Engineered exosomes with atherosclerotic plaque targeting property, preparation method and application thereof

Abstract

The application provides an engineered exosome with atherosclerotic plaque targeting property, a preparation method and application thereof, and belongs to the technical field of biomedical engineering functional materials. The engineered exosome comprises natural exosomes released by vascular endothelial cells under laminar shear stress loading and a targeting polymer HA-PEG-Mal; wherein the targeting polymer is modified on the surface of the natural exosome. The engineered exosome is the exosome released by the vascular endothelial cells under the laminar shear stress loading and modified with the targeting polymer HA-PEG-Mal on the surface, can make the modified targeting polymer bind to the ligand with high affinity at the high expression of CD44 at the atherosclerotic plaque, make the engineered exosome target the macrophage, and act on the atherosclerotic plaque, so as to realize efficient treatment of atherosclerosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering functional materials technology, specifically relating to an engineered exosome with targeting properties for atherosclerotic plaques, its preparation method, and its application. Background Technology

[0002] Cardiovascular disease is one of the leading causes of death and morbidity worldwide. According to the World Health Organization, 8.9 million people died from cardiovascular disease in 2019. Atherosclerosis is one of the main forms of cardiovascular disease, considered a chronic inflammatory disease caused by lipid accumulation in the lumen. Its main pathological features include intimal thickening, smooth muscle proliferation, lipid accumulation, luminal narrowing, and decreased vascular compliance. Currently, the treatment of atherosclerosis mainly includes balloon angioplasty and drug therapy. Balloon stent implantation often causes vascular wall damage such as endothelial detachment, leading to inflammatory responses and poor prognosis. Drug therapy can effectively treat and slow the progression of atherosclerosis, but it also has problems such as significant side effects and difficulty in achieving effective blood drug concentrations.

[0003] Nanoparticle drug delivery can effectively deliver drugs to the lesion site, improving drug bioavailability and reducing toxic side effects. Exosomes, as nanoscale vesicles secreted by all types of cells, naturally possess good biocompatibility, low cytotoxicity, immune inertness, and long-term circulation characteristics. In recent years, exosomes have made considerable progress as nanomedicines in the treatment of atherosclerosis, demonstrating superior potential in this field.

[0004] It is well known that normal physiological laminar shear stress is crucial for maintaining normal vascular function and has a protective effect against atherosclerosis. Endothelial cells, as a unique barrier separating the vessel wall from the blood, directly and highly respond to various hemodynamic forces, including laminar shear stress, and are directly stimulated by laminar shear force. Current research shows that 15 dyn / cm 2 Under normal physiological laminar shear stress loading, endothelial cell-derived exosomes have the potential to influence the pathological process of atherosclerosis. However, the targeting of natural exosomes as nanotherapeutic carriers in vivo is still limited. For example, after use in animal models, they tend to accumulate in the liver, spleen, and lungs, and cannot effectively target the pathological sites of atherosclerosis. Summary of the Invention

[0005] This invention provides an engineered exosome with targeting ability for atherosclerotic plaques, its preparation method and application. It can utilize the targeting polymer HA-PEG-Mal to target the exosomes onto macrophages, thereby helping the exosomes target atherosclerotic plaque sites to achieve the effect of treating atherosclerosis.

[0006] In a first aspect, the present invention provides an engineered exosome with targeting capability for atherosclerotic plaques, comprising: natural exosomes released by vascular endothelial cells under laminar shear stress loading and a targeting polymer HA-PEG-Mal; wherein the targeting polymer is modified on the surface of the natural exosomes.

[0007] Furthermore, the average molecular weight of the targeted polymer HA-PEG-Mal is 7023.67–7170.47, the diameter of the natural exosomes is 124.2–132.1 nm, and the diameter of the engineered exosomes is 143.6–152.2 nm.

[0008] Secondly, the present invention provides a method for preparing engineered exosomes with targeting properties to atherosclerotic plaques, comprising the following steps:

[0009] (1) Under laminar shear stress loading, human umbilical vein endothelial cells release natural exosomes;

[0010] (2) Natural exosomes are linked to the targeted polymer HA-PEG-Mal to obtain engineered exosomes.

[0011] Furthermore, methods for preparing natural exosomes include:

[0012] (a) Applying a size of 5–20 dyn / cm to human umbilical vein endothelial cells 2 The laminar shear stress was maintained for 6–10 hours, and the supernatant was collected afterward.

[0013] (b) The supernatant obtained in step (a) was centrifuged at 4°C and 300g for 20 min, then centrifuged at 4°C and 3000g for 20 min, and then centrifuged at 4°C and 10000g for 30 min to obtain the supernatant.

[0014] (c) Filter the supernatant obtained in step (b) using a filter membrane with a pore size of 0.22 μm to obtain the filtrate;

[0015] (d) Centrifuge the filtrate obtained in step (c) at 4°C and 100,000g for 2 hours in an ultracentrifuge. Take the precipitate and resuspend it by pipetting with ice-cold sterile PBS solution. The resulting resuspended solution is the natural exosomes released by endothelial cells under laminar shear stress loading.

[0016] Furthermore, methods for preparing natural exosomes include:

[0017] (a) Applying a size of 15 dyn / cm to human umbilical vein endothelial cells 2 Laminar shear stress was maintained for 8 hours, and then the supernatant was collected.

[0018] (b) The supernatant obtained in step (a) was centrifuged at 4°C and 300g for 20 min, then centrifuged at 4°C and 3000g for 20 min, and then centrifuged at 4°C and 10000g for 30 min to obtain the supernatant.

[0019] (c) Filter the supernatant obtained in step (b) using a filter membrane with a pore size of 0.22 μm to obtain the filtrate;

[0020] (d) The filtrate obtained in step (c) was centrifuged for 2 hours at 4°C and 100,000g in an ultracentrifuge. The precipitate was resuspended by pipetting with ice-cold sterile PBS solution. The resulting resuspended solution was natural exosomes released by vascular endothelial cells under laminar shear stress loading.

[0021] Furthermore, the methods for preparing engineered exosomes include:

[0022] Add 1 mM TCEP reducing agent to natural exosomes and treat at 37°C for 30 min. Then add 0.5–1 mM of the targeting polymer HA-PEG-Mal and incubate at 25°C for 1 h to allow the natural exosomes to be linked with the targeting polymer HA-PEG-Mal to obtain engineered exosomes.

[0023] Furthermore, the methods for preparing engineered exosomes include:

[0024] 1 mM TCEP reducing agent was added to natural exosomes and treated at 37 °C for 30 min. Then, 1 mM targeting polymer HA-PEG-Mal was added and incubated at 25 °C for 1 h to allow the natural exosomes to be linked with the targeting polymer HA-PEG-Mal to obtain engineered exosomes.

[0025] Furthermore, TCEP reducing agent and targeted polymer were added dropwise using a microsyringe at a rate of 0.5–1.5 mL / h.

[0026] Furthermore, the drop rate of the microsyringe is 1 mL / h.

[0027] Thirdly, the present invention provides the application of engineered exosomes with atherosclerotic plaque targeting properties in the preparation of drugs for treating atherosclerosis.

[0028] This invention provides an engineered exosome with targeting properties for atherosclerotic plaques, its preparation method, and its applications, including:

[0029] This engineered exosome is an exosome released by vascular endothelial cells under laminar shear stress loading of a surface-modified targeting polymer HA-PEG-Mal. It can enable the modified targeting polymer to bind to the CD44 high-affinity ligand highly expressed at atherosclerotic plaques, allowing the engineered exosome to target macrophages and act on atherosclerotic plaques to achieve highly effective treatment of atherosclerosis. Attached Figure Description

[0030] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort and also fall within the protection scope of this application.

[0031] Figure 1 The morphology and particle size distribution of the natural exosomes provided in Example 1 and the engineered exosomes provided in Example 2 are shown.

[0032] Figure 2 The natural exosomes provided in Example 1 and the engineered exosomes provided in Example 2 were used to regulate macrophage polarization in vitro.

[0033] Figure 3 To investigate the in vitro regulation of macrophage polarization by exosomes in static culture.

[0034] Figure 4 The in vivo targeted distribution of natural exosomes provided in Example 1 and engineered exosomes provided in Example 2.

[0035] Figure 5 The natural exosomes provided in Example 1 and the engineered exosomes provided in Example 2 were administered in vivo for a long period of time to alleviate atherosclerosis.

[0036] Figure 6 To provide long-term in vivo administration of statically cultured exosomes to alleviate atherosclerosis.

[0037] Figure 7 The long-term in vivo toxicity of the natural exosomes provided in Example 1 and the engineered exosomes provided in Example 2 is compared. Detailed Implementation

[0038] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0039] This application provides an engineered exosome with targeting capability for atherosclerotic plaques, comprising: natural exosomes released by vascular endothelial cells under laminar shear stress loading and a targeting polymer HA-PEG-Mal; wherein the targeting polymer is modified on the surface of the natural exosomes.

[0040] The targeted polymer HA-PEG-Mal refers to a copolymer of hyaluronic acid, polyethylene glycol, and maleimide. Maleimide has the characteristic of reacting efficiently with thiol groups. In the subsequent processing of the experiment, the disulfide bonds on the surface of exosomes are treated with TCEP reducing agent to reduce a large number of thiol groups and react with maleimide, so that the copolymer HA-PEG-Mal is connected to the exosomes; thereby modifying the surface of natural exosomes with the targeted polymer.

[0041] In this application, the selection of the targeting polymer has the following advantages: Hyaluronic acid (HA) is chosen as a targeting molecule because macrophages in atherosclerotic lesion areas highly express CD44, and HA can bind to CD44's high-affinity ligands. Simultaneously, HA, as a major component of the extracellular matrix, also possesses high non-immunogenicity, biocompatibility, and degradability, which can significantly reduce subsequent biological cascade reactions caused by ligand binding, minimizing unnecessary impacts on target cells. Furthermore, the degradability of hyaluronic acid makes it highly likely to be rapidly degraded and cleared from the bloodstream. Polyethylene glycol (PEG) neutralizes this deficiency of HA, increasing the overall retention time of the targeting polymer in the blood and extending blood circulation duration; simultaneously, PEG also improves the polymer's biocompatibility. Maleimide (Mal) is a powerful click handle whose reaction with thiol groups is faster and more selective than traditional bioconjugation techniques. At the same time, this efficient reaction does not change the natural therapeutic effects of exosomes compared to surface modification methods such as genetic engineering, and does not cause changes in the biological activity of exosomes due to genetic manipulation. As a result, the engineered exosomes obtained have a good therapeutic effect on atherosclerosis.

[0042] Furthermore, the average molecular weight of the targeted polymer HA-PEG-Mal is 7023.67–7170.47, the diameter of the natural exosomes is 124.2–132.1 nm, and the diameter of the engineered exosomes is 143.6–152.2 nm.

[0043] Example 1

[0044] A method for preparing natural exosomes includes:

[0045] (a) Applying a size of 15 dyn / cm to human umbilical vein endothelial cells 2 Laminar shear stress was maintained for 8 hours, and then the supernatant was collected.

[0046] (b) The supernatant obtained in step (a) was centrifuged at 4°C and 300g for 20 min, then centrifuged at 4°C and 3000g for 20 min, and then centrifuged at 4°C and 10000g for 30 min to obtain the supernatant.

[0047] (c) Filter the supernatant obtained in step (b) using a filter membrane with a pore size of 0.22 μm to obtain the filtrate;

[0048] (d) The filtrate obtained in step (c) was centrifuged for 2 hours at 4°C and 100,000g in an ultracentrifuge. The precipitate was resuspended by pipetting with ice-cold sterile PBS solution. The resulting resuspended solution was natural exosomes released by vascular endothelial cells under laminar shear stress loading.

[0049] Example 2

[0050] A method for preparing engineered exosomes includes:

[0051] 1 mM TCEP was added to the exosome suspension obtained in Example 1 and treated at 37°C for 30 min. Then, 1 mM of the targeting polymer HA-PEG-Mal was added and incubated at 25°C for 1 h to allow the natural exosomes to be linked with the targeting polymer HA-PEG-Mal, thereby obtaining engineered exosomes with the targeting polymer modified on the surface of the natural exosomes.

[0052] Experimental Example 1: Morphology and particle size study of exosomes

[0053] The natural exosomes obtained in Example 1 and the engineered exosomes obtained in Example 2 were analyzed by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA), respectively. The morphology and particle size distribution are shown in the figure. Figure 1 ( Figure 1 The left image shows the morphology and particle size distribution of natural exosomes obtained in Example 1. Figure 1 The right side shows the morphology and particle size distribution of the engineered exosomes obtained in Example 2. Figure 1 It can be seen that the particle size of the natural exosomes provided in Example 1 is 127.9 nm, and the particle size of the engineered exosomes provided in Example 2 is 144.1 nm; from Figure 1 As can be seen from the figure on the right, engineered exosomes have a typical double-membrane structure, indicating that the morphology of exosomes was not affected after engineering modification.

[0054] Experimental Example 2: Study on the In Vitro Regulation of Macrophage Polarization by Exosomes

[0055] The natural exosomes obtained in Example 1 and the engineered exosomes obtained in Example 2 were each sampled at a concentration of 5 × 10⁵ m³. 9 Add 4×10 granules6 The exosomes were treated with M1 macrophages at a concentration of 1 / mL for 24 hours. After treatment, the proportion of macrophages with the M1 phenotype in Group 1 and Group 2 was 34.5% and 32.1%, respectively, while the proportion of macrophages with the M2 phenotype was 12.9% and 15.5%, respectively. Therefore, the engineered exosomes prepared by the method of Example 2 have a higher ability to regulate the polarization of macrophages from M1 to M2 type. The results are shown in [Figure 1]. Figure 2 .

[0056] Do not use exosomes or use 5×10 9 Add 4 × 10⁶ statically cultured vascular endothelial cell-derived exosomes to 4 × 10⁶ cells / mL. 6 The macrophages were treated with M1 macrophages at a concentration of [number] cells / mL for 24 hours. After treatment, the percentages of macrophages with the M1 phenotype were 51.9% and 50.8%, respectively, while the percentages with the M2 phenotype were 1.8% and 5.46%, respectively. (See attached table). Figure 3 This indicates that the use of exosomes or statically cultured exosomes cannot effectively regulate the polarization of M1 macrophages toward the M2 phenotype.

[0057] Experimental Example 3: Study on the in vivo targeted distribution of exosomes

[0058] The natural exosomes obtained in Example 1 and the engineered exosomes obtained in Example 2 were each sampled at a rate of 4 × 10⁴ μL. 10 After being labeled with DiI red fluorescence, each particle was injected via tail vein into an ApoE diet for 12 weeks. -\- In mice, after 6 hours, mouse organs and the aorta were harvested and observed for fluorescence distribution under in vivo imaging. The results showed that the fluorescence of Example 1 was rarely distributed in the aorta, while the fluorescence of Example 2 was more widely distributed in the aorta. This indicates that the engineered exosomes provided in Example 2 can better target aortic plaques in vivo. (See results below.) Figure 4 .

[0059] Example 4: Evaluation of the long-term in vivo administration of exosomes to alleviate atherosclerosis

[0060] The natural exosomes obtained in Example 1 and the engineered exosomes obtained in Example 2 were each aspirated at a rate of 1×10⁻⁶. 10 One granule of ApoE was administered via tail vein injection every three days into a high-fat diet for 8 weeks. -\- In mice, after four weeks of euthanasia, the aortic sinus and the entire aortic vessel were isolated, the vessel was dissected, and Oil Red O staining was performed. The results are shown in [Figure number missing]. Figure 5 The results showed that the engineered exosomes provided in Example 2 could effectively reduce the area of ​​atherosclerotic plaques in vivo.

[0061] Do not use exosomes or use 1×10 10ApoE was administered via tail vein injection every three days to a statically cultured vascular endothelial cell-derived exosomes following an 8-week high-fat diet. -\- In mice, after four weeks of euthanasia, the aortic sinus and the entire aortic vessel were isolated, dissected, and stained with Oil Red O. The results... Figure 6 ,from Figure 5 and Figure 6 The comparison shows that the treatment effect of not using exosomes or using statically cultured exosomes is not good for atherosclerosis.

[0062] Experimental Example 5: Long-term in vivo toxicity evaluation of exosomes

[0063] The natural exosomes obtained in Example 1 and the engineered exosomes obtained in Example 2 were each aspirated at a rate of 1×10⁻⁶. 10 One granule was injected via the tail vein into an ApoE diet taken for 8 weeks on a high-fat diet. -\- In mice, the mice were euthanized every three days for four weeks, and major organs were isolated. The heart, liver, spleen, lungs, and kidneys were subjected to H&E pathological staining. (Screenshot shown) Figure 5 It is evident that engineered exosomes administered in vivo have no long-term toxicity to various organs.

Claims

1. A method for preparing engineered exosomes with targeting properties for atherosclerotic plaques, characterized in that, The engineered exosomes comprise: natural exosomes released by vascular endothelial cells under laminar shear stress loading and a targeting polymer HA-PEG-Mal; wherein the targeting polymer is modified on the surface of the natural exosomes; the average molecular weight of the targeting polymer HA-PEG-Mal is 7023.67–7170.47, the diameter of the natural exosomes is 124.2–132.1 nm, and the diameter of the engineered exosomes is 143.6–152.2 nm; The method for preparing the natural exosomes includes: (a) Applying a size of 5–20 dyn / cm to human umbilical vein endothelial cells 2 Laminar shear stress was maintained for 6–10 hours, after which the supernatant was collected. (b) The supernatant obtained in step (a) was centrifuged at 4°C and 300g for 20 min, then centrifuged at 4°C and 3000g for 20 min, and then centrifuged at 4°C and 10000g for 30 min to obtain the supernatant. (c) Filter the supernatant obtained in step (b) using a filter membrane with a pore size of 0.22 μm to obtain the filtrate; (d) The filtrate obtained in step (c) is centrifuged in an ultracentrifuge at 4°C and 100,000g for 2 hours. The precipitate is then resuspended by pipetting with ice-cold sterile PBS solution. The resulting resuspended solution is the natural exosomes released by vascular endothelial cells under laminar shear stress loading. The method for preparing the engineered exosomes includes: 1 mM TCEP reducing agent was added to the natural exosomes and treated at 37°C for 30 min. Then, 0.5–1 mM of the targeting polymer HA-PEG-Mal was added and the mixture was incubated at 25°C for 1 h to allow the natural exosomes to be linked with the targeting polymer HA-PEG-Mal, thereby obtaining the engineered exosomes.

2. The preparation method according to claim 1, characterized in that, The method for preparing the natural exosomes includes: (a) Applying a size of 15 dyn / cm to human umbilical vein endothelial cells 2 Laminar shear stress was maintained for 8 hours, and the supernatant was collected afterward. (b) The supernatant obtained in step (a) was centrifuged at 4°C and 300g for 20 min, then centrifuged at 4°C and 3000g for 20 min, and then centrifuged at 4°C and 10000g for 30 min to obtain the supernatant. (c) Filter the supernatant obtained in step (b) using a filter membrane with a pore size of 0.22 μm to obtain the filtrate; (d) Centrifuge the filtrate obtained in step (c) at 4°C and 100,000g for 2 hours in an ultracentrifuge. Take the precipitate and resuspend it by pipetting with ice-cold sterile PBS solution. The resulting resuspended solution is the natural exosomes released by vascular endothelial cells under laminar shear stress loading.

3. The preparation method according to claim 1, characterized in that, The method for preparing the engineered exosomes includes: 1 mM TCEP reducing agent was added to the natural exosomes and treated at 37°C for 30 min. Then, 1 mM targeting polymer HA-PEG-Mal was added and incubated at 25°C for 1 h to allow the natural exosomes to be linked with the targeting polymer HA-PEG-Mal to obtain the engineered exosomes.

4. The preparation method according to claim 1, characterized in that, The TCEP reducing agent was added dropwise using a microsyringe at a rate of 1 mL / h.

5. The use of the engineered exosomes with atherosclerotic plaque targeting prepared by the preparation method according to any one of claims 1 to 4 in the preparation of a medicament for treating atherosclerosis.

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