Dtrim24 biocompatibility-based Anti-atherosclerotic nanoparticles

Abstract

The present application relates to the technical field of nanocapsules for medical preparations, and particularly relates to dTRIM24 biocompatibility-based anti-atherosclerotic nanoparticles composed of nanoparticles and M1 macrophage membrane vesicles. By using a microfluidic photoporation chip, the nanoparticles are encapsulated by the M1 macrophage membrane vesicles. The nanoparticles are obtained by combining dTRIM24 and Fe3O4 magnetic nanoparticles. The biomimetic nanoparticles of the present application reasonably solve the problems of poor specificity and low efficiency, and have improved anti-atherosclerotic plaque-targeting and biological homology.

Description

Anti-atherosclerosis nanoparticles based on dTRIM24 biocompatibility TECHNICAL FIELD

[0001] The present application relates to the field of nanocapsule for medical formulation, in particular to anti-atherosclerosis nanoparticles based on dTRIM24 biocompatibility. BACKGROUND

[0002] Atherosclerosis (AS) is the main cause of coronary heart disease and ischemic cerebrovascular disease. The current operation and high-dose drug treatment of atherosclerotic disease has limited effect and high risk of adverse reactions. How to more specifically and efficiently eliminate atherosclerotic plaques is the key to preventing and treating atherosclerosis-related cardiovascular and cerebrovascular diseases. Therefore, by constructing more specific biomimetic nanoparticles combined with the transformation of M1 cells into M2 type at the plaque site, the degree of AS can be more efficiently, safely and accurately reduced, and the treatment of AS can be achieved.

[0003] SUMMARY

[0004] The present application provides anti-atherosclerosis nanoparticles based on dTRIM24 biocompatibility; which can reasonably solve the problems of poor specificity and low efficiency, and improve the targeting and biological homology of anti-atherosclerosis plaques.

[0005] In one aspect, the present application provides anti-atherosclerosis nanoparticles based on dTRIM24 biocompatibility (biomimetic nanoparticles), which is composed of nanoparticles and M1 macrophage membrane vesicles; the nanoparticles are wrapped by M1 macrophage membrane vesicles by using a microfluidic optical perforation chip; the nanoparticles are obtained by combining dTRIM24 and Fe3O4 magnetic nanoparticles.

[0006] In one embodiment, the preparation method of the nanoparticles comprises the following steps: combining dTRIM24 and Fe3O4 magnetic nanoparticles by using a double-emulsion solvent evaporation method to obtain nanoparticles.

[0007] In one embodiment, the mass ratio of the nanoparticles and M1 macrophage membrane vesicles is 1-10:1-2.

[0008] In another aspect, the present application provides a preparation method of the above-mentioned anti-atherosclerosis nanoparticles based on dTRIM24 biocompatibility (biomimetic nanoparticles), comprising the following steps:

[0009] Preparation of M1 macrophage membrane vesicles; the nanoparticles are coated by M1 macrophage membrane vesicles by using a microfluidic optical perforation chip to obtain biomimetic nanoparticles.

[0010] In an embodiment, the method for preparing the M1 macrophage membrane vesicles comprises the following steps: destroying M1 macrophages by a hypotonic lysis buffer and repeated freezing and thawing, collecting cell membranes by ultracentrifugation, and extruding the cell membranes by a micro-extruder to obtain the M1 macrophage membrane vesicles.

[0011] In an embodiment, the method for preparing the M1 macrophage membrane vesicles comprises the following steps: destroying M1 macrophages by a hypotonic lysis buffer and repeated freezing and thawing, collecting cell membranes by ultracentrifugation, and extruding the cell membranes by a micro-extruder to obtain the M1 macrophage membrane vesicles.

[0012] The application further provides a use of the biomimetic nanoparticles or the biomimetic nanoparticles prepared by the method in preparation of a product for treating atherosclerosis.

[0013] Compared with the prior art, the anti-atherosclerotic nanoparticles (i.e., biomimetic nanoparticles) based on dTRIM24 biocompatibility provided by the application can solve the problems of poor specificity and low efficiency, greatly improve the targeting and biological homology of anti-atherosclerotic plaques, improve the efficiency of anti-atherosclerosis, and achieve the purpose of preventing and treating the occurrence and development of atherosclerosis-related diseases. Compared with ordinary sensitive modification materials, the metal magnetic nanoparticles have a very small size (particle size of 1-100 nm). The surface effect of the nanomaterials makes the metal magnetic nanoparticle modification material have a very large specific surface area and a very large effective contact area with reactants, which improves the transportation efficiency of drugs in the body. DETAILED DESCRIPTION

[0014] The application will be further described in detail below with reference to specific examples, which are not used to limit the application but only to illustrate the application. Unless otherwise specified, the experimental methods used in the following examples are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial channels.

[0015] In the application:

[0016] The nano-MM is an M1 macrophage membrane vesicle;

[0017] The MNP is a Fe3O4 magnetic nanoparticle;

[0018] The dTRIM24 is a selective bifunctional TRIM24 degradation agent based on the PROTAC technology, and has a CAS number of 2170695-14-2;

[0019] The DMSO refers to dimethyl sulfoxide;

[0020] The PVA refers to polyvinyl alcohol;

[0021] The cell membrane protein extraction kit uses the Biyun Tian cell membrane protein extraction kit.

[0022] LPS refers to lipopolysaccharide (Lipopolysaccharide);

[0023] PMSF refers to phenylmethylsulfonyl fluoride, which is a protease inhibitor;

[0024] PBS refers to PBS buffer;

[0025] BCA refers to BCA protein concentration determination kit (BCA Protein Assay Kit).

[0026] Example 1 Nanoparticle preparation:

[0027] First, 1.35 g of FeCl3·6H2O was added to 40 mL of ethylene glycol and dissolved thoroughly, and then 3.6 g of NaAc was added. The mixed solution was stirred vigorously for 1 h, and then was sealed in a 50 mL stainless steel autoclave lined with Teflon, and was reacted at 200℃ for 8 h. The reaction product was washed with ethanol and deionized water several times, and was Fe3O4 magnetic nanoparticles.

[0028] Take 10 mg of Fe3O4 magnetic nanoparticles dissolved in DMSO and put it into a 10 mL centrifuge tube, add 1 mL of dichloromethane, vortex to dissolve the Fe3O4 magnetic nanoparticles thoroughly, as the oil phase. dTRIM24 is dissolved in DMSO to make 10 mg / mL, take 500 μg of dTRIM24 and add it to the oil phase, use a pipette to take 500 μl of ultrapure water as the water phase, intermittent ultrasonic at 80w power (intermittent mode of 2s on and 1s off), ultrasonic the oil phase for 5 min, while adding the water phase, form the primary emulsion, then slowly drop it into 3% mass of polyvinyl alcohol aqueous solution 3 mL with the same power ultrasonic, ultrasonic for 5 min, form the multiple emulsion. The obtained multiple emulsion is added to 0.3% mass of PVA aqueous solution (10 ml), and is magnetically stirred at room temperature in the dark for 4 h to make the organic solvent fully volatilize. Centrifuge at 12000 rpm for 40 min, and then wash with ultrapure water for 3 times, collect the precipitate after the last washing and centrifugation, and directly store in a-20℃ refrigerator.

[0029] Example 2 Preparation of M1 macrophage membrane vesicles (nano-MM):

[0030] High-sugar medium containing LPS 50 ng / mL was prepared, 150 mm dishes were added, M0 macrophages were seeded in the dishes, and cultured for 24 h to induce M1 macrophages. The medium was discarded, and the M1 macrophages were washed twice with PBS. The M1 macrophages were collected, 1 mL of membrane protein extraction solution A containing PMSF was added, and the mixture was mixed and placed in an ice bath for 15 min. The cell suspension was frozen in liquid nitrogen, thawed at room temperature, and repeatedly frozen and thawed 5 times. Then, the mixture was centrifuged at 700 g at 4°C for 10 min. The obtained supernatant was centrifuged at 14,000 g at 4°C for 30 min. The precipitate was mixed with PBS, and the mixture was ultrasonicated for 5 min. The M1 macrophage membrane was quantified by BCA protein, and the M1 macrophage membrane was stored at -80°C.

[0031] The collected cells were washed with cold PBS and then suspended in a hypotonic solution (a mixture of PBS and deionized water in a volume ratio of 1:4). Then, the M1 macrophages were broken by repeated freeze-thawing 3 times, and then the solution was centrifuged at 2000 g at 4°C for 30 min. The enriched supernatant was further ultracentrifuged at 100,000 g for 40 min to collect the cell membrane. The collected cell membrane was resuspended in PBS and gradually extruded through 800 and 400 nm polycarbonate porous membranes by an Avanti mini extruder to produce M1 macrophage membrane vesicles.

[0032] Example 3 Preparation of biomimetic nanoparticle material:

[0033] To obtain the nanoparticle coated with cell vesicles, 50 μg of the nanoparticles prepared in Example 1 and the M1 macrophage membrane vesicles prepared in Example 2 were injected into a microfluidic optical perforation chip. When the mixture of M1 macrophage membrane vesicles and nanoparticles passed through the optical perforation area at an optimal flow rate of 2 mL / h, the mixture was irradiated with a pulsed laser with an optimal laser energy density of 0.12 J / cm 2 The laser pulse promoted the nanoparticles to enter the M1 macrophage membrane vesicles. The M1 macrophage membrane vesicles and nanoparticles were mixed, and then the mixture was repeatedly extruded through a micro-extruder with a 400 nm pore multiple times. The mixture was ultrasonically fused for 3 min to make the nanoparticles coated with M1 macrophage membrane vesicles, forming biomimetic magnetic nanoparticles (denoted as nano-MM / dTRIM24 / MNP).

[0034] Example 4 Animal experiment

[0035] Atherosclerotic mice were divided into two groups: one group was injected with DiR dye (DiR group), and the other group was injected with DiR dye-containing bioparticles (prepared by replacing dTRIM24 with DiR dye in the manner of Examples 1-3) (nano-MM / MNP / DiR group). Twenty-four hours after injection, the quantitative data of the accumulation of DiR fluorescence signal in the aorta were counted (n = 3). The targeting intensity of bioparticles carrying nano-MM in atherosclerotic plaques was determined by the fluorescence intensity of the dye. The fluorescence intensity of the nano-MM / MNP / DiR group was 2.03 times that of the DiR group.

[0036] Atherosclerotic mice were divided into three groups: a blank drug group with no injection, a dTRIM24 particle group injected with dTRIM24 particles, and a nano-MM / dTRIM24 / MNP group injected with the biomimetic nanoparticles prepared in Example 3. The effect of biomimetic nanoparticles on anti-atherosclerotic plaques was determined by the total area of the plaques stained with oil red. Among them, compared with the blank drug group, the plaque area ratio of the dTRIM24 particle group was 0.81, and that of the nano-MM / dTRIM24 / MNP group was 0.42. Thus, the biomimetic nanoparticles had the best effect on plaque removal, reaching 58%.

[0037] The body weight changes of mice in different experimental groups during treatment were compared. The body weight of mice in each group showed an upward trend during treatment. The WBC, RBC, PLT, ALT, AST, BUN, UA, TG, CHO, LDL, and HDL levels of mice were detected after administration in different experimental groups. Among them, there was no significant difference in the changes of blood routine and liver and kidney function indicators, indicating that different experimental groups had no toxicity in mice.

[0038] In order to further study the formation of lipid deposition in atherosclerotic plaques, the aortic root cross-section was stained with oil red. The results showed that a large amount of lipid (32.13%) was deposited in the saline group. Compared with the saline group, the lipid deposition in the nano-MM / dTRIM24 / MNP group was reduced to 18.94%. It can be seen that biomimetic nanoparticles can significantly reduce lipid deposition.

[0039] Example 5 Cell Experiment

[0040] The biomimetic nanoparticles prepared in Example 3 were detected:

[0041] Transmission electron microscopy showed that the biomimetic nanoparticles were spherical, and their outer surfaces were successfully covered with M1 macrophage membrane vesicles. Dynamic light scattering (DLS) showed that the particle size of the biomimetic nanoparticles was about 83 ± 2.4 nm.

[0042] The zeta potential of the biomimetic nanoparticles changed to about -30 mv. The particle size stability results showed that the particle size of the biomimetic nanoparticles remained stable for 72 h, and in contrast, the Pd particle size gradually increased. Through the observation of DLS and zeta potential, it was shown that the biomimetic nanoparticles had good stability. Through SDS-PAGE and Coomassie blue staining, it was confirmed that the membrane protein existed in the biomimetic nanoparticles, which was consistent with the M1 macrophage membrane vesicle, confirming that the biomimetic nanoparticles successfully encapsulated the cell membrane vesicle.

[0043] The uptake of M1 macrophage vesicles was evaluated using laser scanning confocal microscopy (CLSM) and flow cytometry (FACS) analysis. The fluorescent dye DiD was used instead of dTRIM24 nanoparticles, denoted as nano-MM / MNP / DiD (in the manner of Examples 1-3, the fluorescent dye DiD was used instead of dTRIM24 nanoparticles to prepare nanoparticles), which was tracked, and M1 macrophages were incubated with different amounts of nano-MM / MNP / DiD, with the DiD content in the incubation system being 5 μg / mL, 10 μg / mL, and 15 μg / mL, respectively. The CLSM and FACS results showed that when the DiD content reached 10 μg / mL, the uptake reached saturation.

[0044] For the DiD content of 10 μg / mL in the incubation system, the uptake of M1 macrophages was observed at 1 h, 3 h, 6 h, 12 h, and 24 h, and the cell uptake was the largest at 12 h, and there was no significant increase in uptake with the extension of time.

[0045] The uptake of non-specific cells such as 3T3, H9C2, MLE-12, etc. was evaluated. The CLSM and FACS results showed that the uptake of nano-MM / MNP / DiD by 3T3, H9C2, and MLE-12 cells was weak, indicating that nano-MM / MNP / DiD had selectivity for cell uptake.

[0046] The lysosome escape experiment was performed with DiD instead of dTRIM24, and the cell uptake of nano-MM / MNP / DiD was captured by lysosomes at 3 h. CLSM showed that the red fluorescence of nano-MM / MNP / DiD overlapped with the green fluorescence of lysosome markers. After 9 h, a large number of nano-MM / MNP / DiD separated from the lysosomes.

[0047] Live / dead staining was performed at 24 h and 48 h, and CLSM showed that almost all cells in different groups were Calcein-AM positive (green fluorescence), indicating no toxicity. At 24 and 48 h, respectively, CCK8 was added, and the results were measured by a microplate reader. Analysis showed that the survival rate was more than 90%, proving that the biomimetic nanoparticles had no toxicity.

[0048] DiD, nano-MM / MNP / DiD in vivo 24h pharmacokinetic studies showed that 24h after nano-MM / MNP / DiD in the blood still have 40% of the amount of M1 macrophage membrane vesicle nanoparticles significantly prolonged the time of circulation in vivo, cell membrane vesicle nanoparticles in the extension of the cycle, biocompatibility have good performance. Drug accumulation release results show that nano-MM / MNP / DiD in the acidic environment of PH5.0 release is much greater than the neutral environment of PH7.4, in the inflammatory environment around atherosclerotic plaques more conducive to drug release.

[0049] Realtime PCR detection INOS, Arg1 level. After treating M1 macrophages with different amounts of biomimetic nanoparticles, total RNA was extracted from the cells, and the content of biomimetic nanoparticles in M1 macrophages was 5 μg / mL, 10 μg / mL, 15 μg / mL, respectively. Reverse transcription and real-time fluorescence quantitative analysis showed that with the increase of the concentration of biomimetic nanoparticles, INOS decreased more obviously, and Arg1 increased more obviously. It is shown that dTRIM24 can promote M1 to M2 by knocking down TRIM24 gene.

[0050] After treating M1 macrophages with different concentrations of biomimetic nanoparticles, total protein was extracted for protein electrophoresis, and WB showed results consistent with gene expression.

[0051] After treating M1 macrophages with biomimetic nanoparticles for 24h, protein was extracted for WB analysis, and the results showed that Fe3O4 magnetic nanoparticles loaded with dTRIM24 could be better taken up by cells and had more obvious effect.

[0052] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. A biomimetic nanoparticle, characterized in that, The nanometer particle and the M1 macrophage membrane vesicle are combined by using a microfluidic optical perforation chip.

2. The biomimetic nanoparticle of claim 1, wherein, The preparation method of the nanometer particle comprises the following steps: combining dTRIM24 and Fe3O4 magnetic nanometer particles by using a double-emulsified solvent evaporation method to obtain the nanometer particle.

3. The nanoparticle of claim 2, wherein, The mass ratio of the nanometer particle and the M1 macrophage membrane vesicle is 1-10:1-2.

4. The method of producing a biomimetic nanoparticle according to any one of claims 1 to 3, characterized in that, The preparation method of the M1 macrophage membrane vesicle comprises the following steps: destroying M1 macrophages by using a low-osmotic lysis buffer and repeated freezing and thawing, collecting cell membranes by using ultracentrifugation, and extruding the cell membranes by using a micro-extruder to obtain the M1 macrophage membrane vesicle. The preparation method of the M1 macrophage membrane vesicle comprises the following steps: destroying M1 macrophages by using a low-osmotic lysis buffer and repeated freezing and thawing, collecting cell membranes by using ultracentrifugation, and extruding the cell membranes by using a micro-extruder to obtain the M1 macrophage membrane vesicle.

5. The production method according to claim 4, characterized by, The preparation method of the M1 macrophage membrane vesicle comprises the following steps: destroying M1 macrophages by using a low-osmotic lysis buffer and repeated freezing and thawing, collecting cell membranes by using ultracentrifugation, and extruding the cell membranes by using a micro-extruder to obtain the M1 macrophage membrane vesicle.

6. The production method according to claim 4 or 5, characterized by, 7. The use of the biomimetic nanometer particle of any one of claims 1-3 or the biomimetic nanometer particle prepared by the preparation method of any one of claims 4-6 in the preparation of a product for treating atherosclerosis. ​