Satellite modular structure nanoparticle MEps and preparation method and application thereof

Abstract

The application discloses a satellite modular structure nanoparticle MEps and a preparation method and application thereof, and relates to the specific biological technology field.The application provides a nanoparticle, which comprises a SiO2 carrier, a cell membrane and an exosome, and has stronger cell aging improvement and proliferation promotion capabilities.In addition, compared with the MEPs loaded with common EVs, the MEPs loaded with the IGF1-BMSC EVs significantly improve ApoE ‑ / ‑ Atherosclerosis and vascular aging levels of mice.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a satellite modular structure nanoparticle MEps, its preparation method, and its application. Background Technology

[0002] Atherosclerosis (AS) is a typical chronic inflammatory vascular disease and the common pathological basis for ischemic cardiovascular and cerebrovascular diseases such as cerebrovascular disease, coronary heart disease, and peripheral artery disease, seriously endangering human health. Currently, oral lipid-lowering or anti-inflammatory drugs are commonly used clinically to inhibit the development of AS. However, traditional oral drug therapy suffers from low utilization rate, slow onset of action, and serious toxic side effects. Nanoparticle drug delivery systems can solubilize drugs, increase drug half-life, improve drug distribution in vivo, and reduce toxic side effects, showing great application value and broad market prospects in the clinical diagnosis and treatment of AS. However, traditional nanoparticle drug delivery systems are difficult for normal biological interactions to recognize, inevitably being cleared by the body as "non-self" foreign substances, making it difficult to target drugs to the lesion site. Therefore, constructing a biomimetic nanoparticle drug system with drug sustained-release and targeted drug delivery functions based on the pathological characteristics of AS is key to achieving efficient and safe treatment of AS. Summary of the Invention

[0003] (a) Technical problems to be solved Therefore, one of the main objectives of this invention is to provide a nanoparticle comprising a SiO2 carrier, a cell membrane, and exosomes. The nanoparticle provided by this invention can improve atherosclerosis-related cardiovascular diseases by inhibiting neutrophil infiltration and exerting anti-inflammatory effects.

[0004] (II) Technical Solution To achieve the above objectives, the present invention provides a nanoparticle comprising a SiO2 carrier, a cell membrane, and extracellular vesicles (EVs).

[0005] In one embodiment, the cell membrane is a macrophage cell membrane.

[0006] In one embodiment, the exosomes are mesenchymal stem cell exosomes.

[0007] In one embodiment, the mesenchymal stem cell exosomes are bone marrow mesenchymal stem cell exosomes.

[0008] In one embodiment, the ratio of the SiO2 carrier, cell membrane, and exosomes is (4-8):1:(1-3).

[0009] In another aspect, the present invention provides a method for preparing the above-mentioned nanoparticles, the method comprising: loading the above-mentioned cell membrane and exosomes onto a SiO2 support by electrostatic bonding.

[0010] In another aspect, the present invention also provides nanoparticles obtained by the above preparation method.

[0011] In another aspect, the present invention provides a pharmaceutical composition comprising: (1) A therapeutically effective amount of the above-mentioned nanoparticles; (2) Pharmaceutically or immunologically acceptable carriers or excipients.

[0012] In another aspect, the present invention provides a pharmaceutical preparation comprising the above-described pharmaceutical composition.

[0013] In another aspect, the present invention also provides a product comprising the above-described pharmaceutical composition.

[0014] In another aspect, the present invention also provides the use of the above-mentioned nanoparticles, pharmaceutical compositions, pharmaceutical preparations or products in the preparation of medicaments for the prevention and / or treatment of atherosclerosis-related cardiovascular diseases.

[0015] (III) Beneficial Effects This invention provides a nanoparticle. Compared with the prior art, it has the following advantages: 1. It directly adheres to the surface of damaged ECs through the interaction of ICAM-1 and VCAM-1 receptor-ligand, while inhibiting immune cells from adhering to ECs.

[0016] 2. MEps can precisely deliver engineered mesenchymal stem cell-derived IGF1-EVs to damaged ECs, enabling IGF1-MEps to combine the anti-cellular senescence effect of IGF1-EVs with the targeting effect of macrophage membranes.

[0017] 3. IGF1-EVs significantly inhibited endothelial cell (EC) senescence and delayed the progression of atherosclerotic lesions in mice. Therefore, our work not only provides a new approach to improving endothelial cell function but also offers a potential therapeutic window for treating AS-related cardiovascular diseases. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram of IGF1-MEps preparation.

[0020] Figure 2 This is a graph showing the product characterization results.

[0021] Figure 3 This is a diagram showing the effect of IGF1-MEps on improving endothelial cell aging.

[0022] Figure 4 This is a graph showing the effect of IGF1-MEps on SASP expression in endothelial cells.

[0023] Figure 5 This is a graph verifying the effect of IGF1-MEps in alleviating atherosclerosis. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, 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 scope of protection of the present invention.

[0025] Macrophage membrane (MM) nanomaterials utilize chemokine receptors (such as CCR2) and integrins (α4β1) to directionally accumulate in plaques, enabling precise drug delivery and immune escape (CD47-mediated), and can guide nanoparticles to migrate to atherosclerotic plaques.

[0026] Exosomes (EVs) secreted by bone marrow mesenchymal stromal cells (BMSCs) provide a novel therapeutic strategy for atherosclerosis that combines anti-inflammatory, vascular-protective, and metabolic-regulating effects by regulating inflammation, cell survival, lipid metabolism, and vascular repair at multiple targets, showing great promise in translational medicine. Exosomes are emerging as potential biotherapeutic agents and drug delivery carriers, and can be used to enhance the therapeutic delivery of RNA, peptides, and synthetic drugs.

[0027] Furthermore, insulin-like growth factor 1 (IGF-1) inhibits endothelial cell apoptosis, promotes nitric oxide (NO) production, improves vascular endothelial function, and reduces oxidative stress and the release of inflammatory factors (such as IL-6 and TNF-α) by activating the PI3K / AKT signaling pathway, thereby delaying the progression of atherosclerosis. In this invention, IGF1-overexpressing IGF1-BMSC-EVs were obtained by modifying BMSCs using bioengineering techniques. RNA-seq sequencing results showed that, compared with wild-type mesenchymal stem cells, IGF1 overexpression caused changes in exosome contents, with significant upregulation of cell proliferation-related miRNAs such as miR-21, miR-125, and miR-146. Simultaneously, aging-related signaling pathways underwent significant changes.

[0028] Therefore, we attempted to integrate the targeting capabilities of macrophages with the tissue regeneration capabilities of stem cell-derived EVs. We fused EVs obtained by ultracentrifugation with MMps using sonication, thus constructing a modular satellite-like nanoparticle platform MEps. This platform uses SiO2 nanoparticles as its core, and through electrostatic interactions, it adsorbs IGF1-overexpressing mesenchymal stem cell EVs and MMs onto the particle surface, forming macrophage membrane and exosome fusion particles (nanoparticles with MMs and EVs, MEPs).

[0029] Terms and Definitions As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in this technical field.

[0031] Example 1: Construction of the modular satellite nanoplatform IGF1-MEps: like Figure 1 As shown, bone marrow mesenchymal stem cells overexpressing IGF1 were constructed, and then bone marrow-derived macrophages were isolated and cultured. The macrophages were disrupted by ultrasound, followed by ultracentrifugation to obtain macrophage membranes and exosomes. The macrophage membranes and IGF1 exosomes were then simultaneously loaded onto SiO2 nanoparticles via electrostatic binding.

[0032] MEPs only load macrophage membranes.

[0033] SiO2NPs do not load macrophage membranes or IGF1 exosomes.

[0034] Example 2: Particle characterization of SiO2NPs and MEPs: The SiO2 NPs and MEPs prepared in Example 1 were characterized, and the results are as follows: Figure 2 As shown.

[0035] Figure 2 In the figure, a is a transmission electron microscope image of SiO2NPs and MEps adhesion; b is the particle size analysis result of SiO2NPs and MEps; c is the zeta potential analysis result of SiO2NPs and MEps.

[0036] Example 3: MEps and IGF1-MEps improve endothelial cell senescence and enhance their migration ability: HUVECs were pretreated with H2O2 for 2 hours to induce cell senescence, and then treated with MEps or IGF1-MEps for 24 hours, respectively. HUVECs without H2O2 treatment served as a negative control.

[0037] After 24 hours, all cells were stained with SA-β-gal and the corresponding data were statistically analyzed. The results are as follows: Figure 3 As shown, the scale is 100μm, and n=6.

[0038] Example 4: MEps and IGF1-MEps improve endothelial cell senescence and inhibit SASP expression HUVECs were pretreated with H2O2 for 2 hours to induce cell senescence, and then treated with MEps or IGF1-MEps for 24 hours, respectively. HUVECs without H2O2 treatment served as a negative control.

[0039] Cells were collected 24 hours later. Western blot was used to detect cellular senescence-related proteins and NF-κB-related proteins, and QT-qPCR was used to detect the expression of SASP (cellular senescence-associated secretory factor phenotype) related genes. The results are as follows: Figure 4 As shown.

[0040] Example 5: MEps and IGF1-MEps improve vascular lesions in atherosclerotic mice: ApoE - / - Mice were injected with PBS, MEPs (50 μg), or IGF1-MEps (50 μg) three times a week. After four weeks of injection, the aortic plaques in each group of mice were evaluated.

[0041] The results are as follows Figure 5As shown, (a) Oil Red O staining and Masson staining of the aortic sinus of mice in each group; (bc) Statistical analysis of Oil Red O staining and Masson staining data. Bar charts represent mean ± SEM. ns indicates no significant difference. This indicates that p < 0.05. This indicates that p < 0.01.

[0042] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0043] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nanoparticle, characterized in that, The nanoparticles include SiO2 carriers, cell membranes, and exosomes.

2. The nanoparticles according to claim 1, characterized in that, The ratio of cell membrane to exosomes in the SiO2 carrier is (4-8):1:(1-3).

3. The nanoparticles according to claim 1 or 2, characterized in that, The cell membrane in question is the macrophage cell membrane.

4. The nanoparticles according to claim 1 or 2, characterized in that, The exosomes are mesenchymal stem cell exosomes.

5. The nanoparticles according to claim 4, characterized in that, The mesenchymal stem cell exosomes are bone marrow mesenchymal stem cell exosomes.

6. The nanoparticles according to claim 1, 2, 4, or 5, characterized in that, The exosomes overexpress insulin-like growth factor 1.

7. The method for preparing the nanoparticles according to any one of claims 1-6, characterized in that, The preparation method includes: loading the cell membrane of claim 1 or 2 or 3 and the exosomes of any one of claims 1 or 2 or 4-6 onto the SiO2 support of claim 1 or 2 by electrostatic binding.

8. A nanoparticle, characterized in that, It is obtained by the preparation method described in claim 7.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (1) A therapeutically effective amount of the nanoparticles according to any one of claims 1-6 or claim 8; (2) Pharmaceutically or immunologically acceptable carriers or excipients.

10. The use of any one of claims 1-6, or the nanoparticles of claim 8, or the pharmaceutical composition of claim 9, in the preparation of a medicament for the prevention and / or treatment of atherosclerosis-related cardiovascular diseases.

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