A glutathione-responsive iron-based nanozyme for anti-atherosclerosis and its preparation method and application

Abstract

This invention relates to the field of targeted drug delivery systems, specifically to a glutathione-responsive iron-based nanozyme for anti-atherosclerosis, its preparation method, and its application. The iron-based nanozyme prepared by this invention uses iron oxide nanoparticles as the core, a glutathione-responsive delivery carrier as the intermediate layer, and cationic lipids and platelet membranes as the outer shell. It can effectively escape lysosomes and, in response to the action of glutathione, release the encapsulated iron oxide nanoparticles. In the cytoplasmic environment, it exerts CAT-like enzyme activity, reduces reactive oxygen species levels, and the generated oxygen can alleviate hypoxia in atherosclerotic plaques, thus helping to reduce plaque inflammation, improve plaque stability, and has a positive therapeutic effect on atherosclerosis.

Description

Technical Field

[0001] The present invention relates to the field of drug targeted delivery systems, and in particular to a glutathione-responsive iron-based nanozyme for resisting atherosclerosis, and a preparation method and application thereof. Background Art

[0002] As the first discovered nanozyme, the enzyme-like activity of iron oxide nanoparticles (IONPs) has been fully recognized and developed. The enzyme-like activity of IONPs is regulated by the environment. Under acidic conditions, IONPs exhibit peroxidase (POD) activity, that is, catalyzing hydrogen peroxide to produce hydroxyl radicals; under neutral or weakly alkaline conditions, they exhibit catalase (CAT) activity, that is, catalyzing hydrogen peroxide to produce oxygen and water. The POD-like activity of IONPs is widely used in tumors. The acidic microenvironment of the tumor, especially the acidic lysosomal environment, provides conditions for POD activity. The generated hydroxyl radicals have a killing effect and can effectively kill tumor cells. However, in other diseases, such as atherosclerosis, cell death is often avoided. For example, the death of foam cells in atherosclerotic plaques may lead to the formation of a necrotic core of the plaque, increasing the vulnerability of the plaque.

[0003] The CAT-like activity of IONPs has a positive therapeutic effect on atherosclerosis. CAT-like activity can remove hydrogen peroxide, and the oxygen produced can relieve plaque hypoxia, which is beneficial to reduce plaque inflammation and improve plaque stability.

[0004] In order to exert the CAT-like activity of IONPs and avoid POD-like activity, IONPs were wrapped with glutathione-responsive silicon nanoparticles to shield the enzymatic activity of IONPs. Platelet membranes were then wrapped on the surface of the nanoparticles to improve the targeting of atherosclerotic plaques. When the nanoparticles reached the plaques and were taken up by foam cells, the nanoparticles were trapped in the cell lysosomes. In order for the nanoparticles to escape from the lysosomes, lipid 5 with a tertiary amine was mixed into the platelet membrane to give the nanoparticles the ability to escape from the lysosomes. After escaping the lysosomes, the nanoparticles entered the neutral cytoplasmic environment. At this time, glutathione in the cytoplasm degraded the silicon nanoparticles, exposing the internal IONPs and exerting CAT-like activity. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a glutathione-responsive iron-based nanozyme for anti-atherosclerosis and its preparation method and application in view of the deficiencies of the prior art. The iron-based nanozyme created by the present invention specifically targets foam cells in atherosclerotic plaques, exerts catalase activity through lysosomal escape and glutathione degradation, and reduces oxidative stress in foam cells.

[0006] In order to solve the above technical problems, the present invention discloses a glutathione-responsive iron-based nanozyme for anti-atherosclerosis and its preparation method and application. The specific technical scheme is as follows:

[0007] A glutathione-responsive iron-based nanozyme IONPs@OSiNPs@PM / L5 for anti-atherosclerosis, wherein the iron-based nanozyme comprises iron oxide nanoparticles, a glutathione-responsive delivery carrier, cationic lipids and platelet membranes; the iron oxide nanoparticles are used as a core, the glutathione-responsive delivery carrier is used as a middle layer, and the cationic lipids and platelet membranes are used as an outer shell.

[0008] Wherein, the iron-based nanozyme has a particle size of 70-80 nm. Preferably, the particle size of the iron-based nanozyme is 79.6 nm.

[0009] The iron oxide nanoparticles have a particle size of 8-15 nm, preferably 10 nm.

[0010] Wherein, the glutathione-responsive delivery carrier is a glutathione-responsive organosilicon, preferably a composition of bis-[3-(triethoxysilyl)propyl]-disulfide and ethyl silicate.

[0011] More preferably, the glutathione-responsive organosilicon is a composition of bis-[3-(triethoxysilyl)propyl]-disulfide and ethyl silicate, and the volume ratio of bis-[3-(triethoxysilyl)propyl]-disulfide to ethyl silicate is 1:2-8, preferably 1:2.5.

[0012] Wherein, the cationic lipid is lipid 5. The lipid 5 is a composition of heptadecan-9-yl-8-((2-hydroxyethyl)(8-nonyloxy)-8-oxooctylamino)octanoate and octanoic acid 8-((2-hydroxyethyl)(8-nonyloxy-8-((oxooctyl)amino)1-octylnonyl ester. Preferably, the lipid 5 is purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0013] In a second aspect, the present invention provides a method for preparing the iron-based nanozyme according to the first aspect, comprising the following steps:

[0014] (1) mixing iron oxide nanoparticles with a glutathione-responsive delivery carrier to obtain particles formed by the delivery carrier coating the iron oxide nanoparticles;

[0015] (2) The particles formed after the delivery carrier is coated with the iron oxide nanoparticles are mixed with cationic lipids and platelet membranes to obtain.

[0016] Wherein, in step (1), the glutathione-responsive delivery carrier is a composition of bis-[3-(triethoxysilyl)propyl]-disulfide and ethyl silicate; the volume ratio of bis-[3-(triethoxysilyl)propyl]-disulfide and ethyl silicate in the composition is 1:2-8; preferably, the volume ratio is 1:2.5.

[0017] The mixing system includes: iron oxide nanoparticles, bis-[3-(triethoxysilyl)propyl]-disulfide and ethyl silicate, and the amount of the iron oxide nanoparticles added is calculated by adding 50-150 mg of iron oxide nanoparticles per mL of bis-[3-(triethoxysilyl)propyl]-disulfide; the mixing temperature is 50-80°C. Preferably, 50 mg of iron oxide nanoparticles are added per mL of bis-[3-(triethoxysilyl)propyl]-disulfide, and the mixing temperature is 70°C. The particles formed after the delivery carrier is coated with the iron oxide nanoparticles are IONPs@OSiNPs. The iron oxide nanoparticles are used in the form of a mixture of iron oxide nanoparticles, and the mixture of iron oxide nanoparticles is obtained by dispersing the iron oxide nanoparticles in chloroform, and then adding NaOH solution and water.

[0018] Wherein, in step (2), the amount of cationic lipid added is calculated as 0.1-3 mg of cationic lipid mixed with each mg of delivery carrier coated with iron oxide nanoparticles; the amount of platelet membrane added is calculated as 2×10 8 -6×10 8 Preferably, 0.5 mg of cationic lipid is mixed with IONPs@OSiNPs per mg of delivery vehicle coated with iron oxide nanoparticles; 5×10 8 Platelet membrane meter.

[0019] In a third aspect, the present invention provides the use of the iron-based nanozyme described in the first aspect in the preparation of anti-atherosclerotic drugs.

[0020] Beneficial effects:

[0021] In order to achieve the enzyme-like activity regulation of iron oxide nanoparticles (IONPs) in foam cells, the present invention provides an organosilicon nanoparticle (IONPs@OSiNPs@PM) with an IONPs core wrapped in a platelet membrane (PM); in order to achieve lysosomal escape, lipid 5 (L5) is inserted into PM to obtain iron-based nanozyme IONPs@OSiNPs@PM / L5. The IONPs@OSiNPs@PM / L5 can effectively aggregate into atherosclerotic plaques; further, the IONPs@OSiNPs@PM / L5 is taken up by foam cells in the plaques and escapes from the lysosomes, exerting the CAT enzyme activity of IONPs instead of the POD enzyme activity, reducing the level of reactive oxygen, and the generated oxygen can alleviate the hypoxia of atherosclerotic plaques, which is beneficial to reduce plaque inflammation, improve the stability of plaques, and have a positive therapeutic effect on atherosclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0023] Figure 1 Schematic diagram of the structure of IONPs@OSiNPs@PM / L5.

[0024] Figure 2 Transmission electron microscopy image of IONPs@OSiNPs@PM / L5 treated with glutathione. Figure 2 A is the transmission electron microscopy image of IONPs@OSiNPs@PM / L5 before glutathione treatment; Figure 2 B is the transmission electron microscopy image of IONPs@OSiNPs@PM / L5 after glutathione treatment.

[0025] Figure 3 This is the enzyme activity test diagram of IONPs@OSiNPs@PM / L5 nanoparticles. Figure 3 A is the absorption spectrum of rhodamine B; Figure 3 B is the graph of oxygen content changes.

[0026] Figure 4 The intracellular reactive oxygen levels of RAW264.7 cells after different treatments.

[0027] Figure 5 The cytotoxicity of different concentrations of IONPs@OSiNPs@PM or IONPs@OSiNPs@PM / L5 on RAW264.7 cells. DETAILED DESCRIPTION

[0028] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0029] Example 1 Preparation of IONPs@OSiNPs@PM / L5 Nanoparticles

[0030] In this example, IONPs@OSiNPs@PM / L5 nanoparticles were prepared, and the preparation method included the following steps:

[0031] (1) Mixing iron oxide nanoparticles with a glutathione-responsive delivery vehicle to obtain particles IONPs@OSiNPs formed after the delivery vehicle coated the iron oxide nanoparticles: 10 mg of iron oxide nanoparticles (IONPs, purchased from Nanjing Xianfeng Nanomaterials Technology Co., Ltd., size ~10 nm) were dispersed in 0.5 mL of chloroform, and then 0.3 mL of NaOH solution (2M) and 45 mL of water were added to obtain a mixture of iron oxide nanoparticles. The iron oxide nanoparticle mixture was heated to 70°C, and 0.5 mL of ethyl silicate (TEOS) and 0.2 mL of bis-[3-(triethoxysilyl)propyl]-disulfide (BTSD) were added to obtain a suspension. After the suspension was cooled to room temperature, the nanoparticles were collected by magnetic force and washed three times with anhydrous ethanol to obtain particles IONPs@OSiNPs formed after the delivery vehicle coated the iron oxide nanoparticles.

[0032] (2) 10 mg of IONPs@OSiNPs prepared in step (1), 5 mg of lipid 5 (heptadecan-9-yl-8-((2-hydroxyethyl)(8-nonyloxy)-8-oxooctylamino)octanoate and octanoic acid 8-((2-hydroxyethyl)(8-nonyloxy-8-((oxooctyl)amino)1-octylnonyl ester composition, purchased from Shanghai MacLean Biochemical Technology Co., Ltd.) and 2 mL of platelet membrane (approximately 5×10 9 Platelets were obtained by repeated freezing and thawing of human platelet plasma, centrifuging at 4000×g, 4°C for 5 min, removing the supernatant, and washing twice with saline. The mixture was sonicated in an ultrasonic bath for 2 min and extruded 10 times through a polycarbonate membrane (nominal pore size 200 nm). Finally, the iron-based nanozyme IONPs@OSiNPs@PM / L5 was collected by magnetic force. The schematic diagram of its structure is shown in the figure. Figure 1 As shown in Figure 2, the prepared iron-based nanozyme IONPs@OSiNPs@PM / L5 has a size of 79.6 nm and a surface potential of -3.7 mV.

[0033] Example 2

[0034] In order to detect the responsive degradation of glutathione, 1 mL of the iron-based nanozyme IONPs@OSiNPs@PM / L5 aqueous solution (0.1 mg / mL) prepared in Example 1 was placed in a dialysis membrane (Mw 1000) and dialyzed against 20 mL of PBS (pH = 7.4) containing 10 mM glutathione at 37°C. After 6 hours of dialysis, the samples were collected and their morphological changes were observed by TEM ( Figure 2 ).from Figure 2 It can be seen that after the iron-based nanozyme IONPs@OSiNPs@PM / L5 was treated with glutathione, the structure of the delivery carrier OSiNPs was destroyed and IONPs were exposed, proving the glutathione responsiveness of IONPs@OSiNPs@PM / L5.

[0035] Example 3

[0036] In order to test the enzyme-like activity of glutathione-stimulated iron-based nanozymes, 1 mL of the IONPs@OSiNPs@PM / L5 aqueous solution (0.1 mg / mL) prepared in Example 1 was placed in a dialysis membrane (Mw 1000) and dialyzed against 20 mL of PBS containing 10 mM glutathione at 37°C for 6 hours.

[0037] (1) To verify the POD-like enzyme activity, the samples before and after dialysis were added to a 100 mM hydrogen peroxide solution (pH 5.0) containing 1 mM rhodamine B. After standing at room temperature for 4 hours, the absorption spectrum of rhodamine B in the samples was detected.

[0038] (2) To verify the activity of CAT-like enzymes, the samples before and after dialysis were added to 100 mM hydrogen peroxide solution (pH 7.4), and the oxygen content in the samples was detected every 30 seconds using a dissolved oxygen meter.

[0039] from Figure 3 It can be seen that the iron-based nanozyme IONPs@OSiNPs@PM / L5 without glutathione treatment neither exhibits POD enzyme activity ( Figure 3 A has an absorption peak at 562nm) and does not show CAT-like enzyme activity ( Figure 3 The oxygen content in B did not change); however, the iron-based nanozyme IONPs@OSiNPs@PM / L5 treated with glutathione showed POD-like enzyme activity at pH 5.0 ( Figure 3 A has no absorption peak at 562 nm), and exhibits CAT-like enzyme activity at pH 7.4 ( Figure 3 The oxygen content in B increased from the initial 2.6 mg / L to 13.5 mg / L).

[0040] Example 4

[0041] Mouse macrophages RAW264.7 were inoculated in glass-bottomed plates and incubated at 37°C for 12 hours. After the cells adhered, 100 mM hydrogen peroxide solution (20 μL) was added. After incubation for 2 hours, the culture medium was removed and 100 μL IONPs@OSiNPs@PM aqueous solution (1 mg / mL) or 100 μL IONPs@OSiNPs@PM / L5 aqueous solution (1 mg / mL) was added and cultured for another 4 hours. The level of intracellular reactive oxygen species was then detected using the reactive oxygen species probe DCFH-DA. Figure 4 As shown, the level of reactive oxygen species in cells treated with hydrogen peroxide increased. After incubation with IONPs@OSiNPs@PM, the level of reactive oxygen species did not change significantly, while after incubation with IONPs@OSiNPs@PM / L5, the level of reactive oxygen species decreased significantly. The results show that IONPs@OSiNPs@PM cannot escape from the lysosome, so it exerts a POD-like enzyme activity, catalyzing hydrogen peroxide into hydroxyl radicals, so the level of reactive oxygen species has hardly changed; while IONPs@OSiNPs@PM / L5 can escape from the lysosome and reach the cytoplasm, exerting a CAT-like enzyme activity, converting hydrogen peroxide into oxygen, resulting in a significant decrease in the level of reactive oxygen species.

[0042] Example 5

[0043] Mouse macrophages RAW264.7 were inoculated in a 96-well plate (8000 cells / well) and cultured overnight at 37°C. 50 μM hydrogen peroxide (10 μL) was added and cultured with the cells for 4 hours, and then 10 μL of IONPs@OSiNPs@PM aqueous solution or IONPs@OSiNPs@PM / L5 aqueous solution of different concentrations (1.25-40 μg / mL) was added and cultured with the cells for 24 hours. After adding 10 μL of CCK-8 reagent and culturing at 37°C for 2 hours, the absorbance was measured at 450 nm using a microplate reader to evaluate cell viability. Figure 5 As shown, at the same concentration, the cytotoxicity of IONPs@OSiNPs@PM is higher than that of IONPs@OSiNPs@PM / L5. At low concentrations, IONPs@OSiNPs@PM has almost no cytotoxicity. Since IONPs@OSiNPs@PM cannot escape from the lysosome, it exerts a POD-like enzyme activity, and the generated hydroxyl radicals kill the cells. IONPs@OSiNPs@PM / L5 can quickly escape from the lysosome and enter the cytoplasm, exerting a CAT-like enzyme activity, with low cytotoxicity. After culturing cells with 20μg / mL of IONPs@OSiNPs@PM / L5 for 24 hours, the cell activity still remains above 50%.

[0044] In summary, IONPs@OSiNPs@PM / L5 can effectively escape from lysosomes and respond to the action of glutathione to release the encapsulated IONPs, exerting CAT enzyme-like activity in the cytoplasmic environment and reducing the level of reactive oxygen species.

[0045] The present invention provides a glutathione-responsive iron-based nanozyme for anti-atherosclerosis and a preparation method and application thereof. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented using existing technologies.

Claims

1. A glutathione-responsive iron-based nanozyme for anti-atherosclerosis, characterized in that: The iron-based nanozyme comprises iron oxide nanoparticles, a glutathione-responsive delivery carrier, cationic lipids and platelet membranes; the iron oxide nanoparticles are used as the core, the glutathione-responsive delivery carrier is used as the middle layer, and the cationic lipids and platelet membranes are used as the outer shell; The iron oxide nanoparticles have a particle size of 8-15 nm; The glutathione-responsive delivery carrier is a glutathione-responsive organosilicon; the glutathione-responsive organosilicon is a composition of bis-[3-(triethoxysilyl)propyl]-disulfide and ethyl silicate; the volume ratio of bis-[3-(triethoxysilyl)propyl]-disulfide and ethyl silicate in the composition is 1:2-8; The cationic lipid is lipid 5; the lipid 5 is a composition of heptadecan-9-yl-8-((2-hydroxyethyl)(8-nonyloxy)-8-oxooctylamino)octanoate and octanoic acid 8-((2-hydroxyethyl)(8-nonyloxy-8-((oxooctyl)amino)1-octylnonyl ester.

2. The iron-based nanozyme according to claim 1, characterized in that The iron-based nanozyme has a particle size of 70-80nm.

3. The method for preparing the iron-based nanozyme according to any one of claims 1 to 2, characterized in that: The steps include: (1) mixing iron oxide nanoparticles with a glutathione-responsive delivery carrier to obtain particles formed by coating the iron oxide nanoparticles with the delivery carrier; the glutathione-responsive delivery carrier is a composition of bis-[3-(triethoxysilyl)propyl]-disulfide and ethyl silicate; the volume ratio of bis-[3-(triethoxysilyl)propyl]-disulfide to ethyl silicate in the composition is 1:2-8; (2) The particles formed after the delivery carrier is coated with the iron oxide nanoparticles are mixed with cationic lipids and platelet membranes to obtain the product.

4. The preparation method according to claim 3, characterized in that: In step (1), the mixing system comprises: iron oxide nanoparticles, bis-[3-(triethoxysilyl)propyl]-disulfide and ethyl silicate, and the amount of iron oxide nanoparticles added is calculated by adding 50-150 mg of iron oxide nanoparticles per mL of bis-[3-(triethoxysilyl)propyl]-disulfide; and the mixing temperature is 50-80°C.

5. The preparation method according to claim 3, characterized in that: In step (2), the amount of cationic lipid added is calculated as 0.1-3 mg of cationic lipid mixed with each mg of the delivery carrier coated with iron oxide nanoparticles; the amount of platelet membrane added is calculated as 2×10 8 -6×10 8 Platelet membrane meter.

6. Use of the iron-based nanozyme according to any one of claims 1 to 2 in the preparation of anti-atherosclerotic drugs.

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