Nanometer preparation loaded with nucleic acid medicine, preparation method and application

Abstract

The invention discloses a nano preparation loaded with nucleic acid drugs, a preparation method and application. Comprising a composite carrier material and nucleic acid drugs. The composite carrier material comprises an active oxygen responsive and scavenging carrier material, an amphiphilic biological material and polyethyleneimine (PEI); the nucleic acid medicine is a nucleic acid medicine for resisting atherosclerosis. The mass ratio of the active oxygen responsiveness and clearance carrier material to the amphiphilic biological material is (1: 1)-(10: 1), the mass ratio of the active oxygen responsiveness and clearance carrier material to the polyethyleneimine is (100: 1)-(5: 1), and the mass ratio of the active oxygen responsiveness and clearance carrier material to the nucleic acid medicine is (10: 1)-(10000: 1). The particle size of the nano preparation is between 10 nm and 500 nm. The pharmaceutical composition can be applied to treatment of various acute and chronic cardiovascular diseases or oxidative stress related diseases, and the administration modes comprise intravenous injection, oral administration, subcutaneous injection, intramuscular injection and any combination of the administration modes. The nano preparation has a remarkable prevention and treatment effect on cardiovascular diseases such as atherosclerosis.

Description

Technical Field

[0001] The present invention relates to the medical field, and in particular to the composition and preparation method of a nano preparation loaded with nucleic acid drugs and its application in preventing and treating atherosclerosis and various acute and chronic cardiovascular diseases or oxidative stress-related diseases. Background Art

[0002] Atherosclerosis (AS) is caused by the increase in the generation of oxygen free radicals and their peroxidation derivatives, reactive oxygen species (ROS), which in turn leads to an imbalance in the body's oxidation / antioxidation function and oxidative stress damage. [1,2] The oxidative damage of ROS leads to endothelial dysfunction and promotes the production of inflammatory factors. Chronic inflammation caused by oxidative damage leads to the deposition of lipoproteins in the blood under the damaged endothelium. [3,4] After being oxidized and modified, it becomes oxidized low-density lipoprotein (oxLDL). After being activated by oxLDL, endothelial cells express proinflammatory cytokines, chemokines and adhesion molecules, recruiting leukocytes and monocytes [5] Under the stimulation of colony stimulating factor, monocytes enter the intima through activated endothelial cells, differentiate and mature into macrophages, and differentiate into foam cells by phagocytizing oxLDL, inducing the formation of lipid streaks and then forming AS plaques. In addition, the inflammation of the AS vascular wall is driven by both adaptive and innate immune responses. [6] Regulatory T cells (Tregs) have attracted considerable attention as immune regulators in cardiovascular diseases. [7] Treg cells are a type of CD4 + T lymphocyte subsets, whose number and function are impaired, are associated with some autoimmune diseases. Treg cells play an important role in maintaining the body's immune balance. The number of Treg cells in AS patients is significantly reduced, and the imbalance with helper T cells will lead to an increase in pro-inflammatory factors and a decrease in anti-inflammatory factors, which in turn causes plaque instability. The role of Treg cells in cardiovascular diseases has attracted widespread attention from researchers. [8] . Because it is involved in controlling the progression of autoimmune diseases, inhibiting the body's inflammatory response, and maintaining autoimmune tolerance. Treg cells can inhibit the proliferation of autoreactive T cells and reduce the secretion of proinflammatory cytokines by secreting cytokines such as TGF-β, IL-4, and IL-10, thereby regulating the immune response during inflammatory diseases of the nervous system, reducing the inflammatory damage to cells and tissues caused by AS, and helping to maintain the body's immune homeostasis. [9,10]Therefore, by removing ROS, anti-inflammatory and anti-oxidation, the formation of oxLDL and its abnormal accumulation in macrophages can be inhibited, the lipid balance of macrophages can be promoted, and their transformation into foam cells can be inhibited, which is of great significance in the prevention and treatment of AS. [11-14] .

[0003] MicroRNA (miRNA) is a type of endogenous, single-stranded, non-coding RNA. Each miRNA can have multiple target genes, and several miRNAs can regulate the same gene. Therefore, as a very important post-transcriptional regulatory factor, understanding and utilizing its regulatory mechanism is very meaningful for the occurrence and prevention of diseases. Domestic and foreign scholars have found that multiple miRNAs can regulate ABCA1. [15,16] In macrophages and AS animal models, miR-33 [17,18] 、miR758

[19] 、miR-106b

[20] 、miR-27

[21] and miR-145

[22] MiRNAs such as ABCA1 can target and bind to ABCA1, regulating the role of ABCA1 and regulating AS. In addition, overexpression of miRNAs in macrophages and hepatocytes can reduce the efflux of cholesterol to ApoA1, indicating that inhibition of miRNAs may have a protective effect against AS. This suggests that miRNAs have become an important target for the prevention and treatment of AS. [23-25] .

[0004] Based on the existing research background, inspired by oxidative stress and inflammatory diseases, a large number of studies have constructed ROS-responsive drug delivery systems using ROS-responsive groups, such as peroxyoxalates, polythioetherketones, and aromatic borate-containing polymers. [26,27] ROS-responsive drug delivery systems are further used for the diagnosis and treatment of tumors, cardiovascular diseases, and other diseases related to oxidative stress or inflammation.

[28] Therefore, it is very important to select a suitable ROS-responsive drug delivery system to effectively deliver miRNA to the AS lesion site to exert its effect. Summary of the invention

[0005] In view of the above research background and the shortcomings of the prior art, the purpose of the present invention is to provide a composition and preparation method of a nano preparation loaded with nucleic acid drugs. Another purpose of the present invention is to verify the advantages of the nano preparation in preventing and treating atherosclerosis and other diseases related to inflammation or oxidative stress injury by clearing local ROS, targeting atherosclerotic plaque sites and synergistic effects of miRNA.

[0006] To achieve the above-mentioned purpose, the technical solution adopted by the present invention is a nano-preparation loaded with nucleic acid drugs, including a composite carrier material and a nucleic acid drug. The composite carrier material includes an active oxygen responsive and scavenging carrier material, an amphiphilic biomaterial and polyethyleneimine (PEI); the nucleic acid drug is an anti-atherosclerotic nucleic acid drug. The mass ratio of the active oxygen responsive and scavenging carrier material to the amphiphilic biomaterial is between 1:1 and 10:1, the mass ratio of the active oxygen responsive and scavenging carrier material to polyethyleneimine is between 100:1 and 5:1, the mass ratio of the active oxygen responsive and scavenging carrier material to the nucleic acid drug is between 10:1 and 10000:1, and the particle size of the nano-preparation is between 10-500nm.

[0007] Specifically, the amphiphilic biomaterial is selected from one or more of poloxamer 407, poloxamer 188, lecithin and DSPE-PEG2000.

[0008] Specifically, the nucleic acid drug is a nucleic acid drug for targeted treatment of atherosclerosis selected from one or more of the antisense oligonucleotides of miR-33, anti-miR33, miRNA-126 and miRNA-145.

[0009] Specifically, the molecular weight of the polyethyleneimine is 0.6 kDa, 1.8 kDa, 10 kDa or 25 kDa.

[0010] Specifically, the chemical structural formula of the above active oxygen responsive and scavenging carrier material is:

[0011]

[0012] The present invention also provides a composition and preparation method of a nano preparation loaded with nucleic acid drugs, comprising the following steps: firstly, adding an active oxygen responsive and scavenging carrier material and polyethyleneimine into an organic solvent, stirring to fully dissolve them, and preparing an organic phase 1 (0 1 ); then add the nucleic acid drug to sterile ultrapure water to fully dissolve it to prepare an aqueous phase 1 (W 1 ); W 1 Add O 1 The ultrasonic probe is inserted into the water-in-oil (W 1 / O 1 ), then add the amphiphilic biomaterial into sterile ultrapure water, stir and disperse evenly, then 1 / O 1 Slowly add and ultrasonically emulsify for 3 to 7 minutes to make water-in-oil-in-water (W 1 / O 1 / W 2) double nanoemulsion; volatilize at room temperature with magnetic stirring for 3 to 4 hours, remove the organic solvent, centrifuge, collect the nanoparticles, then disperse and wash with deionized water, and freeze-dry to obtain the nanoparticle preparation loaded with nucleic acid drugs.

[0013] Specifically, the concentration of the polyethyleneimine in the aqueous phase is 0.1 to 10 mg / mL, and the ratio of the organic phase to the aqueous phase is between 1:1 and 1:20.

[0014] The organic solvent is selected from dichloromethane, chloroform, tetrahydrofuran or a mixed solvent, wherein the mixed solvent is dichloromethane / chloroform, dichloromethane / DMSO, dichloromethane / tetrahydrofuran, and the ratio is 1 mL: 1 to 10 mL.

[0015] The present invention also includes the use of nano-preparations loaded with nucleic acid drugs in the preparation of drugs for preventing and / or treating atherosclerosis and various acute and chronic cardiovascular diseases or oxidative stress-related diseases.

[0016] The present invention also includes the use of nano-preparations loaded with nucleic acid drugs in the preparation of drugs for increasing the content of ABCA1, ABCG1 and / or increasing the content of Treg cells.

[0017] The administration methods of the above-mentioned drugs include oral administration, intravenous injection, subcutaneous injection, intramuscular injection, external application to the skin, and any combination of the above administration methods.

[0018] The present invention uses atherosclerosis as a model to verify the role of the nanoformulation of the present invention in preventing and treating inflammation-related diseases. The beneficial effects of the present invention are:

[0019] (1) The anti-atherosclerosis nanoformulation loaded with nucleic acid drugs described in the present invention has a good AS plaque targeting effect.

[0020] (2) The anti-atherosclerosis nanoformulation loaded with nucleic acid drugs described in the present invention can achieve effective loading of nucleic acid drugs.

[0021] (3) The anti-atherosclerosis nanoformulation loaded with nucleic acid drugs described in the present invention has good in vivo biocompatibility, is degradable in the body, and the degradation products have no toxic side effects on the body.

[0022] (4) After intravenous administration, the anti-atherosclerotic nanoformulation loaded with nucleic acid drugs described in the present invention can respond to the high ROS microenvironment of the local inflammatory lesions, and at the same time, through active and passive targeting, it can target macrophages and neutrophils in the AS plaque area.

[0023] (5) After administration, the anti-atherosclerosis nanoformulation loaded with nucleic acid drugs described in the present invention can exert its efficacy through targeting, scavenging local reactive oxygen species and the synergistic effect of miRNA, and its therapeutic effect on atherosclerosis is significantly better than that of other small molecule drug control groups, free miRNA control groups and unloaded blank reactive oxygen response and scavenging carrier material control groups.

[0024] (6) The size of the anti-atherosclerosis nanoformulation loaded with nucleic acid drugs described in the present invention can be regulated by the preparation process parameters. The preparation method is relatively simple and low in cost, and it is easy to realize the industrialization of the nanoformulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 are the transmission electron microscopy, scanning electron microscopy, size distribution, and surface potential images of nucleic acid-loaded anti-atherosclerotic nanoformulations;

[0026] Figure 2 OVE-ANM NP is a nanoparticle loaded with anti-miR-33 oligonucleotide anti-miR33 in 10 mM H 2 O 2 or its release profile in PBS;

[0027] Figure 3 This is the result of the inhibitory effect of OVE-ANM NPs on foam cell production;

[0028] Figure 4 The effects of OVE-ANM NPs on the mRNA expression of ABCA1 and ABCG1 in macrophages were detected by fluorescence quantitative PCR, and the effects of OVE-ANM NPs on the protein content of ABCA1 and ABCG1 in macrophages were determined by Western blot;

[0029] Figure 5 Intravenous injection of OVE-ANM NPs to treat ApoE in AS model - / - After the mice were grown, the results of oil red staining of the whole aorta, brachiocephalic artery and aortic root of the mice were shown;

[0030] Figure 6 Intravenous injection of OVE-ANM NPs to treat ApoE in AS model - / - Immunohistochemical staining results of HE, Masson, CD68, MMP9, INOS, and Arg1 in the aortic root of mice after operation;

[0031] Figure 7 Intravenous injection of OVE-ANM NPs to treat ApoE in AS model - / -Immunofluorescence staining results of cholesterol crystals, ABCA1, ABCG1, FoxP3 and DHE in the aortic root of mice. DETAILED DESCRIPTION

[0032] The content of the invention of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the embodiments of the present invention are only used to illustrate the present invention rather than to limit the present invention. Without departing from the technical concept of the present invention, various replacements and changes made according to common technical knowledge and customary means in the art should be included in the scope of the present invention. The present invention is described in detail below in conjunction with non-limiting embodiments.

[0033] Example 1

[0034] In an ice bath, 2 mg of active oxygen responsive and scavenging carrier material OVE (obtained by esterification of vitamin E with oxalyl chloride) and 0.1 mg of PEI with a molecular weight of 1.8 kDa were added to dichloromethane and stirred to fully dissolve to prepare an organic phase 1 (O 1 ); secondly, 5 nmol anti-miR33 was added to sterile ultrapure water to fully dissolve it to prepare an aqueous phase 1 (W 1 );W1 is added to O 1 The ultrasonic probe was inserted into the diaphragm and the power was 117W to perform ultrasonic emulsification to make water-in-oil (W1 / O 1 ), then add 1 mg DSPE-PEG into sterile ultrapure water, stir and disperse evenly, then 1 / O 1 Slowly add and ultrasonically emulsify for 3 minutes to make water-in-oil-in-water (W 1 / O 1 / W 2 ) double nanoemulsion; volatilize at room temperature with magnetic stirring for 3 to 4 hours, remove the organic solvent, centrifuge, collect the nanoparticles, disperse and wash with deionized water, and freeze-dry to obtain the nanoformulation OVE-ANM NP loaded with nucleic acid drugs with targeted anti-inflammatory and antioxidant effects.

[0035] Example 2

[0036] In an ice bath, 2 mg of active oxygen responsive and scavenging carrier material OVE (obtained by esterification of vitamin E with oxalyl chloride) and 0.4 mg of PEI with a molecular weight of 0.6 kDa were added to dichloromethane and stirred to fully dissolve to prepare an organic phase 1 (O 1 ); secondly, 5 nmol anti-miR33 was added to sterile ultrapure water to fully dissolve it to prepare an aqueous phase 1 (W 1 ); W 1 Add O 1The ultrasonic probe was inserted into the diaphragm and the power was 117W to perform ultrasonic emulsification to make oil-in-water (W 1 / O 1 ), then add 2 mg DSPE-PEG into sterile ultrapure water, stir and disperse evenly, then 1 / O 1 Slowly add and ultrasonically emulsify for 3 minutes to make water-in-oil-in-water (W 1 / O 1 / W 2 ) double nanoemulsion; volatilize at room temperature with magnetic stirring for 3 to 4 hours, remove the organic solvent, centrifuge, collect the nanoparticles, disperse and wash with deionized water, and freeze-dry to obtain a nano-preparation with targeted anti-inflammatory and antioxidant effects loaded with nucleic acid drugs.

[0037] Example 3

[0038] In an ice bath, 4 mg of active oxygen responsive and scavenging carrier material OVE (obtained by esterification of vitamin E with oxalyl chloride) and 0.4 mg of PEI with a molecular weight of 10 kDa were added to chloroform and stirred to fully dissolve to prepare an organic phase 1 (O 1 ); secondly, 5 nmol of anti-miR33 was added to sterile ultrapure water to fully dissolve it to prepare an aqueous phase 1 (W 1 ); W 1 Add O 1 The ultrasonic probe was inserted into the diaphragm and the power was 117W to perform ultrasonic emulsification to make oil-in-water (W 1 / O 1 ), then add 2 mg DSPE-PEG into sterile ultrapure water, stir and disperse evenly, then 1 / O 1 Slowly add and ultrasonically emulsify for 3 minutes to make water-in-oil-in-water (W 1 / O 1 / W 2 ) double nanoemulsion; volatilize at room temperature with magnetic stirring for 3 to 4 hours, remove the organic solvent, centrifuge, collect the nanoparticles, disperse and wash with deionized water, and freeze-dry to obtain a nano-preparation with targeted anti-inflammatory and antioxidant effects loaded with nucleic acid drugs.

[0039] Example 4

[0040] In an ice bath, 4 mg of active oxygen responsive and scavenging carrier material OVE (obtained by esterification of vitamin E with oxalyl chloride) and 0.2 mg of PEI with a molecular weight of 25 kDa were added to chloroform and stirred to fully dissolve to prepare an organic phase 1 (O 1 ); secondly, 5 nmol anti-miR33 was added to sterile ultrapure water to fully dissolve it to prepare an aqueous phase 1 (W 1 ); W1 Add O 1 The ultrasonic probe was inserted into the diaphragm and the power was 117W to perform ultrasonic emulsification to make oil-in-water (W 1 / O 1 ), then add 1 mg of DSPE-PEG into sterile ultrapure water, stir and disperse evenly, then 1 / O 1 Slowly add and ultrasonically emulsify for 3 minutes to make water-in-oil-in-water (W 1 / O 1 / W 2 ) double nanoemulsion; volatilize at room temperature with magnetic stirring for 3 to 4 hours, remove the organic solvent, centrifuge, collect the nanoparticles, disperse and wash with deionized water, and freeze-dry to obtain a nano-preparation with targeted anti-inflammatory and antioxidant effects loaded with nucleic acid drugs.

[0041] Example 5

[0042] In an ice bath, 2 mg of active oxygen responsive and scavenging carrier material OVE (obtained by esterification of vitamin E with oxalyl chloride) and 0.1 mg of PEI with a molecular weight of 25 kDa were added to dichloromethane and stirred to fully dissolve to prepare an organic phase 1 (O 1 ); secondly, 5 nmol of anti-miR33 was added to sterile ultrapure water to fully dissolve it to prepare an aqueous phase 1 (W 1 ); W 1 Add O 1 The ultrasonic probe was inserted into the diaphragm and the power was 117W to perform ultrasonic emulsification to make oil-in-water (W 1 / O 1 ), then add 1 mg of DSPE-PEG into sterile ultrapure water, stir and disperse evenly, then 1 / O 1 Slowly add and ultrasonically emulsify for 3 minutes to make water-in-oil-in-water (W 1 / O 1 / W 2 ) double nanoemulsion; volatilize at room temperature with magnetic stirring for 3 to 4 hours, remove the organic solvent, centrifuge, collect the nanoparticles, disperse and wash with deionized water, and freeze-dry to obtain a nano-preparation with targeted anti-inflammatory and antioxidant effects loaded with nucleic acid drugs.

[0043] Example 6

[0044] In an ice bath, 4 mg of active oxygen responsive and scavenging carrier material OVE (obtained by esterification of vitamin E with oxalyl chloride) and 0.1 mg of PEI with a molecular weight of 1.8 kDa were added to tetrahydrofuran and stirred to fully dissolve to prepare an organic phase 1 (O 1); secondly, 5 nmol anti-miR33 was added to sterile ultrapure water to fully dissolve it to prepare an aqueous phase 1 (W 1 ); W 1 Add O 1 The ultrasonic probe was inserted into the diaphragm and the power was 117W to perform ultrasonic emulsification to make oil-in-water (W 1 / O 1 ), then add 1 mg of DSPE-PEG into sterile ultrapure water, stir and disperse evenly, then 1 / O 1 Slowly add and ultrasonically emulsify for 3 minutes to make water-in-oil-in-water (W 1 / O 1 / W 2 ) double nanoemulsion; volatilize at room temperature with magnetic stirring for 3 to 4 hours, remove the organic solvent, centrifuge, collect the nanoparticles, disperse and wash with deionized water, and freeze-dry to obtain a nano-preparation with targeted anti-inflammatory and antioxidant effects loaded with nucleic acid drugs.

[0045] Example 7

[0046] In an ice bath, 4 mg of active oxygen responsive and scavenging carrier material OVE (obtained by esterification of vitamin E with oxalyl chloride) and 0.1 mg of PEI with a molecular weight of 1.8 kDa were added to tetrahydrofuran and stirred to fully dissolve to prepare an organic phase 1 (O 1 ); secondly, 5 nmol anti-miR33 was added to sterile ultrapure water to fully dissolve it to prepare an aqueous phase 1 (W 1 );W1 is added to O 1 The ultrasonic probe was inserted into the diaphragm and the power was 117W to perform ultrasonic emulsification to make oil-in-water (W 1 / O 1 ), then add 1 mg of poloxamer 407 into sterile ultrapure water, stir and disperse evenly, then 1 / O 1 Slowly add and ultrasonically emulsify for 5 minutes to make water-in-oil-in-water (W 1 / O 1 / W 2 ) double nanoemulsion; volatilize at room temperature with magnetic stirring for 3 to 4 hours, remove the organic solvent, centrifuge, collect the nanoparticles, disperse and wash with deionized water, and freeze-dry to obtain a nano-preparation with targeted anti-inflammatory and antioxidant effects loaded with nucleic acid drugs.

[0047] Example 8

[0048] In an ice bath, 10 mg of active oxygen responsive and scavenging carrier material OVE (obtained by esterification of vitamin E with oxalyl chloride) and 0.1 mg of PEI with a molecular weight of 1.8 kDa were added to tetrahydrofuran and stirred to fully dissolve to prepare an organic phase 1 (O 1 ); secondly, 5 nmol anti-miR33 was added to sterile ultrapure water to fully dissolve it to prepare an aqueous phase 1 (W 1 ); W 1 Add O 1 The ultrasonic probe was inserted into the diaphragm and the power was 117W to perform ultrasonic emulsification to make oil-in-water (W 1 / O 1 ), then add 1 mg of lecithin into sterilized ultrapure water, stir and disperse evenly, then 1 / O 1 Slowly add and ultrasonically emulsify for 3 minutes to make water-in-oil-in-water (W 1 / O 1 / W 2 ) double nanoemulsion; volatilize at room temperature with magnetic stirring for 3 to 4 hours, remove the organic solvent, centrifuge, collect the nanoparticles, disperse and wash with deionized water, and freeze-dry to obtain a nano-preparation with targeted anti-inflammatory and antioxidant effects loaded with nucleic acid drugs.

[0049] Example 9

[0050] In an ice bath, 4 mg of active oxygen responsive and scavenging carrier material OVE (obtained by esterification of vitamin E with oxalyl chloride) and 0.1 mg of PEI with a molecular weight of 1.8 kDa were added to tetrahydrofuran and stirred to fully dissolve to prepare an organic phase 1 (O 1 ); Secondly, 5nmol miRNA-126 was added to sterile ultrapure water to fully dissolve it to prepare an aqueous phase 1 (W 1 );W1 is added to O 1 The ultrasonic probe was inserted into the diaphragm and the power was 117W to perform ultrasonic emulsification to make oil-in-water (W 1 / O 1 ), then add 2 mg of lecithin into sterilized ultrapure water, stir and disperse evenly, then slowly add W1 / O1, and after ultrasonic emulsification for 5 minutes, make water-in-oil-in-water (W 1 / O 1 / W 2 ) double nanoemulsion; volatilize at room temperature with magnetic stirring for 3 to 4 hours, remove the organic solvent, centrifuge, collect the nanoparticles, disperse and wash with deionized water, and freeze-dry to obtain a nano-preparation with targeted anti-inflammatory and antioxidant effects loaded with nucleic acid drugs.

[0051] Example 10

[0052] In an ice bath, 4 mg of active oxygen responsive and scavenging carrier material OVE (obtained by esterification of vitamin E with oxalyl chloride) and 0.1 mg of PEI with a molecular weight of 1.8 kDa were added to tetrahydrofuran and stirred to fully dissolve to prepare an organic phase 1 (O 1 ); secondly, 5 nmol miRNA-145 was added to sterile ultrapure water to fully dissolve it to prepare an aqueous phase 1 (W 1 ); W 1 Add O 1 The ultrasonic probe was inserted into the diaphragm and the power was 117W to perform ultrasonic emulsification to make oil-in-water (W 1 / O 1 ), then add 1 mg of lecithin into sterilized ultrapure water, stir and disperse evenly, then 1 / O 1 Slowly add and ultrasonically emulsify for 7 minutes to make water-in-oil-in-water (W 1 / O 1 / W 2 ) double nanoemulsion; volatilize at room temperature with magnetic stirring for 3 to 4 hours, remove the organic solvent, centrifuge, collect the nanoparticles, disperse and wash with deionized water, and freeze-dry to obtain a nano-preparation with targeted anti-inflammatory and antioxidant effects loaded with nucleic acid drugs.

[0053] Embodiment 11

[0054] In an ice bath, 4 mg of active oxygen responsive and scavenging carrier material OVE (obtained by esterification of vitamin E with oxalyl chloride) and 0.1 mg of PEI with a molecular weight of 25 kDa were added to tetrahydrofuran and stirred to fully dissolve to prepare an organic phase 1 (O 1 ); secondly, 5 nmol anti-miR33 was added to sterile ultrapure water to fully dissolve it to prepare an aqueous phase 1 (W 1 ); W 1 Add O 1 The ultrasonic probe was inserted into the diaphragm and the power was 117W to perform ultrasonic emulsification to make oil-in-water (W 1 / O 1 ), then add 1 mg of poloxamer 188 into sterile ultrapure water, stir and disperse evenly, then 1 / O 1 Slowly add and ultrasonically emulsify for 7 minutes to make water-in-oil-in-water (W 1 / O 1 / W 2 ) double nanoemulsion; volatilize at room temperature with magnetic stirring for 3 to 4 hours, remove the organic solvent, centrifuge, collect the nanoparticles, disperse and wash with deionized water, and freeze-dry to obtain a nano-preparation with targeted anti-inflammatory and antioxidant effects loaded with nucleic acid drugs.

[0055] Figure 1 The invention discloses an anti-atherosclerotic nanoparticle OVE-ANM NP loaded with nucleic acid drugs prepared by the above method, wherein the anti-inflammatory and antioxidant active oxygen response and scavenging carrier material is OVE, which is obtained by esterification of vitamins and oxalyl chloride under the catalysis of N,N-diisopropylethylamine (DIPEA), the cationic non-viral transfection carrier is PEI with a molecular weight of 1.8 kDa, the organic solvent is dichloromethane, the miRNA targeting atherosclerotic plaques is miR33 antagonist nucleotide anti-miR33, and the prepared OVE-ANM NP is a relatively regular spherical shape with uniform morphology and size and uniform dispersion. The particle size measurement results show that the average particle size of OVE-ANM NP is 191.6±2.26nm, and the surface potential is 21.37±1.10mV.

[0056] Figure 2 OVE-ANM NP is a nanoparticle loaded with anti-miR-33 oligonucleotide anti-miR33 in 10 mM H 2 O 2 Or its release curve in PBS. The results show that the anti-atherosclerosis nanoformulation loaded with nucleic acid drugs prepared by the above method has significant ROS responsiveness and can slowly release the loaded nucleic acid drugs to exert their effects.

[0057] Figure 3 This is the result of the inhibitory effect of OVE-ANM NP on foam cell formation. The brief experimental steps are as follows: After stimulating the cells of each group except the Control group with LPS for 24 hours, anti-miR33 (ANM33), OVENP and OVE-ANM NP solution prepared in complete culture medium were added to the cells for 2 hours. The Model group only added culture medium, and then oxLDL was added to treat the cells for 48 hours. The cells of each group were stained with oil red and observed under a light microscope for photography. The results show that OVE-ANM NP can inhibit the generation of foam cells derived from macrophages and vascular smooth muscle, inhibit the development of atherosclerosis, and have an inhibitory effect on atherosclerosis.

[0058] Figure 4The effect of OVE-ANM NP on the mRNA expression of ABCA1 and ABCG1 in macrophages was detected by fluorescence quantitative PCR, and the effect of OVE-ANM NP on the protein content of ABCA1 and ABCG1 in macrophages was determined by Western blot. The results show that the mRNA expression of ABCA1 and ABCG1 (Figure A and Figure B) increased significantly after OVE-ANM NP treatment, and the effect was significantly better than the free anti-miR33 group and the single reactive oxygen response and scavenging carrier material group. At the same time, Western blot detection also showed that the protein expression of ABCA1 and ABCG1 (Figure C) increased significantly after OVE-ANM NP treatment relative to the Control group, and the effect was significantly better than the free anti-miR33 group and the single reactive oxygen response and scavenging carrier material group. The results show that OVE-ANM NP can play a therapeutic role in AS by upregulating the expression of ABCA1 and ABCG1. The Western blot and PCR experiments in the present invention all adopt conventional experimental operation steps in the art.

[0059] Figure 5 OVE-ANM NPs were injected into the tail vein to treat ApoE in AS model - / - After the mice were treated, the oil red staining and quantitative statistical results of the whole aorta, brachiocephalic artery and aortic root of the mice were performed (Figure A and Figure B). The brief experimental steps are as follows: ApoE - / - After the mice were fed a high-fat diet for 1 month, they were given ANM33, OVE NP or OVE-ANM NP for 2 months while continuing to be fed a high-fat diet. The mice were euthanized and the whole aorta of the mice was dissected for oil red staining. The brachiocephalic artery and aortic root were frozen and stained with oil red, and then observed and photographed under a light microscope. The results showed that after OVE-ANM NP treatment, compared with the model group, OVE-ANM NP could significantly reduce the plaque area of ​​the whole aorta, brachiocephalic artery and aortic root of AS mice, indicating that it has a significant targeted inhibitory effect on AS.

[0060] Figure 6 OVE-ANM NPs were injected into the tail vein to treat ApoE in AS model - / - The immunohistochemical staining results of HE, Masson, CD68, MMP-9, INOS and Arg1 in the aortic root of mice were shown in Figure 2. - / -After paraffin sectioning of the mouse aorta root, immunohistochemical staining of HE, Masson, CD68, MMP-9, INOS or Arg1 was performed, and the sections were observed and photographed under a light microscope. The results showed that compared with the model group and the free miRNA group, OVE-ANM NP treatment could significantly reduce the necrotic core and plaque infiltration, increase the collagen content in the plaque site, effectively reduce the macrophage content and matrix metalloproteinase content in the plaque site, and significantly reduce the INOS content and increase the Arg1 content. The above results all indicate that OVE-ANM NP has a significant inhibitory effect on AS.

[0061] Figure 7 OVE-ANM NPs were injected into the tail vein to treat ApoE in AS model - / - The immunofluorescence staining results of FoxP3 (green), ABCA1 (red), ABCG1 (red), cholesterol crystals (white) and DHE (red) in the aortic root of mice were shown in Figure 2. - / - The aortic root of mice was fixed by frozen sections, and FoxP3, ABCA1 and ABCG1 antibodies were used as primary antibodies, and DAPI staining was performed after fluorescent labeling of secondary antibodies, and CLSM observation and photography were performed; DHE staining was performed by staining the sections with a DHE solution at a concentration of 2μM, and CLSM observation and photography were performed; the cholesterol crystals in the aortic root were directly observed and photographed by CLSM reflected light technology using frozen sections of the aortic root. The above results show that after OVE-ANM NP treatment, compared with the model group, it can significantly reduce the formation of cholesterol crystals in the aortic root, increase the content of ABCA1, ABCG1 and the content of Treg cells, and inhibit the generation of ROS in the aortic root. The above results all indicate that OVE-ANM NP can delay the development of AS and has a significant inhibitory effect on AS.

[0062] That is to say, the anti-atherosclerosis nanoformulation loaded with nucleic acid drugs of the present invention can reach the AS lesion site through its ROS responsive group and miRNA to play a targeting role together, release anti-inflammatory antioxidant drugs and miRNA under local high active oxygen conditions, reduce the possibility of drug leakage in the body circulation, and enhance the targeting of the nanoformulation. At the same time, the anti-atherosclerosis nanoformulation loaded with nucleic acid can exert its efficacy through the synergistic effect of clearing local active oxygen, targeting inflammatory cells and anti-inflammatory antioxidant drug immunomodulatory Treg cells, and the therapeutic effect on asthma, heart failure, myocardial ischemia / reperfusion injury, ischemic stroke, inflammatory bowel inflammation, non-alcoholic fatty liver, gallbladder disease and atherosclerosis is significantly better than other small molecule drug control groups, free miRNA control groups and unloaded blank active oxygen response and scavenging carrier material control groups.

[0063] References

[0064] [1]H. Li, Horke S., U. Oxidative stress in vascular disease and its pharmacological prevention[J]. Trends in pharmacological sciences, 2013, 34(6): 313 - 319.

[0065] [2]U. Nitric oxide and oxidative stress in vascular disease[J]. Pflugers Archiv: European journal of physiology, 2010, 459(6): 923 - 939.

[0066] [3]C. Chrysohoou, Kollia N., Tousoulis D. The link between depression and atherosclerosis through the pathways of inflammation and endothelium dysfunction[J]. Maturitas, 2018, 109: 1 - 5.

[0067] [4]R. Stocker, Keaney J. F., Jr. Role of oxidative modifications in atherosclerosis[J]. Physiological reviews, 2004, 84(4): 1381 - 1478.

[0068] [5]A. V. Poznyak, Bharadwaj D., Prasad G., et al. Anti - inflammatory therapy for atherosclerosis: focusing on cytokines[J]. International journal of molecular sciences, 2021, 22(13).

[0069] [6]A. Arce - Sillas, -Luquín D.D.,Tamaya-Domínguez B.,etal.Regulatory T cells:molecular actions on effector cells in immuneregulation[J].Journal of immunology research,2016,2016:1720827.

[0070] [7]A.J.Ali,Makings J.,Ley K.Regulatory T cell stability andplasticity in atherosclerosis[J].Cells,2020,9(12).2665:1-15.

[0071] [8]X.Meng,Yang J.,Dong M.,et al.Regulatory T cells in cardiovasculardiseases[J].Nature reviews Cardiology,2016,13(3):167-179.

[0072] [9]H.Ait-Oufella,Taleb S.,Mallat Z.,et al.Cytokine network and T cellimmunity in atherosclerosis[J].Seminars in immunopathology,2009,31(1):23-33.

[0073]

[10] Z.Mallat,Tedgui A.Immunomodulation to combat atherosclerosis:thepotential role of immune regulatory cells[J].Expert opinion on biologicaltherapy,2004,4(9):1387-1393.

[0074]

[11] S.Yang,Yuan H.Q.,Hao Y.M.,et al.Macrophage polarization inatherosclerosis[J].Clinica chimica acta;international journal of clinicalchemistry,2020,501:142-146.

[0075]

[12] X.Feng,Chen W.,Ni X.,et al.Metformin,macrophagedysfunction andAtherosclerosis[J].Frontiers in immunology,2021,12:682853.

[0076]

[13] G.J.Koelwyn,Corr E.M.,Erbay E.,et al.Regulation of macrophageimmunometabolismin atherosclerosis[J].Nature immunology,2018,19(6):526-537.

[0077]

[14] L.Groh,Keating S.T.,Joosten L.A.B.,et al.Monocyte and macrophageimmunometabolism in atherosclerosis[J].Seminars in immunopathology,2018,40(2):203-214.

[0078]

[15] S.Kalayinia,Arjmand F.,Maleki M.,et al.MicroRNAs:roles incardiovascular development and disease[J].Cardiovascular pathology:theofficial journal of the Society for Cardiovascular Pathology,2021,50:107296.

[0079]

[16] S.S.Zhou,Jin J.P.,Wang J.Q.,et al.miRNAS in cardiovasculardiseases:potential biomarkers,therapeutic targets and challenges[J].Actapharmacologica Sinica,2018,39(7):1073-1084.

[0081]

[17] M.S.Afonso,Sharma M.,Schlegel M.,et al.miR-33 silencingreprograms the immune cell landscape in atherosclerotic Plaques[J].Circulation research,2021,128(8):1122-1138.

[0082]

[18] M.Ouimet,Koster S.,Sakowski E.,et al.Mycobacterium tuberculosisinduces the miR-33 locus to reprogram autophagy and host lipid metabolism[J].Nature immunology,2016,17(6):677-686.

[0085]

[19] I.J.Teng,Tsai M.C.,Shih S.F.,et al.Chalcone Derivatives enhanceATP-Binding cassette transporters A1 in human THP-1 macrophages[J].Molecules(Basel,Switzerland),2018,23(7).

[0087]

[20] A.Finardi,Diceglie M.,Carbone L.,et al.Mir106b-25 and Mir17-92are crucially involved in the development of experimental neuroinflammation[J].Frontiers in neurology,2020,11:912.

[0089]

[21] M.Zhang,Wu J.F.,Chen W.J.,et al.MicroRNA-27a / b regulates cellularcholesterol efflux,influx and esterification / hydrolysis in THP-1 macrophages[J].Atherosclerosis,2014,234(1):54-64.

[0091]

[22] M.H.Kang,Zhang L.H.,Wijesekara N.,et al.Regulation of ABCA1protein expression and function in hepatic and pancreatic islet cells by miR-145[J].Arteriosclerosis,thrombosis,and vascular biology,2013,33(12):2724-2732.

[0093]

[23] M.W.Feinberg,Moore K.J.MicroRNA regulation of atherosclerosis[J].Circulation research,2016,118(4):703-720.

[0094]

[24] Y.Lu,Thavarajah T.,Gu W.,et al.Impact of miRNA in Atherosclerosis[J].Arteriosclerosis,thrombosis,and vascular biology,2018,38(9):e159-e170.

[0096]

[25] F.Zhang,Zhang R.,Zhang X.,et al.Comprehensive analysis of circRNAexpression pattern and circRNA-miRNA-mRNA network in the pathogenesis ofatherosclerosis in rabbits[J].Aging,2018,10(9):2266-2283.

[0098]

[26] B.L.Allen,Johnson J.D.,Walker J.P.Encapsulation and enzyme-mediated release of molecular cargo in polysulfide nanoparticles[J].ACS nano,2011,5(6):5263-5272.

[0099]

[27] D.S.Wilson,Dalmasso G.,Wang L.,et al.Orally delivered thioketalnanoparticles loaded with TNF-α-siRNA target inflammation and inhibit geneexpression in the intestines[J].Nature materials,2010,9(11):923-928.

[0101]

[28] G.Saravanakumar,Lee J.,Kim J.,et al.Visible light-induced singletoxygen-mediated intracellular disassembly of polymeric micelles co-loadedwith a photosensitizer and an anticancer drug for enhanced photodynamictherapy[J].Chemical communications(Cambridge,England),2015,51(49):9995-9998.

Claims

1. A nanoparticle preparation loaded with nucleic acid drugs, characterized in that: Active oxygen responsiveness and scavenging carrier material, amphiphilic biomaterial, polyethyleneimine and nucleic acid drugs, the mass ratio of the active oxygen responsiveness and scavenging carrier material to the amphiphilic biomaterial is between 1:1 and 10:1, the mass ratio of the active oxygen responsiveness and scavenging carrier material to polyethyleneimine is between 100:1 and 5:1, the mass ratio of the active oxygen responsiveness and scavenging carrier material to the nucleic acid drugs is between 10:1 and 10000:1, and the particle size of the nano preparation is between 10-500nm.

2. A nanoparticle preparation loaded with nucleic acid drugs according to claim 1, characterized in that: The amphiphilic biomaterial is selected from one or more of poloxamer 407, poloxamer 188, lecithin and DSPE-PEG2000.

3. The nanoparticle preparation loaded with nucleic acid drugs according to claim 1, characterized in that: The nucleic acid drug is a nucleic acid drug for targeted treatment of atherosclerosis and is selected from one or more of the antisense oligonucleotide anti-miR33 of miR-33, miRNA-126 and miRNA-145.

4. The nanoparticle preparation loaded with nucleic acid drugs according to claim 1, characterized in that: The molecular weight of the polyethyleneimine is 0.6 kDa, 1.8 kDa, 10 kDa or 25 kDa.

5. The nanoparticle preparation loaded with nucleic acid drugs according to claim 1, characterized in that: The chemical structural formula of the active oxygen responsive and scavenging carrier material is:

6. The method for preparing the nanoformulation loaded with nucleic acid drugs according to any one of claims 1 to 5, characterized in that: The following steps are involved: Active oxygen responsive and scavenging carrier materials and polyethyleneimine are added to an organic solvent, stirred to fully dissolve them, and an organic phase 1 (O1) is prepared; nucleic acid drugs are added to sterile ultrapure water, and fully dissolved to prepare an aqueous phase 1 (W1); W1 is added to O1, and an ultrasonic probe is inserted to perform ultrasonic emulsification to prepare oil-in-water (W1 / O1), and then the amphiphilic biomaterial is added to the sterile ultrapure water, stirred and dispersed evenly, and then W1 / O1 is slowly added, and ultrasonic emulsification is performed for 3 to 7 minutes to prepare a water-in-oil-in-water (W1 / O1 / W2) double nanoemulsion; magnetic stirring is performed at room temperature for 3 to 4 hours to evaporate, the organic solvent is removed, and the nanoparticles are collected by centrifugation, and then dispersed and washed with deionized water, and freeze-dried to obtain a nano-preparation loaded with nucleic acid drugs.

7. The method for preparing a nanoparticle preparation loaded with nucleic acid drugs according to claim 6, characterized in that: The concentration of the polyethyleneimine in the aqueous phase is 0.1-10 mg / mL, and the ratio of the organic phase to the aqueous phase is between 1:1 and 1:

20.

8. The method for preparing a nanoparticle preparation loaded with nucleic acid drugs according to claim 6, characterized in that: The organic solvent is selected from dichloromethane, chloroform, tetrahydrofuran or a mixed solvent, and the mixed solvent is dichloromethane / chloroform, dichloromethane / DMSO, dichloromethane / tetrahydrofuran, and the ratio is 1 mL: 1-10 mL.

9. Use of the nanoformulation loaded with nucleic acid drugs according to any one of claims 1 to 5 in the preparation of drugs for preventing and / or treating atherosclerosis and various acute and chronic cardiovascular diseases or oxidative stress-related diseases.

10. Use of the nanoformulation loaded with nucleic acid drugs according to any one of claims 1 to 5 in the preparation of drugs for increasing the content of ABCA1, ABCG1 and / or increasing the content of Treg cells.

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