MM@TIIA-NPs on macrophage oxidative stress and the release of inflammatory factors; among which, Figure 11 In the figure, 'a' represents the level of 8-OHdG, a biomarker of oxidative damage. Figure 11 In the figure, b represents the level of the lipid peroxidation marker 8-iso-PGF2α. Figure 11 In the figure, c represents the release level of the pro-inflammatory factor TNF-α; Figure 11 In this context, d represents the release level of the chemokine MCP-1; Figure 11 The 'e' in the graph represents the release level of the pro-inflammatory factor IL-1β.

[0038] Figure 12 This is a diagram illustrating the inhibitory effect of MM@TIIA-NPs on macrophage apoptosis; where, Figure 12 The image in Figure 'ae' represents the apoptosis of macrophages in each group as detected by flow cytometry. Figure 12 f in the figure represents a quantitative analysis of macrophage apoptosis rate.

[0039] Figure 13The image shows the inhibition of oxLDL uptake by macrophages by MM@TIIA-NPs; where, Figure 13 In the image, 'a' represents a confocal microscopy image showing the uptake of DiI-oxLDL by macrophages; Figure 13 In the figure, b represents the quantitative analysis of the average fluorescence intensity of oxLDL uptake.

[0040] Figure 14 This is a diagram showing the inhibition of oxLDL internalization by MM@TIIA-NPs in vascular smooth muscle cells; among them, Figure 14 In the image, 'a' is a fluorescence micrograph showing the internalization of DiI-oxLDL by VSMCs; Figure 14 In the figure, b represents the quantitative analysis of the average fluorescence intensity of oxLDL uptake.

[0041] Figure 15 Representative flow cytometry images of intracellular reactive oxygen species generation in different treatment groups.

[0042] Figure 16 A diagram showing the inhibition of foaming in macrophages and vascular smooth muscle cells by MM@TIIA-NPs (n=3); among them, Figure 16 In the diagram, 'a' represents the identification of foam cell formation using Oil Red O staining. Figure 16 In the figure, b represents the quantitative analysis of the relative area of ​​lipid droplets in foam cells derived from macrophages; Figure 16 In the figure, 'c' represents a quantitative analysis of the relative area of ​​lipid droplets within foam cells derived from vascular smooth muscle cells.

[0043] Figure 17 A schematic diagram of the experimental procedure for inducing atherosclerosis in zebrafish larvae using a high-cholesterol, high-sugar diet.

[0044] Figure 18 Map showing how MM@TIIA-NPs improve lipid metabolism and reduce vascular lipid accumulation in zebrafish (scale bar = 250 μm); among them, Figure 18 In the image 'a', there is a representative image of triglyceride accumulation in the blood vessels of zebrafish (Oil Red O staining). Figure 18 b in the image represents a representative image of cholesterol accumulation in the blood vessels of zebrafish. Figure 18 In the figure, c represents the quantitative analysis diagram of the fluorescence intensity of oleotriester accumulation; Figure 18 In the figure, d represents the quantitative analysis of cholesterol accumulation fluorescence intensity.

[0045] Figure 19 A diagram showing the inhibition of inflammatory cell infiltration in zebrafish blood vessels by MM@TIIA-NPs (scale bar = 250 μm); among which, Figure 19 In the image, 'a' represents a representative image of neutrophil infiltration. Figure 19 In the image, b represents a representative image of macrophage infiltration; Figure 19 In the graph, 'c' represents the quantitative analysis of neutrophil count. Figure 19In the graph, d represents the quantitative analysis of macrophage count.

[0046] Figure 20 Map showing how MM@TIIA-NPs improve vascular function and reduce oxidative stress in zebrafish (scale bar = 250 μm); among which, Figure 20 In the image, 'a' is a microscopic image of the vascular endothelial cell layer in the fundus of a zebrafish. Figure 20 In this context, 'b' represents the quantitative analysis of blood flow velocity in zebrafish larvae. Figure 20 In the figure, 'c' represents a quantitative analysis of reactive oxygen species levels in zebrafish larvae.

[0047] Figure 21 For MM@TIIA-NPs in ApoE - / - Graph showing the targeted delivery characteristics in mice and its inhibitory effect on aortic atherosclerotic plaques; among which, Figure 21 'a' in ApoE - / - Schematic diagram of mouse experiments and treatment protocols; Figure 21 In the figure, b represents the pharmacokinetic curve; Figure 21 In the figure, c represents the fluorescence intensity analysis of blood samples at different time points; Figure 21 In this context, d represents fluorescence imaging of the isolated aorta (48 h). Figure 21 The image showing 'e' in the diagram represents the quantitative analysis of fluorescence signals in the aorta. Figure 21 f in the image represents a representative image of Oil Red O staining of the aorta. Figure 21 The graph in 'g' represents a quantitative analysis of the aortic lesion region. Figure 21 The 'h' in the figure represents the trend of mouse weight change during the treatment period.

[0048] Figure 22 MM@TIA-NPs for multi-target regulation of ApoE - / - Analysis of plaque stability and inflammatory response in the aortic root of mice; among which... Figure 22 'a' in ApoE - / - Histochemical staining analysis of the root of the mouse aorta; Figure 22 In the diagram, b represents the percentage of the necrotic core area. Figure 22 In the figure, 'c' represents the area ratio of macrophages. Figure 22 In the figure, d represents the percentage of MMP2 expression area. Figure 22 In the figure, 'e' represents the percentage of MMP9 expression area. Figure 22 In the figure, f represents the area ratio of collagen. Figure 22 In the figure, g represents the area percentage of VSMC; Figure 22 In the figure, h represents the percentage of TNF-α expression area; Figure 22 In the figure, i represents the percentage of MCP-1 expression area.

[0049] Figure 23 For ApoE - / -Figure showing the results of detecting inflammatory factor levels in mouse serum; where, Figure 23 In the figure, 'a' represents the serum tumor necrosis factor-α level in each group. Figure 23 b in the figure represents the serum interleukin-6 level in each group; Figure 23 In the graph, b represents the serum C-reactive protein level of each group.

[0050] Figure 24 ApoE after long-term intervention with different formulations - / - Safety assessment of typical hematological parameters and biochemical markers related to liver, kidney, and cardiac function in mouse serum (n=3); Liver function indicators: alanine aminotransferase, aspartate aminotransferase, total bilirubin, albumin; Myocardial injury markers: creatine kinase, creatine kinase isoenzyme MB, lactate dehydrogenase, lactate dehydrogenase-1; Kidney function indicators: blood urea nitrogen, creatinine, uric acid; Blood lipid parameters: total cholesterol, triglycerides; Complete blood count parameters: white blood cell count, red blood cell count, platelet count.

[0051] Figure 25 For ApoE - / - Observation results of H&E staining sections of major organs of mice (scale bar = 500 μm). Detailed Implementation

[0052] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0053] In the following embodiments, all results involving numerical values ​​are expressed as mean ± standard error, *P<0.05; **P<0.01.

[0054] Example 1: Synthesis, Characterization, and Analysis of an M2 Macrophage Membrane-Encapsulated ROS-Responsive Biomimetic Nanodelivery System

[0055] 1. Synthesis of M2 macrophage membrane-encapsulated drug-loaded ROS-responsive biomimetic nanodelivery system: A schematic diagram of the synthesis process of the M2 macrophage membrane-encapsulated drug-loaded ROS-responsive biomimetic nanodelivery system (MM@TIIA-NPs) is shown below. Figure 1 As shown, the specific synthesis method is as follows:

[0056] (1) Synthesis of ROS-responsive nanoparticles (Oxi-COS): 5 mmol of 4-hydroxyphenylboronic acid pinacol ester was reacted with 25 mmol of carbonyl diimidazole (CDI) in 15 mL of dry dichloromethane. The crude product was washed with deionized water (3 × 20 mL) and saturated NaCl solution (10 mL), and dried with MgSO4 for later use. 5 mmol of chitosan oligosaccharide was dissolved in 10 mL of anhydrous dimethyl sulfoxide, 10 mmol of 4-dimethylaminopyridine was added, followed by the addition of 10 mmol of the borate-imidazole activation product. The reaction was carried out for 10 h, and Oxi-COS was obtained by dialysis with deionized water for 72 h.

[0057] (2) Synthesis of ROS-responsive nanoparticles (TIIA-NPs) of tanshinone IIA: 30 mg of Oxi-COS was dissolved in 300 mL of deionized water and stirred for 1 hour. Oxi-COS and tanshinone IIA (TIIA) were added to dimethyl sulfoxide (DMSO) at a mass ratio of 90:10. After dissolution, the solution was added dropwise to water and stirred continuously. TIIA-NPs were obtained by dialysis purification. The drug encapsulation efficiency (DEE) of TIIA-NPs was 96.5% and the drug loading (DLC) was 2%.

[0058] (3) M2 macrophage membrane extraction: M2 macrophages were centrifuged and resuspended in TM buffer containing 1% PMSF. o Incubate at C for 2 hours. Mix the cell suspension with 1 M sucrose and incubate at 4°C. o Centrifuge twice at 2000 × g for 15 minutes at C. Centrifuge again at 3000 × g, collect the supernatant, and wash the cell membrane twice with 0.25 M sucrose TM buffer for purification.

[0059] (4) Synthesis of MM@TIIA-NPs: TIIA-NPs and M2 macrophage membranes were mixed in a buffer solution at a volume ratio of 2:1 and stirred evenly. The mixture was then co-extruded through a 200 nm polycarbonate filter membrane to achieve uniform membrane encapsulation and obtain a drug-loaded ROS-responsive biomimetic nanodelivery system (MM@TIIA-NPs) encapsulated in M2 macrophage membrane.

[0060] 2. Characterization tests of MM@TIIA-NPs

[0061] pass 1¹H NMR and FT-IR spectroscopy were used to characterize the structure of Oxi-COS and its precursors, verifying the successful synthesis of MM@TIIA-NPs using the above method. Transmission electron microscopy (TEM) was used to observe the morphology of TIIA-NPs, MM, and MM@TIIA-NPs. Dynamic light scattering (DLS) was used to verify their size and dispersion. Zeta potential analysis was used to analyze the surface properties of the M2 macrophage membrane, and Western blotting was used to assess the biological function of the M2 macrophage membrane. In vitro simulation experiments were conducted to evaluate the ROS responsiveness and drug release of the synthesized MM@TIIA-NPs.

[0062] 3. Characterization test results

[0063] Depend on 1 H NMR spectroscopy ( Figure 2 a and b in the text), FT-IR spectral analysis ( Figure 2 As shown in result c), the above method successfully synthesized Oxi-COS. TEM results ( Figure 3 ) and DLS results ( Figure 4 As shown in a), TIIA-NPs exhibit a spherical structure with uniform size and an average diameter of approximately 150 nm, showing no aggregation. MM@TIIA-NPs exhibit a core-shell structure, with the nanoparticle core surrounded by a film approximately 10 nm thick, and the average diameter increasing from 150 nm to 200 nm. The Zeta potential measurement results are as follows... Figure 4 As shown in b, MM@TIIA-NPs exhibit a reduced negative zeta potential compared to free TIIA-NPs, reflecting the surface properties of the M2 macrophage membrane.

[0064] Depend on Figure 5 As shown in Figure ae, Western blot results indicate that key macrophage markers TLR4 (LPS receptor), integrin β1, CCR2 (MCP-1 receptor in atherosclerotic plaques), and SRA (oxLDL receptor) are all significantly expressed on MM@TIIA-NPs. This suggests that MM@TIIA-NPs successfully retain the original proteins of the macrophage membrane and possess the biological functions of the M2 macrophage membrane.

[0065] ROS responsiveness, such as Figure 5 As shown in f, when exposed to simulated ROS conditions (1.00 mM H2O2), the nanoparticles degrade and release TIIA. Drug release curves ( Figure 5 The results (g) indicate that in an environment with excessive ROS, the drug release reaches approximately 60% within 24 hours, compared to only 18% under non-ROS conditions, confirming the ROS-responsive release mechanism.

[0066] Example 2: In vitro performance study of MM@TIIA-NPs

[0067] 1. Cell Culture and Processing

[0068] RAW264.7 macrophages, vascular smooth muscle cells (VSMCs), and human umbilical vein endothelial cells (HUVECs) used in the experiment were all provided and identified by the Guangdong Academy of Traditional Chinese Medicine. Cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2.

[0069] 2. Testing Methods

[0070] 2.1 Cytotoxicity test

[0071] RAW264.7, VSMCs, and HUVECs were each used at 5×10⁻⁶ pores per well. 3 Cells were seeded at a density of 1000 μg / mL in 96-well plates and cultured for 24 h until adherence. Then, different concentrations of TIIA, TIIA-NPs, and MM@TIIA-NPs (0, 5, 25, and 50 μg / mL) were added. After 12 h and 24 h of treatment, 10 μL of CCK-8 solution was added to each well, and incubation continued for 2 h. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated.

[0072] 2.2 Cell uptake experiment

[0073] RAW264.7 macrophages were used at a rate of 2 × 10⁻⁶. 5 VSMCs and HUVECs cells were used at 3 × 10⁶ cells / well. 5 Cells were seeded per well and cultured for 24 h. The experiment included an inflammatory stimulation group and a non-inflammatory group (n=3). The inflammatory stimulation group was pretreated for 12 h with culture medium containing lipopolysaccharide (LPS, 100 ng / mL) and interferon-γ (IFN-γ, 100 IU / mL). Each group was then treated with Cy5.5-labeled TIIA-NPs or MM@TIIA-NPs (5 μg / mL) and cultured for different durations (0.5, 2, 6, 12 h). After culture, cells were washed with PBS, nuclei were stained with DAPI (blue), and late endosomes and lysosomes were labeled with LysoTracker Green (green). The intracellular distribution of nanoparticles was observed under a confocal microscope (Cy5.5 red fluorescence), and the mean fluorescence intensity (MFI) was quantitatively analyzed using flow cytometry.

[0074] 2.3 Intracellular reactive oxygen species (ROS) scavenging and oxidative damage treatment capacity test

[0075] ROS clearance capacity: RAW264.7 cells were pretreated with 5 μg / mL TIIA, TIIA-NPs, and MM@TIIA-NPs for 2 h, respectively, and then an inflammation model was induced by stimulation with 100 ng / mL LPS and 100 IU / mL IFN-γ. Cells were then incubated with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) in fresh culture medium for 30 min, washed, and intracellular ROS levels were measured by flow cytometry.

[0076] Detection of oxidative damage markers: An oxidative damage model was established by treating RAW264.7 cells with 1.00 mM H2O2. The concentrations of intracellular DNA oxidation marker 8-hydroxy-2′-deoxyguanosine (8-OHdG) and lipid peroxidation marker 8-iso-prostaglandin F2α (8-iso-PGF2α) were determined by ELISA.

[0077] 2.4 Apoptosis Experiment

[0078] RAW264.7 cells were pretreated with different agents at 5 μg / mL for 2 h, and then an apoptosis model was induced by 1.00 mM H2O2. The apoptosis status of RAW264.7 macrophages was assessed by flow cytometry.

[0079] 2.5 Effects on inflammatory factors

[0080] RAW264.7 cells were stimulated with 100 ng / mL LPS and 100 IU / mL IFN-γ. After intervention with different preparations, cell supernatants were collected, and flow cytometry was used to evaluate whether MM@TIIA-NPs could reduce the inflammatory response.

[0081] 2.6 Impact on oxLDL internalization

[0082] RAW264.7 cells and VSMCs were pre-incubated with 5 μg / mL TIIA, TIIA-NPs, and MM@TIIA-NPs for 2 hours, respectively, followed by co-culturing with DiI-oxLDL for 4 hours to assess the effect of MM@TIIA-NPs on the uptake of oxLDL by RAW264.7 macrophages and VSMCs. Cell nuclei were labeled blue with DAPI, and cell membranes were labeled green with DiO. Fluorescence intensity was observed using confocal microscopy and quantitatively analyzed by flow cytometry.

[0083] 2.7 Effects on foam cell formation

[0084] RAW264.7 cells were pretreated with LPS (100 ng / mL) and IFN-γ (100 IU / mL) for 24 hours, followed by co-incubation for 2 hours with 5 μg / mL TIIA, TIIA-NPs, and MM@TIIA-NPs (n=5). Then, oxLDL (50 μg / mL) was added and incubated for 48 hours. After washing and fixation, sections were stained with Oil Red O (ORO) to assess the effect of MM@TIIA-NPs on foam cell development.

[0085] 3. Experimental Results

[0086] 3.1 Cytotoxicity test results: The results are as follows Figure 6 As shown, at the highest tested concentration (50 μg / mL), the cell viability of the above three cell types was greater than 95%, confirming the excellent biocompatibility and safety of MM@TIIA-NPs in therapeutic applications.

[0087] 3.2 Results of Cell Uptake Experiment

[0088] RAW264.7 macrophage uptake results: Under non-inflammatory stimuli, such as Figure 7 As shown in a, cells treated with TIIA-NPs exhibited a strong red fluorescent signal, indicating that they were taken up in large quantities by macrophages; while the fluorescence intensity of the MM@TIIA-NPs group was significantly reduced, confirming that the membrane coating of M2 macrophages endowed them with immune escape ability. Figure 7 c and Figure 7 The data in Figure d show that the uptake of TIIA-NPs continuously increased with prolonged culture time, but MM@TIIA-NPs maintained a low uptake level at all time points. Under inflammatory stimuli, such as... Figure 7 As shown in b, macrophages further enhanced their internalization of TIIA-NPs, while the uptake of MM@TIIA-NPs remained largely unchanged. Figure 7 e and Figure 7 Quantitative analysis further showed that the uptake of TIIA-NPs continued to increase with the extension of culture time, while MM@TIIA-NPs maintained a low uptake level for 0.5-12 hours, further verifying its immune escape characteristics.

[0089] VSMCs and HUVECs uptake results: Under non-inflammatory conditions, Figure 8 a, c (HUVECs) and Figure 8 The results from e and g (VSMCs) showed that the uptake of MM@TIIA-NPs by both cell types was significantly stronger than that by TIIA-NPs. Under inflammatory stimulation ( Figure 8 b, d and Figure 8In the f and h groups, the uptake of MM@TIIA-NPs by both cell types was further enhanced and significantly higher than that by the TIIA-NPs group. This indicates that the encapsulation of the M2 macrophage membrane endows MM@TIIA-NPs with penetrability and enables them to specifically target HUVECs and VSMCs within atherosclerotic plaques, thereby increasing the accumulation of the drug in the local lesions.

[0090] 3.3 Results of ROS scavenging and oxidative damage treatment ability test: After treatment with MM@TIIA-NPs, the average fluorescence signal of ROS decreased, indicating that MM@TIIA-NPs can reduce ROS production. Figure 9 a in Figure 9 (b) Flow cytometry analysis confirmed that MM@TIIA-NPs effectively inhibited ROS levels in LPS / IFN-γ-stimulated macrophages ( Figure 10 a in Figure 10 (b) Measurements of intracellular 8-OHdG and 8-iso-PGF2α concentrations showed that exposure to H2O2 significantly increased the levels of 8-OHdG and 8-iso-PGF2α in RAW264.7 cells. Pretreatment with TIIA or TIIA-NPs and MM@TIIA-NPs significantly reduced the levels of these biomarkers. Figure 11 a in Figure 11 (b) and MM@TIIA-NPs showed a stronger protective effect than TIIA or TIIA-NPs, highlighting their potential as a superior therapeutic agent for alleviating ROS-induced oxidative damage.

[0091] 3.4 Apoptosis Experiment Results: The results showed that pretreatment with 5 μg / mL MM@TIIA-NPs effectively inhibited H2O2-triggered apoptosis in RAW264.7 cells. Figure 12 (af in the text)

[0092] 3.5 Effects of Inflammatory Factors: Experimental Results: The results showed a significant increase in the secretion of pro-inflammatory cytokines: tumor necrosis factor-α (TNF-α), chemokine-1 (MCP-1), and interleukin-1β (IL-1β). Figure 11 (ce in the text). After pretreatment with 5 μg / mL MM@TIIA-NPs for 2 hours, the levels of these factors in RAW264.7 cells were significantly reduced, indicating that MM@TIIA-NPs can alleviate inflammation in macrophages by reducing intracellular ROS production, highlighting their potential therapeutic effects in reducing inflammatory responses associated with atherosclerosis.

[0093] 3.6 Results of oxLDL internalization experiment: such as Figure 13As shown in a, RAW264.7 cells exhibited significant red fluorescence in the cytoplasm. Pretreatment with TIIA moderately reduced intracellular DiI-oxLDL uptake, while pre-incubation with MM@TIIA-NPs significantly reduced the fluorescence signal from DiI-oxLDL in RAW264.7 cells. Figure 13 (b) in the example. Similarly, as in... Figure 14 As shown, pretreatment with MM@TIIA-NPs significantly inhibited the uptake of oxLDL in VSMCs. Fluorescence-based observations were further validated by flow cytometry analysis, showing a significant reduction in the presence of DiI-oxLDL in the MM@TIIA-NPs-treated group compared to the TIIA-NPs or TIIA group. Figure 15 These findings highlight the superior efficacy of MM@TIIA-NPs in inhibiting oxLDL uptake in RAW264.7 cells and VSMCs.

[0094] 3.7 Effects on foam cell formation: Experimental results as follows: Figure 16 Figure a shows that, upon stimulation with oxLDL, RAW264.7 cells exhibited significant intracellular lipid accumulation and marked foam cell formation. In contrast, pretreatment with MM@TIIA-NPs inhibited foam cell formation. This is consistent with the results of intracellular ORO quantification analysis. Figure 16 (b) indicates that foam cell formation was significantly reduced after treatment with MM@TIIA-NPs. Similarly, VSMCs treated with MM@TIIA-NPs also showed a significant reduction in oxLDL-induced foam cell formation ( Figure 16 (a) These results are consistent with the observed reduction in oxLDL uptake ( Figure 16 c) indicates that MM@TIIA-NPs can reduce foam cell formation.

[0095] Example 3. Therapeutic effect of zebrafish atherosclerosis model

[0096] 1. Modeling and drug administration methods

[0097] (1) Experimental animals: Wild-type AB albino zebrafish and three transgenic strains, Tg(fli-1:EGFP), Tg(mpeg1:EGFP), and Tg(mpx:EGFP), were used in this study and were purchased from Hangzhou Huante Biotechnology Co., Ltd. Before the experiment, the zebrafish were acclimatized to the environment in the standard system water for at least one week (28℃, pH 6.5-8.5).

[0098] (2) Establishment of animal models: such as Figure 17As shown in a, zebrafish were modeled by administering a high-fat, high-sugar diet (0.15% egg yolk powder solution during the day and 3% glucose solution at night) via water-soluble administration. The zebrafish were treated at 28°C for 2 days to induce an atherosclerosis model.

[0099] (3) Grouping and Drug Administration: After successful modeling, zebrafish of various strains were randomly divided into 5 groups (n=10), including a control group (healthy zebrafish), a model group (atherosclerosis model), a TIIA group, a TIIA-NPs group, and a MM@TIIA-NPs group. The control group received no drug administration, while the model group received saline treatment. The TIIA group, TIIA-NPs group, and MM@TIIA-NPs group were all exposed to the drug-containing system via water bath administration at a concentration of 5 μg / mL for 24 hours. After treatment, relevant indicators were measured to evaluate the therapeutic effect of the drug on atherosclerosis.

[0100] 2. Testing Methods

[0101] 2.1 Lipid Level Test in Zebrafish

[0102] Wild-type AB albino zebrafish atherosclerosis models were treated with 0.03% ethyl-3-aminobenzoic acid methanesulfonate solution and then stained with ORO to observe lipid deposition in the vascular region. Low-density lipoprotein (LDL) cholesterol levels in the zebrafish samples were quantitatively measured using a commercial kit (Nanjing Jiancheng, China).

[0103] 2.2 Inflammation Level Test in Zebrafish

[0104] The level of inflammation in zebrafish was detected by transgenic zebrafish Tg(mpeg1:EGFP) and Tg(mpx:EGFP). The fluorescence intensity and number of macrophages and neutrophils were observed and counted using fluorescence microscopy.

[0105] 2.3 Endothelial Protection and Blood Flow Recovery Test

[0106] Optical sections of the ocular vascular system of transgenic zebrafish Tg(fli1:EGFP) were used to quantify the thickness of the endothelial cell layer. The green fluorescent protein (GFP) signal emitted by the endothelial cell layer was detected using the green fluorescence channel, thus enabling visual observation of the endothelial cell structure.

[0107] 2.4 ROS Level Test in Zebrafish

[0108] An atherosclerosis model was constructed using wild-type AB zebrafish, and the level of reactive oxygen species (ROS) was quantitatively measured using the commercial CM-H2DCFDA kit (Invitrogen, USA).

[0109] 3. Experimental Results

[0110] 3.1 Effects of lipid levels in zebrafish

[0111] The results showed that, compared with the control group, a large amount of triglycerides and cholesterol accumulation was observed near the tail of the blood vessels in the model group. Administration of TIIA, TIIA-NPs and MM@TIIA-NPs significantly reduced triglyceride and cholesterol deposition in zebrafish, and the difference between MM@TIIA-NPs and the model group was the most significant. Figure 18 (ad in the text). In addition, such as... Figure 18 As shown in e, compared with the control group, the low-density lipoprotein cholesterol (LDL-C) level in the model group was significantly increased, while MM@TIIA-NPs significantly reduced the LDL-C level.

[0112] 3.2 Effects on inflammation levels in zebrafish

[0113] like Figure 19 As shown in the ad, the results indicate that macrophage and neutrophil infiltration was significant in the blood vessels of the transgenic zebrafish atherosclerosis model group, and MM@TIIA-NPs treatment significantly reduced the infiltration of neutrophils and macrophages in the blood vessels.

[0114] 3.3 Endothelial protection and blood flow restoration results

[0115] In zebrafish with atherosclerosis, the thickness of the endothelial cell layer of the retinal blood vessels is increased. Figure 20 (a) Compared with the Tg(fli-1:EGFP) zebrafish model group, MM@TIIA-NPs treatment significantly reduced the thickness of the retinal vascular endothelium. Figure 20 As shown in b, MM@TIIA-NPs treatment can improve blood flow velocity, further highlighting its potential in improving hemodynamic conditions in atherosclerotic vessels.

[0116] 3.4 Effects on ROS levels in zebrafish

[0117] Figure 20 As shown in c, the results indicate that the ROS level in the model group was significantly higher than that in the control group, and MM@TIIA-NPs treatment significantly reduced the ROS level.

[0118] Example 4 ApoE - / - Therapeutic effect in mouse model of atherosclerosis

[0119] 1. Modeling and drug administration methods

[0120] (1) Experimental animals: 6-8 week old male C57BL / 6 wild-type mice and ApoE mice weighing 30g. - / - Mice. All mice were acclimatized to the environment and subjected to non-experimental stress for one week prior to the experiment.

[0121] (2) Establishment of animal models:

[0122] The process of constructing an atherosclerosis model is as follows: Figure 21 As shown in a, all mice simultaneously received a high-cholesterol, high-fat diet for a total of 12 weeks; ApoE was initiated from week 4. - / - The mice began to form atherosclerotic plaques, indicating successful model establishment. Continuing the high-cholesterol, high-fat diet until week 12 showed significant plaque accumulation.

[0123] (3) Grouping and drug administration: Mice were randomly divided into 5 groups (n=3), including a control group (healthy C57BL / 6 wild-type mice), a model group (ApoE), and a control group (healthy C57BL / 6 wild-type mice). - / - A mouse model of atherosclerosis was established, comprising the TIIA group, TIIA-NPs group, and MM@TIIA-NPs group. Intervention began in mice on week 4 of a high-cholesterol, high-fat diet. The TIIA, TIIA-NPs, and MM@TIIA-NPs groups received intravenous administration of 0.175 mg / kg once weekly. The model groups received an equal volume of saline, while the control group received no injections and served as a baseline reference. All groups continued the high-cholesterol, high-fat diet until week 12 (a total treatment period of 8 weeks). The therapeutic efficacy and safety of MM@TIIA-NPs were evaluated, and its pharmacokinetics were analyzed.

[0124] 2. Testing Methods

[0125] 2.1 Pharmacokinetic Testing

[0126] To determine the optimal timing for in vivo plaque targeting studies, the pharmacokinetics of Cy5.5-labeled MM@TIIA-NPs (MM@Cy5.5-NPs) and Cy5.5-labeled TIIA-NPs (Cy5.5-NPs) were evaluated using an in vivo imaging system (IVIS). Blood samples were collected from mice in each group at 0 h, 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, and 48 h after intravenous injection of the corresponding formulations for fluorescence intensity analysis.

[0127] 2.2 Treatment efficacy test

[0128] 2.2.1 Patch Load Detection

[0129] The overall plaque area of ​​the aorta was quantitatively assessed by ORO staining, and changes in mouse body weight were recorded periodically during the experiment.

[0130] 2.2.2 Patch stability evaluation

[0131] Observation of necrotic core area and tissue structure: H&E staining was used to analyze plaque morphology and the proportion of necrotic area;

[0132] Detection of inflammatory cell infiltration: Immunohistochemistry or immunofluorescence was used to detect the infiltration of macrophages in the plaque and the expression levels of matrix metalloproteinases MMP-9 and MMP-2;

[0133] Collagen content determination: The collagen deposition in the fiber cap was assessed using the Masson trichrome staining method;

[0134] Smooth muscle cell content detection: The distribution and accumulation of vascular smooth muscle cells (VSMCs) in the plaque were assessed by α-SMA immunostaining.

[0135] 2.2.3 Measurement of systemic and local inflammatory factors

[0136] Mouse serum was extracted, and the levels of pro-inflammatory cytokines (such as TNF-α, MCP-1, and IL-1β) were detected using methods such as ELISA; at the same time, inflammation-related staining or immunohistochemical analysis was performed on aortic root sections.

[0137] 2.3 In vivo safety assessment

[0138] 2.3.1 Hematological and Biochemical Indicator Detection

[0139] (1) Hematological analysis

[0140] Detect the levels of white blood cell count (WBC), red blood cell count (RBC), and platelet count (PLT);

[0141] (2) Safety assessment of the levels of biochemical markers related to liver and kidney function and cardiac function

[0142] Liver function indicators: alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), albumin (ALB);

[0143] Myocardial injury markers: creatine kinase (CK), creatine kinase isoenzyme MB (CKMB), lactate dehydrogenase (LDH), lactate dehydrogenase-1 (LDH-1);

[0144] Kidney function indicators: blood urea nitrogen (BUN), creatinine (Cr), uric acid (UA); blood lipid parameters: total cholesterol (TC), triglycerides (TG);

[0145] 2.3.2 Histopathological examination

[0146] Organ toxicity testing: Major organs (heart, liver, spleen, lung, kidney, brain, intestine and muscle) were paraffin-embedded, sectioned and H&E stained to observe their tissue morphology and structure to assess the potential toxicity of the drug.

[0147] 3. Experimental Results

[0148] 3.1 Pharmacokinetics

[0149] TIIA-NPs (Cy5.5-NPs) showed a significant decrease in fluorescence intensity 24 hours after administration and were almost completely cleared after 48 hours. Figure 21 b in Figure 21 (c) In contrast, MM@TIIA-NPs (MM@Cy5.5-NPs) showed a longer circulation time compared to TIIA-NPs (Cy5.5-NPs). Therefore, aortas from all treatment groups were collected and examined for fluorescence 48 hours after injection. The aortas of the MM@TIIA-NPs (MM@Cy5.5-NPs) group showed higher fluorescence intensity than those of the TIIA-NPs (Cy5.5-NPs) group, indicating superior accumulation in aortic tissue (c). Figure 21 d in Figure 21 (e). The above results indicate that MM@TIIA-NPs have a much longer retention time in the blood than ordinary nanoparticles, significantly prolonging the circulation time. At the same time, they can actively enhance the enrichment of nanoparticles into atherosclerotic plaques, improving the plaque-targeting accumulation ability.

[0150] 3.2 Treatment Efficacy

[0151] 3.2.1 The plaque load was significantly reduced.

[0152] The results showed that the plaque area in the model group mice was significantly enlarged, while the plaque area in the mice treated with MM@TIIA-NPs was significantly reduced, and the effect was better than that in the TIIA-NPs group and the TIIA group. Figure 21 f and Figure 21 In the g), MM@TIIA-NPs had the least effect on the change in body weight in treated mice. Figure 21 (h in the text), this result directly proves that MM@TIIA-NPs can effectively inhibit lipid deposition and delay the progression of atherosclerosis.

[0153] 3.2.2 Significantly improved plaque stability

[0154] Shrinkage of necrotic core: such as Figure 22 a and Figure 22H&E staining showed that the plaques in the model group were mainly composed of lipid-rich necrotic cores, while the necrotic area in the MM@TIIA-NPs treatment group was significantly reduced, indicating that the plaques tended to stabilize. Simultaneously, MM@TIIA-NPs treatment significantly reduced macrophage infiltration, accompanied by downregulation of matrix metalloproteinases MMP-9 and MMP-2 expression.

[0155] Increased collagen content: Masson trichrome staining confirmed increased collagen deposition around plaques in the MM@TIIA-NPs treatment group, promoting thickening of the fibrous cap. Figure 22 a and Figure 22 In addition, MM@TIIA-NPs increased the accumulation of vascular smooth muscle cells (VSMCs) within plaques. Figure 22 g in (the middle part).

[0156] The results showed that VSMC proliferation and collagen synthesis synergistically enhanced plaque structure, while macrophage reduction and MMP inhibition reduced the risk of plaque rupture.

[0157] 3.2.3 Effective relief of systemic and local inflammation

[0158] The results showed that MM@TIIA-NPs treated ApoE - / - The levels of pro-inflammatory cytokines in mouse aortic root sections and serum were significantly reduced. Figure 22 h and Figure 22 i in Figure 23 (ac). In contrast, the model group mice showed elevated cytokine levels. These findings further support the claim that MM@TIIA-NPs effectively reduce systemic inflammation and inflammation within atherosclerotic plaques.

[0159] 3.3 In vivo safety assessment

[0160] 3.3.1 Hematological toxicity and liver and kidney function tests

[0161] like Figure 24 As shown, routine blood tests revealed no abnormalities in red blood cell, white blood cell, or platelet counts in mice treated with MM@TIIA-NPs, indicating no hematological toxicity. Furthermore, clinical biochemical analyses of liver and kidney function showed no significant changes, indicating that MM@TIIA-NPs treatment did not induce organ dysfunction. Lipid profile assessment showed that, compared to the model group, MM@TIIA-NPs treatment reduced total cholesterol and triglyceride levels.

[0162] 3.3.2 Organ toxicity test

[0163] like Figure 25As shown, no significant histopathological abnormalities or damage were detected in the MM@TIIA-NPs treatment group, indicating good treatment tolerance and no significant organ damage. Intravenous injection of MM@TIIA-NPs is well-tolerated and safe. MM@TIIA-NPs had no adverse effects on hematological parameters, liver and kidney function, or blood lipid levels, and no significant organ damage was observed.

[0164] In vivo safety assessment results show that MM@TIIA-NPs possess both lipid-lowering activity and high biocompatibility, providing a safe and effective targeted strategy for the treatment of atherosclerosis.

[0165] In summary, MM@TIIA-NPs, as a ROS-responsive biomimetic nanodelivery system encapsulated in the membrane of M2 macrophages, successfully retains key M2 macrophage proteins such as TLR4, integrin β1, CCR2, and SRA in its core-shell structure, endowing it with the ability to actively target atherosclerotic plaques. Through membrane camouflage, it achieves immune evasion, significantly reducing non-specific macrophage phagocytosis while enhancing specific targeting of VSMCs and HUVECs. In pathological environments, MM@TIIA-NPs respond to the high levels of reactive oxygen species (ROS) within plaques, degrading and releasing TIIA, achieving a release rate of 60% within 24 hours (compared to only 18% in non-ROS environments). It effectively clears intracellular ROS, inhibits oxLDL uptake and foam cell formation, and significantly reduces the levels of pro-inflammatory factors (TNF-α, MCP-1, IL-1β) and oxidative damage markers (8-OHdG, 8-iso-PGF2α). In in vivo models, it exhibits prolonged circulation time and enhanced plaque accumulation, significantly reducing plaque burden, stabilizing plaque structure, and simultaneously improving blood lipids and systemic inflammation. Safety assessments confirmed that it has no adverse effects on hematological parameters, liver and kidney function, and no pathological damage to major organs. It possesses excellent biocompatibility and therapeutic potential, providing a novel biomimetic nanodelivery system with intelligent response and multi-dimensional therapeutic functions for atherosclerosis.

[0166] The present invention has been further described above with reference to specific embodiments. However, it should be understood that the specific description herein should not be construed as limiting the nature and scope of the present invention. Various modifications made to the above embodiments by those skilled in the art after reading this specification are all within the scope of protection of the present invention.

Claims

1. A ROS-responsive biomimetic nanodelivery system encapsulated in M2 macrophage membranes, characterized in that, It consists of ROS-responsive nanoparticles carrying therapeutic drugs and M2 macrophage membranes encapsulating the ROS-responsive nanoparticles.

2. The ROS-responsive biomimetic nanodelivery system as described in claim 1, characterized in that, The thickness of the M2 macrophage membrane ranges from 5 to 20 nm.

3. The ROS-responsive biomimetic nanodelivery system encapsulated in an M2 macrophage membrane according to claim 1, characterized in that, The ROS-responsive biomimetic nanoparticles are particles containing phenylboronic acid ester bonds.

4. A method for preparing a ROS-responsive biomimetic nanodelivery system encapsulated in an M2 macrophage membrane, characterized in that, include: By using the ROS-responsive nanoparticles as described in claim 1 to carry therapeutic drugs, ROS-responsive nanoparticles carrying drugs are obtained. The drug-carrying ROS-responsive nanoparticles were encapsulated using an M2 macrophage membrane to obtain the M2 macrophage membrane-encapsulated ROS-responsive biomimetic nanodelivery system.

5. The method for preparing the ROS-responsive biomimetic nanodelivery system as described in claim 4, characterized in that, The method for synthesizing the ROS-responsive biomimetic nanoparticles includes: dissolving 4-hydroxyphenylboronic acid pinacol ester and carbonyl diimidazole in dichloromethane at a molar ratio of 1:4-6, reacting at 20-30℃ for 3-6 hours to obtain the activated product of borate ester-imidazole. Using chitosan oligosaccharide and the activated product of borate ester-imidazole as raw materials, 4-dimethylaminopyridine as catalyst, and dimethyl sulfoxide as solvent, the reaction was carried out at 20-30℃ for 8-16 h, and then purified by dialysis to obtain ROS-responsive biomimetic nanoparticles; the molar ratio of chitosan oligosaccharide to the activated product of borate ester-imidazole was 1:1.5-2.

5.

6. The method for preparing the ROS-responsive biomimetic nanodelivery system as described in claim 4, wherein the M2 macrophage membrane and the drug-carrying ROS-responsive nanoparticles are fused by co-extrusion to obtain the ROS-responsive biomimetic nanodelivery system encapsulated by the M2 macrophage membrane.

7. The method for preparing the ROS-responsive biomimetic nanodelivery system as described in claim 4, characterized in that, The therapeutic drugs include: drugs for treating atherosclerosis.

8. The ROS-responsive biomimetic nanodelivery system encapsulated in M2 macrophage membranes prepared by the preparation method according to any one of claims 4 to 7.

9. The use of the ROS-responsive biomimetic nanodelivery system encapsulated by the M2 macrophage membrane as described in any one of claims 1 to 3, or the ROS-responsive biomimetic nanodelivery system encapsulated by the M2 macrophage membrane prepared by the preparation method as described in any one of claims 4 to 7, in the preparation of drugs for treating atherosclerosis.

10. A drug for treating atherosclerosis, characterized in that, The active ingredient and pharmaceutically acceptable excipients of the drug; The active ingredient includes: an M2 macrophage membrane-encapsulated ROS-responsive biomimetic nanodelivery system prepared by the preparation method according to any one of claims 4 to 7.

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