Magnetic nanoparticles for the treatment of atherosclerosis

Abstract

The application discloses a kind of magnetic nanoparticles for atherosclerosis diagnosis and treatment.It is core-shell structure, the inner core is Fe₃O₄ magnetic nano material, shell is the drug-loading layer of biodegradable polymer material, the drug-loading layer is encapsulated with analog movement polypeptide K271la, and the surface of nano particle is covalently coupled with CD36 targeting antibody.Its preparation method mainly includes: by complex emulsification-solvent volatilization method, Fe₃O₄ nanoparticle and K271la polypeptide are co-encapsulated in one step, form nanoparticle, and then CD36 antibody is modified on the surface of particle by chemical coupling.The nanoparticle of the application has the ability of active targeting (CD36 antibody) and passive targeting (magnetic response), can be efficiently enriched in plaque, and deliver therapeutic polypeptide to macrophages, activate lipophagy to remove lipid;It can be used as magnetic resonance imaging contrast agent, realizes diagnosis and treatment integration, and provides a new strategy for solving the problem of low efficiency of atherosclerosis targeted therapy and lack of effective monitoring means.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of biomedicine and nanotechnology, specifically to a magnetic targeted nanoparticle for the diagnosis and treatment of atherosclerosis. Background Technology

[0002] Atherosclerosis (AS) is a chronic inflammatory disease characterized by lipid deposition in the arterial wall, inflammatory cell infiltration, and fibrous cap formation, ultimately leading to atherosclerotic plaques. Plaque instability or rupture can trigger acute thrombotic events, such as myocardial infarction or stroke. Therefore, current clinical intervention strategies for AS mainly revolve around two core aspects: first, systematically lowering blood lipid levels to reduce lipid deposition at its source; and second, stabilizing plaques and preventing rupture and thrombosis.

[0003] In recent years, non-pharmacological interventions, especially regular aerobic exercise, have been confirmed by numerous basic and clinical studies as key measures to delay the progression of atherosclerosis (AS) and improve cardiovascular prognosis. Their benefits lie not only in improving systemic lipid metabolism, reducing insulin resistance, and lowering blood pressure, but also in their direct regulatory effect on the local atherosclerotic lesions. Studies have shown that aerobic exercise can induce epigenetic modifications, promoting the interaction between lipid droplets and lysosomes within macrophages, thereby enhancing the selective autophagy process of lipophage, actively clearing intracellularly accumulated lipids, and achieving reverse lipid transport and inflammation relief within plaques. This mechanism of targeting lesions by activating endogenous lipid clearance pathways is not available in current conventional drug therapies and has significant application potential.

[0004] However, for the vast majority of elderly patients, those with weak constitutions, those with severe joint diseases, or those with poor exercise tolerance due to heart failure, it is difficult to benefit from regular exercise. This contradiction greatly limits the clinical application of exercise as an efficient and natural intervention. Therefore, developing a drug or therapy that can mimic the beneficial effects of aerobic exercise, especially its activation of macrophage lipophagus function, has significant clinical need and scientific value.

[0005] To address this need, research has identified bioactive peptides with motion-mimicking effects, such as the MeCP2 K271la peptide. These peptides can mimic motion-induced epigenetic regulatory signals, activating the lipophageal function of macrophages at the cellular level and promoting lipid clearance within plaques, thus demonstrating an effect of delaying the progression of ankylosing spondylitis (AS) in animal models. However, efficiently and precisely delivering these therapeutic peptides to macrophages located deep within AS plaques in the blood vessel wall is a key challenge in translating them into clinically effective therapies. Systemic administration faces challenges such as widespread systemic drug distribution, low target lesion enrichment efficiency, rapid clearance, and potential off-target side effects.

[0006] To improve delivery efficiency, nanocarrier technology has been extensively studied. Among them, magnetic nanoparticles (such as Fe3O4) have attracted much attention due to their unique properties: on the one hand, their surfaces are easy to functionalize and can be coupled with targeting molecules (such as antibodies and peptides) and therapeutic drugs; on the other hand, their inherent magnetism allows for physical targeting under the guidance of an external magnetic field; in addition, Fe3O4 serves as an excellent contrast agent for T2-weighted magnetic resonance imaging (MRI), enabling integrated diagnosis and treatment.

[0007] Although the aforementioned magnetic nanoparticle-based targeted delivery systems have shown potential in theoretical design and preliminary experiments, several significant technical shortcomings and challenges remain when it comes to practical clinical translational applications: 1. Insufficient efficiency in deep tissue targeted delivery: AS plaques are located in the intima or media of blood vessels. Nanoparticles must cross multiple biological barriers, including the intact endothelial cell layer and subendothelial matrix, to reach the macrophages at the core of the lesion. Current strategies rely on external static magnetic fields, which have limited penetration depth and whose strength decreases sharply with increasing tissue depth in vivo. This results in insufficient control over nanoparticles within the deep vascular wall, making it difficult to achieve precise cross-barrier delivery and spatial positioning.

[0008] 2. Issues with the precision and uniformity of magnetic field manipulation: In in vitro experiments or small animal models, good control can be achieved through a carefully designed uniform magnetic field. However, in the complex anatomical structure of the human body, it is difficult to establish a stable, uniform, and sufficiently strong targeted magnetic field. Uneven magnetic field distribution can lead to uneven distribution of nanoparticles within the lesion, potentially creating therapeutic "cold zones," affecting overall efficacy, and potentially posing risks due to non-specific aggregation in other areas.

[0009] 3. Translation from Laboratory to Clinical: Nanoparticles synthesized on a small scale in the laboratory typically exhibit high uniformity in terms of particle size, morphology, drug loading, and surface modification, achieving ideal results in a controlled, simple biological environment. However, ensuring high-quality batch-to-batch reproducibility during large-scale production remains a significant challenge. More importantly, given the complex physiological and pathological microenvironment of the human body and the substantial individual differences, developing personalized and precise treatment plans and effectively regulating drug release kinetics at the target site to reproduce laboratory efficacy currently lacks comprehensive and systematic preclinical and clinical data.

[0010] Therefore, there is an urgent need in this field to develop a new targeted therapy strategy for atherosclerosis that can overcome the barriers to deep tissue delivery, achieve higher targeting precision and controllability, and is easy to translate into clinical applications, in order to address the aforementioned shortcomings of existing motion-simulation therapy delivery systems. Summary of the Invention

[0011] To address the problems of low targeting efficiency, uneven treatment effects, and limited clinical applicability in existing technologies, this invention proposes a magnetic nanoparticle for the treatment of atherosclerosis, aiming to achieve more efficient and precise targeted therapy and imaging monitoring of macrophages within atherosclerotic plaques.

[0012] In a first aspect, the present invention provides magnetic nanoparticles for treating atherosclerosis.

[0013] The magnetic nanoparticles are CD36@FeK271la; The core-shell structure, from the inside out, includes: 1. Core: Composed of magnetic nanomaterials, serving as a magnetic targeting drive unit and imaging contrast agent.

[0014] 2. Polymer shell: A biodegradable polymer material layer covering the core for loading therapeutic agents.

[0015] 3. Therapeutic agent: A bioactive substance encapsulated within the polymer shell that can mimic aerobic exercise and activate the lipophagus function of macrophages.

[0016] 4. Targeting ligands: Molecules coupled to the surface of nanoparticles that can specifically bind to markers on the surface of macrophages within atherosclerotic plaques.

[0017] Preferably, the magnetic nanomaterial is Fe3O4 nanoparticles.

[0018] Preferably, the biodegradable polymer material is a polylactic acid-hydroxyacetic acid copolymer PLGA-COOH with carboxyl groups at the ends.

[0019] Preferably, the therapeutic agent is a MeCP2 K271la motion-mimicking polypeptide.

[0020] Preferably, the targeting ligand is the specific antibody CD36.

[0021] Secondly, the present invention provides a method for preparing the above-mentioned magnetic nanoparticles.

[0022] This method mainly includes the following steps: S1: Organic phase preparation: Biodegradable polymer materials are dissolved in volatile organic solvents to form an organic solution; then, magnetic nanomaterials, therapeutic agents, bovine serum albumin (BSA) and optional imaging agents are sequentially added to and dispersed in the organic solution, and thoroughly mixed to form an oil phase loaded with functional components.

[0023] S2: Construction of the double emulsion: First, the organic phase is primary emulsified to form an oil-in-water primary emulsion; then, the primary emulsion is transferred into the aqueous phase and secondary emulsified under mechanical stirring to finally form a stable water-in-oil-in-water double emulsion system.

[0024] S3: Solidification and collection of nanoparticles: Under continuous stirring, the volatile organic solvents in the emulsion are completely evaporated, causing the coating polymer material to solidify and precipitate, thereby forming solid nanoparticles; the nanoparticles are collected by centrifugation and purified by washing with deionized water or buffer solution multiple times.

[0025] S4: Surface targeting modification: The purified nanoparticles are redispersed in a suitable buffer solution, and the targeting ligands are covalently fixed to the surface of the nanoparticles through chemical coupling, finally obtaining magnetic nanoparticles with active targeting function.

[0026] Preferably, the volatile organic solvent is chloroform or dichloromethane; Preferably, the aqueous phase in step S2 is an aqueous solution of ethylene alcohol; Thirdly, the present invention provides the use of the magnetic nanoparticles as described in the first aspect in the preparation of medicaments for the prevention and / or treatment of atherosclerosis.

[0027] Preferably, the drug is administered via intravenous injection and can accumulate at the site of atherosclerotic lesions under the guidance of an external magnetic field.

[0028] Fourthly, the present invention provides the use of the magnetic nanoparticles as described in the first aspect in the preparation of contrast agents for the imaging diagnosis of atherosclerosis.

[0029] Preferably, the imaging diagnosis includes magnetic resonance imaging and / or near-infrared fluorescence imaging.

[0030] Compared with the prior art, the technical solution provided by the present invention has the following outstanding substantive features and significant progress: This invention constructs a regulatory mechanism using magnetic nanoparticles to simulate aerobic exercise. Through a dual targeting mechanism of CD36 antibody and external magnetic field, peptide drugs are delivered across the fibrous cap, acting on macrophages within the plaque to promote lipid clearance. This represents a significant innovation compared to traditional drugs that inhibit the progression of atherosclerotic plaques, and may even reverse atherosclerotic plaques. It significantly improves drug accumulation at the lesion site, reduces systemic drug distribution, and lowers side effects, fundamentally improving the pathological process of atherosclerosis. Simultaneously, the use of biodegradable PLGA as a carrier reduces toxicity to the body. Combined with magnetic resonance imaging (MRI), real-time monitoring of lesions and evaluation of treatment efficacy are achieved, supporting personalized treatment. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the magnetic nanoparticle preparation process of the present invention; Figure 2 This is a transmission electron microscope image of the magnetic nanoparticles of the present invention. Figure 3 This is a dynamic light scattering (DLS) diagram of the magnetic nanoparticles of the present invention; Figure 4 This is a potential diagram of the ZETA magnetic nanoparticles of the present invention; Figure 5 This is a Prussian blue staining image of the magnetic nanoparticles of the present invention; Figure 6 Nuclear magnetic resonance imaging of the magnetic nanoparticles of the present invention entering atherosclerotic plaques; Figure 7 This is a small animal in vivo imaging image of the magnetic nanoparticles of the present invention; Figure 8 Prussian blue staining image of the magnetic nanoparticles of the present invention entering atherosclerotic plaques; Figure 9 This is a hemolysis experiment diagram of the magnetic nanoparticles of the present invention; Figure 10 CCK-8 diagram showing the effects of the magnetic nanoparticles of the present invention on the proliferation of macrophages (BMDMs), cardiac fibroblasts (CFs), and umbilical vein endothelial cells (HUVECs); Figure 11 This is a fluorescence detection image showing the effect of the magnetic nanoparticles of the present invention on the life and death of macrophages (BMDMs), cardiac fibroblasts (CFs), and umbilical vein endothelial cells (HUVECs). Figure 12 The effect of the magnetic nanoparticles of this invention on biochemical indicators in mice; Figure 13 The effect of the magnetic nanoparticles of this invention on the morphology of major organs in mice; Figure 14 The effect of the magnetic nanoparticles of this invention in treating atherosclerosis is shown in the figure. Detailed Implementation

[0032] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings. The embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0033] Example 1: Preparation of magnetic nanoparticles The specific steps are as follows: 1. Preparation of organic phase: 100 mg PLGA-COOH was completely dissolved in 4 mL chloroform and sonicated in an ice-water bath for 10 minutes. 5 mg of pre-prepared and dried Fe3O4 nanoparticles were added to the PLGA solution and vortexed for 1 minute to disperse them initially.

[0034] 2. Co-loading of drug and dye: Add 4 mg MeCP2 K271la-peptide, 0.5 mg IR780 fluorescent dye, and 8 ml BSA (30 mg / ml) sequentially to the above dispersion. Sonicate the mixture for 5 minutes to ensure the peptide and dye are fully dispersed and dissolved in the organic phase, forming the organic phase.

[0035] 3. Preparation of the double emulsion: The organic phase was dispersed in 10 mL of 2% (w / v) PVA aqueous solution pre-cooled in an ice bath. The mixture was then treated with a probe-type ultrasonic disruptor (200W) for 30 seconds under ice-water bath conditions to form an oil-in-water emulsion.

[0036] 4. Solidification and Collection of Nanoparticles: The above emulsion was rapidly transferred to a 50 mL three-necked round-bottom flask. At room temperature, the mixture was continuously stirred at 500 rpm for 3 hours to allow complete chloroform evaporation and PLGA solidification into solid nanoparticles. Subsequently, the resulting nanoparticle suspension was centrifuged at 15,000 rpm for 15 minutes at 4°C to collect the precipitate. The nanoparticles were washed and centrifuged three times with deionized water to thoroughly remove residual PVA and free drug. The purified nanoparticles were redispersed in sterile phosphate-buffered saline (PBS) or ultrapure water and sterilely filtered through a 0.22 μm microporous membrane to obtain the final product, which was stored at 4°C protected from light for later use.

[0037] 5. Surface Targeting Modification (CD36 Antibody Conjugation): 4 μg of CD36 antibody was dissolved in 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) solution for 15 minutes to activate the carboxyl groups. This solution was then mixed with the prepared nanoparticles and a solution of 1 mg N-carboxysuccinimide at 37°C for 2 hours. The prepared nanoparticles were washed by centrifugation to obtain magnetic nanoparticles CD36@FeK271la. Figure 1 ).

[0038] Example 2 The ratio of PLGA-COOH to Fe3O4 nanoparticles was varied, for example, PLGA-COOH: 150 mg, Fe3O4 nanoparticles: 3 mg, while the ratios of other materials remained unchanged. The experimental steps of Example 1 were repeated, and the therapeutic effects were compared.

[0039] Example 3 The dosage of MeCP2 K271la-peptide was varied, for example, PLGA-COOH: 100mg, Fe3O4 nanoparticles: 5mg, MeCP2 K271la-peptide: 6mg, while the proportions of other materials remained unchanged. The experimental steps of Example 1 were repeated to compare the therapeutic effects.

[0040] Test Example 1 Particle size and morphology testing The morphology and size of nanoparticles were observed using transmission electron microscopy (TEM). Figure 2 Dynamic light scattering (DLS) for detecting the hydrodynamic size of nanoparticles. Figure 3 ) and ZETA potential ( Figure 4 This ensures its stability and dispersion.

[0041] Test Example 2 Targeted testing Bone marrow-derived macrophages were extracted and cultured in vitro for 7 days. After stimulating the macrophages with oxidized low-density lipoprotein (ox-LDL) for 24 hours, CD36@FeK271la nanoparticles were co-incubated with the bone marrow-derived macrophages for another 24 hours. Prussian blue staining was used to observe the cellular uptake of the nanoparticles, confirming that the nanoparticles could enter the macrophages. Figure 5 ).

[0042] In a mouse model, APOE was administered. - / - Mice were fed a high-fat diet for 16 weeks. IR780-labeled CD36@FeK271la nanoparticles were injected via the tail vein, followed by an external magnetic field at the heart for 15 minutes to induce nanoparticle entry into aortic plaques. Magnetic resonance imaging (MRI) was performed before and 24 hours after injection to detect the presence of magnetic nanoparticles. T2-weighted MRI showed smooth aortic arches before re-injection, and nanoparticles were visible at the aortic arch 24 hours after injection. Figure 6 The heart and aorta were isolated and placed in a small animal in vivo imaging system, indicating that the magnetic nanoparticles were mainly distributed in the aortic arch of the mouse heart. Figure 7 Frozen sections of the aortic sinus region were examined, and Prussian blue staining was used to detect the level of nanoparticle penetration into the plaque. The results confirmed that magnetic nanoparticles could penetrate atherosclerotic plaques. This demonstrates that magnetic nanoparticles can be used as a contrast agent in MRI to visualize the size of atherosclerotic plaques and to target plaque macrophages. Figure 8 ).

[0043] Test Example 3 Biocompatibility Hemolysis test: Mouse peripheral blood erythrocytes were isolated and co-cultured with different concentrations of magnetic nanoparticles CD36@FeK271la. Hemolysis was observed in the positive control group, while no hemolysis was observed in the magnetic nanoparticle groups at different concentrations. Figure 9 ).

[0044] Effects on cell viability: Bone marrow-derived macrophages (BMDM), cardiac fibroblasts (CF), and human umbilical vein endothelial cells (HUVEC) were co-cultured with different concentrations of magnetic nanoparticles CD36@FeK271la in vitro. Cell viability in all three groups remained above 90% as determined by the Cell Counting Kit-8 (CCK-8). Figure 10 Simultaneously, staining with live and dead cells confirmed that different concentrations of magnetic nanoparticles had no effect on cell death in three cell types. These results confirm that the magnetic nanoparticles possess good biocompatibility. Figure 11 ).

[0045] Test Example 4 In vivo safety assessment Give APOE - / - Mice were subjected to a high-fat diet for 16 weeks. At week 8, they were divided into four groups, receiving weekly intravenous injections of saline, CD36@Fe, a polypeptide (K271la-pe), and magnetic nanoparticles CD36@FeK271la, respectively. An external magnetic field was applied to the heart for 15 minutes after each injection. The injections were continued for 8 weeks. Peripheral blood plasma was separated from the mice, and plasma biochemical indicators were detected, confirming that CD36@FeK271la did not affect liver and kidney function or blood lipids in mice. Figure 12 Major organs, including the heart, liver, spleen, lungs, and kidneys, were isolated. HE staining of the major organ morphology confirmed that CD36@FeK271la had no effect on the major organs of mice. Figure 13 ).

[0046] Test Example 5 The ability to treat atherosclerosis Give APOE - / - Mice were subjected to a high-fat diet for 16 weeks. At week 8, they were divided into four groups, receiving weekly intravenous injections of saline, CD36@Fe, a polypeptide (K271la-pe), and magnetic nanoparticles CD36@FeK271la, respectively. An external magnetic field was applied to the heart for 15 minutes after each injection. The injections lasted for 8 weeks. Frozen sections of the mouse aortic sinus were prepared and pathologically stained, including Oil Red O stain, HE stain, and Masson stain. These results confirmed that the CD36@FeK271la nanoparticles significantly reduced the area of ​​atherosclerotic plaques and increased their stability. Figure 14 ).

[0047] In summary, we have successfully constructed magnetic nanoparticles CD36@FeK271la for the treatment of atherosclerosis. These nanoparticles are non-toxic to cells and mice and exhibit good biocompatibility. The magnetic nanoparticles can target atherosclerotic regions through both CD36 antibody and in vitro magnetic field induction. Furthermore, the Fe3O4 in the magnetic nanoparticles provides MRI capabilities, enabling the constructed magnetic nanoparticles to combine therapeutic and diagnostic functions, thus achieving integrated diagnosis and treatment for atherosclerosis-related diseases.

[0048] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A magnetic nanoparticle for the diagnosis and treatment of atherosclerosis, characterized in that, The nanoparticles have a core-shell structure and include: The core is composed of magnetic nanomaterials; A biodegradable polymer shell encapsulating the core; A polypeptide therapeutic agent encapsulated within the shell; Targeting ligands coupled to the surface of nanoparticles that can specifically bind to markers on the surface of macrophages within atherosclerotic plaques.

2. The magnetic nanoparticles according to claim 1, characterized in that, The magnetic nanomaterial is a superparamagnetic Fe3O4 nanoparticle.

3. The magnetic nanoparticles according to claim 1, characterized in that, The biodegradable polymer material is a polylactic acid-hydroxyacetic acid copolymer with carboxyl groups at the ends.

4. The magnetic nanoparticles according to claim 1, characterized in that, The therapeutic agent is a MeCP2 K271la motion-mimicking peptide.

5. The magnetic nanoparticles according to claim 1, characterized in that, The targeting ligand is an antibody that targets the CD36 receptor on the surface of macrophages.

6. The magnetic nanoparticles according to claim 1, characterized in that, The shell also encapsulates a near-infrared fluorescent imaging agent.

7. A method for preparing magnetic nanoparticles as described in any one of claims 1-6, characterized in that, Includes the following steps: S1: Dissolve biodegradable polymer materials in a volatile organic solvent, add magnetic nanomaterials, therapeutic agents, and optionally near-infrared fluorescent imaging agents, and mix to form an organic phase; S2: First, the organic phase is subjected to primary emulsification to form an oil-in-water primary emulsion; then, the primary emulsion is added to the second aqueous phase for secondary emulsification to form an oil-in-water secondary emulsion. S3: Stir the emulsion to evaporate the organic solvent and solidify the polymer material to form solid nanoparticles, which are then collected after centrifugation and washing. S4: The targeting ligand is chemically coupled to the surface of the solid nanoparticles to obtain the magnetic nanoparticles.

8. The method according to claim 7, characterized in that, The volatile organic solvent is chloroform or dichloromethane; the aqueous phase contains a polyvinyl alcohol solution.

9. The use of the magnetic nanoparticles as described in any one of claims 1-6 in the preparation of a medicament for the prevention and / or treatment of atherosclerosis.

10. The use of the magnetic nanoparticles as described in any one of claims 1-6 in the preparation of contrast agents for the imaging diagnosis of atherosclerosis.

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