Use of a drug-loaded nanoparticle in the preparation of a drug for preventing or treating atherosclerosis

Abstract

The application belongs to the field of medicine, and specifically provides application of drug-loaded nanoparticles in preparation of medicine for preventing or treating atherosclerosis, wherein the drug-loaded nanoparticles comprise: (1) a polyamino acid carrier, wherein a nitric oxide donor is grafted on the polyamino acid carrier; and (2) a nucleic acid drug for silencing Camk2g gene expression, which is encapsulated in the polyamino acid carrier. The drug-loaded nanoparticles target the nucleic acid drug for silencing Camk2g gene expression to be delivered into plaque macrophages, restore the phagocytosis function of the macrophages, and then improve the microenvironment inside the plaque; meanwhile, the drug-loaded nanoparticles can also deliver the nitric oxide donor to the macrophages, and the nitric oxide donor reacts with inducible nitric oxide synthase in the macrophages to generate nitric oxide. Nitric oxide gas molecules diffuse to the vicinity of damaged endothelial cells, repair the endothelial cell barrier, restore the integrity of the vascular endothelial barrier, and play a combined treatment role, thereby achieving efficient treatment of atherosclerosis.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to the application of drug-loaded nanoparticles in the preparation of medicines for preventing or treating atherosclerosis. Background Art

[0002] In recent years, the global prevalence of cardiovascular disease has become increasingly severe. Characterized by high prevalence, disability, and mortality rates, cardiovascular diseases pose a serious threat to human health. There are many types of cardiovascular disease, such as coronary heart disease, myocardial infarction, heart failure, and stroke, all of which can lead to serious life and health consequences. However, these diseases all share a key and fundamental cause: atherosclerosis.

[0003] The pathogenesis of atherosclerosis is complex, and macrophages play a crucial role in the pathological progression of atherosclerosis. In the early stages of the disease, monocytes differentiate into macrophages in large numbers, phagocytizing low-density lipoproteins (oxLDL) in the intima and excreting them. However, when macrophages consume too much lipid and are unable to metabolize and excrete it in a timely manner, they transform into foam cells, which undergo apoptosis or necrosis, ultimately forming a growing "necrotic core" in the intima composed of cholesterol esters, cholesterol crystals, and cellular debris. In the middle and late stages of the disease, occlusive intraluminal thrombosis may develop, leading to serious consequences such as myocardial infarction, stroke, and sudden cardiac death. Furthermore, lymphocytes such as T cells and B cells also participate in various stages of atherosclerosis development. While effective drug treatment can effectively inhibit disease progression in the early stages, maintaining the stability of the plaque at the lesion becomes more important in the middle and late stages of atherosclerosis treatment, a key component of atherosclerosis treatment.

[0004] Current clinical treatments primarily target lipid-lowering and anti-inflammatory effects. Among lipid-lowering drugs, statins, such as atorvastatin, rosuvastatin, and lovastatin, are commonly used clinically. They are 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, whose primary mechanism of action is to lower total cholesterol and low- and medium-density lipoproteins (LDLs). They can also reduce triglycerides to a certain extent, thereby achieving an anti-atherosclerotic effect. In terms of anti-inflammatory treatment, monoclonal antibodies and protease inhibitors are commonly used clinically. For example, canakinumab, an interleukin-1β (IL-1β) antagonist, has been shown to significantly improve symptoms in patients with atherosclerosis after intervention with canakinumab. Dapansutrile, an inhibitor of the inflammasome NLRP3, has demonstrated safety and tolerability in patients with heart failure and reduced left ventricular ejection fraction in a phase I randomized trial.

[0005] However, after long-term clinical medication, the side effects of statins are constantly emerging, such as myopathy, liver adverse reactions, gastrointestinal reactions, etc., in addition, the drug efficacy of statins is short, and it needs to be taken for life, and it cannot be cured. These disadvantages make the use of statins gradually restricted. Monoclonal antibodies, protease inhibitors and other drugs also have potential hazards such as affecting the immune function of patients and causing inflammatory storm, and the cost of monoclonal antibodies is high, which many patients cannot afford.

[0006] In recent years, in order to avoid the shortcomings of lipid-lowering and anti-inflammatory treatment methods, researchers are also constantly exploring new treatment methods and means. Maintaining the stability of the plaque by regulating the microenvironment of the plaque, or even eliminating the plaque is a very promising research direction. In the microenvironment of atherosclerosis, due to the damaged endothelial barrier, the continuous accumulation of oxLDL, the continuous phagocytosis of oxLDL by macrophages, the transformation of macrophages into foam cells, and finally the apoptosis or necrosis of macrophages, the necrotic core continues to increase. Therefore, regulating macrophages, including inhibiting the recruitment of monocytes and their differentiation into macrophages, inhibiting the proliferation of macrophages, promoting the apoptosis of macrophages, restoring the phagocytic function of macrophages, etc., is gradually becoming an important target for treating atherosclerosis. However, single regulation of macrophages cannot solve the root problem of oxLDL accumulation. How to design and construct a new type of drug-loaded nanoparticle with the function of remodeling the internal microenvironment of the plaque, and improve the effect of treating atherosclerosis, is the technical problem to be solved and the goal to be achieved of the present application. SUMMARY

[0007] In order to overcome the technical problem that the existing drugs have poor treatment effect on atherosclerosis, the present application provides an application of a drug-loaded nanoparticle in the preparation of a drug for preventing or treating atherosclerosis. The drug-loaded nanoparticle targets the delivery of a nucleic acid drug silencing Camk2g gene expression into plaque macrophages, the nucleic acid drug silences Camk2g gene expression, restores the phagocytic function of macrophages, and improves the internal microenvironment of the plaque. At the same time, a nitric oxide donor can also be delivered to macrophages to react with inducible nitric oxide synthase (iNOS) in macrophages to produce nitric oxide. Nitric oxide gas molecules diffuse to the vicinity of damaged endothelial cells, repair the endothelial cell barrier, play a role in combination therapy, and achieve efficient treatment of atherosclerosis.

[0008] The present application provides an application of a drug-loaded nanoparticle in the preparation of a drug for preventing or treating atherosclerosis, wherein the drug-loaded nanoparticle comprises:

[0009] (1) a polyamino acid carrier, wherein the polyamino acid carrier is grafted with a nitric oxide donor;

[0010] (2) a nucleic acid drug silencing Camk2g gene expression, which is encapsulated in the polyamino acid carrier.

[0011] Further, the nitric oxide donor is selected from one or more of arginine, azobenzene glycol enolate, nitroso thiol.

[0012] Among them, azobenzene glycol enolate and isopentyl nitrite are existing known nitric oxide donors, for example, can be defined as any one of CN113244245A specification.

[0013] Further, the nucleic acid drug is siRNA, preferably, the nucleic acid drug is selected from one or more of siCamk2g, siCCR2, siEpsin1 / 2, siScr, siGFP. These nucleic acid drugs can be purchased.

[0014] Further, the polyamino acid carrier has the following structural formula as shown in formula (I):

[0015]

[0016] Among them, m, n is the number average degree of polymerization, y is selected from an integer from 2 to 16;

[0017]

[0018]

[0019] Preferably, n is 10-500, m is 10-200, and n≥m.

[0020] Further, the preparation method of the polyamino acid carrier comprises the following steps:

[0021] (1) lysine with amino acid protecting group is reacted with triphosgene or phosgene in the presence of a solvent to obtain L-lysine-N-carboxyl cyclic anhydride with amino acid protecting group;

[0022] (2) L-lysine-N-carboxyl cyclic anhydride with amino acid protecting group is ring-opening polymerized with monomer as shown in formula (II) in the presence of a solvent, and then the amino acid protecting group is removed to obtain polylysine with alkyl chain;

[0023] (3) arginine with amino acid protecting group is used as grafting monomer to react with amide catalyst in the presence of a solvent, and the reaction product is grafted and polymerized with polylysine with alkyl chain in the presence of an acid binding agent, and then the amino acid protecting group is removed;

[0024] (4) the polymeric product obtained by removing the amino acid protecting group in step (3) is modified with small molecule acid anhydride as shown in formula (III) to obtain the polyamino acid carrier;

[0025]

[0026] wherein, y is selected from an integer from 2 to 16, R2, R3 are independently selected from H or C1-C6 alkyl;

[0027] for or Preferably, the small molecule anhydride is selected from one or more of maleic anhydride (MA), succinic anhydride (SSA) or dimethyl maleic anhydride (DMA).

[0028] Further, the small molecule anhydride is dimethyl maleic anhydride (DMA); wherein, n is from 60 to 120, the grafting ratio of arginine is greater than 0 and less than or equal to 100%; preferably from 25% to 100%, the modification ratio of DMA is greater than 0 and less than or equal to 100%; preferably from 10% to 100%; more preferably, n is 90, the grafting ratio of arginine is 50%, and the modification ratio of DMA is 100%.

[0029] The grafting ratio of arginine is greater than 0 and less than or equal to 100%, including but not limited to 10%, 20%, 30%, 50%, 80%, 100%. The modification ratio of dimethyl maleic anhydride is greater than 0 and less than or equal to 100%, including but not limited to 10%, 20%, 30%, 50%, 80%, 100%.

[0030] Further, the molar ratio between the nitrogen element in the polyamino acid carrier and the phosphorus element in the nucleic acid drug is 0.25-16:1 (preferably 4:1); and / or, the average particle size of the polyamino acid carrier-based nucleic acid drug delivery system is 40-80 nm (preferably 50-60 nm).

[0031] Further, the preparation method of the drug-loaded nanoparticles comprises mixing the polyamino acid carrier and the nucleic acid drug in the presence of an aqueous solution; preferably, the mass ratio of the polyamino acid carrier to the nucleic acid drug is 0.25 μg-0.03 mg:80-500 pmol; preferably, the mixing time is 30 s-5 min.

[0032] Further, the aqueous solution is water, a PBS solution, a glucose solution or a culture medium.

[0033] Further, the medicine further comprises other pharmaceutically acceptable carriers; preferably, the pharmaceutically acceptable excipients are selected from at least one of pharmaceutically acceptable solvents, solubilizers, co-solvents, emulsifiers, coloring agents, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, thickening agents, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retardants, high molecular skeleton materials and film-forming materials.

[0034] Further, the dosage form of the medicine is selected from injection, oral preparation or external preparation; preferably, the injection is selected from injection solution or powder injection; the oral preparation is selected from tablets, solutions, capsules, powders, pills, granules, syrups, suspensions or oral sustained-release preparations; and the external preparation is selected from ointments, sprays or patches.

[0035] Further, the molecular weight of the polyamino acid carrier is 1500-8000 g / mol, for example, the molecular weight of the polyamino acid carrier prepared in Example 1 below is 32397 g / mol.

[0036] Compared with the prior art, the above technical scheme of the present application has the following advantages:

[0037] 1. The application provides a use of drug-loaded nanoparticles in the preparation of a medicine for preventing or treating atherosclerosis, wherein the drug-loaded nanoparticles comprise: (1) a polyamino acid carrier, wherein the polyamino acid carrier is grafted with a nitric oxide donor; and (2) a nucleic acid medicine for silencing Camk2g gene expression, wherein the nucleic acid medicine is encapsulated in the polyamino acid carrier. The drug-loaded nanoparticles target the nucleic acid medicine for silencing Camk2g gene expression into plaque macrophages, the nucleic acid medicine silences the expression of the Camk2g gene, and the phagocytosis function of the macrophages is restored. The macrophages with restored phagocytosis function can remove the necrotic core, thereby improving the microenvironment inside the plaque; meanwhile, the nitric oxide donor can be delivered to the macrophages, and reacts with inducible nitric oxide synthase (iNOS) in the macrophages to produce nitric oxide. The nitric oxide gas molecules diffuse to the vicinity of damaged endothelial cells, repair the endothelial cell barrier, restore the integrity of the vascular endothelial barrier, and have a combined therapeutic effect, thereby achieving efficient treatment of atherosclerosis.

[0038] 2. The application provides the application of the drug-loaded nanoparticles in the preparation of medicines for preventing or treating atherosclerosis, the polyamino acid carrier has the structural formula shown in the following formula (I), the polyamino acid carrier has higher drug loading capacity, and provides arginine as a nitric oxide donor to release nitric oxide in macrophages, so that the drug-loaded nanoparticles have good prevention or treatment capacity for atherosclerosis.

[0039] 3. The application provides the application of the drug-loaded nanoparticles in the preparation of medicines for preventing or treating atherosclerosis, compared with other options of the application, when R1 is selected from

[0040] or the small molecule anhydride is DMA, the transfection efficiency is obviously improved; the reason is that succinic anhydride lacks acid responsiveness, the acid responsiveness of maleic anhydride is weak, the acid responsiveness of dimethyl maleic anhydride is the strongest, and finally the DMA modified PLL-Arg has the strongest lysosome escape capacity and the best transfection performance.

[0041] 4. The application provides the application of the drug-loaded nanoparticles in the preparation of medicines for preventing or treating atherosclerosis, through the research on the influence of the number average polymerization degree of polylysine, the arginine grafting rate and the modification proportion of dimethyl maleic anhydride on the transfection efficiency of the polylysine gene carrier, it is found that the transfection efficiency of the polylysine gene carrier prepared under the condition that the L-lysine polymerization degree is 90, the arginine grafting rate is 50%, and the DMA modification proportion is 100% is the best.

[0042] The average particle size of the nucleic acid drug delivery system based on the polyamino acid carrier is 40-80 nm (preferably 50-60 nm), which is significantly smaller than the particle size (>100 nm) reported in the literature. BRIEF DESCRIPTION OF DRAWINGS

[0043] In order to make the content of the application more easily understood, the application will be further described in detail below according to specific embodiments of the application and in combination with the drawings, in which:

[0044] Figure 1 The nuclear magnetic spectrum of the polyamino acid carrier LAD with different DMA grafting proportions prepared for experimental example 1 is shown in the figure;

[0045] Figure 2 The particle size distribution diagram of the drug-loaded nanoparticles LAD@siCamk2g is shown in the figure; the vertical coordinate Concentration is concentration;

[0046] Figure 3 The transmission electron microscope diagram of the drug-loaded nanoparticles LAD@siCamk2g is shown in the figure;

[0047] Figure 4UV absorption spectrum of polyamino acid carrier LAD, nucleic acid drug siCamk2g and drug-loaded nanoparticle LAD@siCamk2g; ordinate Absorbance: absorbance; abscissa Wavelength: wavelength;

[0048] Figure 5 Gel electrophoresis map of drug-loaded nanoparticles prepared by loading different N / P ratios of nucleic acid drugs based on polyamino acid carriers LAD with different DMA grafting ratios in experimental example 2;

[0049] Figure 6 Confocal imaging map of nitric oxide produced by drug-loaded nanoparticles in macrophages in experimental example 3;

[0050] Figure 7 Detection results of Western-blot for detecting the gene silencing efficiency of drug-loaded nanoparticles;

[0051] Figure 8 Detection results of the effect of drug-loaded nanoparticles on promoting endothelial cell migration;

[0052] Figure 9 Detection results of the in vivo targeting effect of drug-loaded nanoparticles on atherosclerotic mice;

[0053] Figure 10 Detection results of the in vivo treatment effect of drug-loaded nanoparticles on atherosclerotic mice;

[0054] Figure 11 Detection results of in situ editing of macrophages of atherosclerotic mice by drug-loaded nanoparticles;

[0055] Figure 12 Detection results of the repair effect of the endothelial barrier of atherosclerotic mice by drug-loaded nanoparticles in vivo; wherein the ordinate intensity of EB in aorta in B figure is the intensity of EB in the aorta, and * indicates p<0.05. DETAILED DESCRIPTION

[0056] The technical solutions of the present application will be described clearly and completely below in combination with the drawings. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0057] The preparation method of the polyamino acid carrier provided in the present application comprises the following steps:

[0058] (1) Take 0.5-20 g of Nε-benzyloxycarbonyl-L-lysine, preferably 8 g; dissolve in 4-100 mL of anhydrous tetrahydrofuran, preferably 40 mL; then take 0.4-15 g of triphosgene, preferably 4 g; dissolve in 4-100 mL of anhydrous tetrahydrofuran, preferably 20 mL; add dropwise at a constant rate using a constant pressure dropping funnel, and condense backflow at 50-60 °C, preferably 50 °C; when the reaction solution becomes clear and transparent, precipitate with n-hexane, filter the obtained solid, recrystallize from THF / n-hexane = 1:1-4, preferably 1:2; and dry the obtained recrystallized solid at room temperature under reduced pressure to obtain Nε-benzyl-benzyloxycarbonyl-L-lysine-(N-carboxylic cyclic anhydride) (i.e., ZLL(NCA)).

[0059] (2) Take 1-100 mg of solid hexadecylamine in a round-bottom flask, preferably 7 mg; then add 0.3-30 g of ZLL(NCA) solid, preferably 1 g; and 1-50 mL of anhydrous DMF, preferably 5 mL; react at 30-40 °C for 48-96 h, preferably 37 °C for 72 h; then precipitate the reaction product with ice ethyl ether twice to obtain a solid, and dry under vacuum overnight to obtain hexadecylamine-PZLL 90 . Then take the obtained hexadecylamine-PZLL 90 , add 1-50 mL of trifluoroacetic acid to dissolve it thoroughly, preferably 5 mL; then add 2-20 mL of 33% HBr / acetic acid solution, preferably 3 mL; hydrolyze in an ice water bath for 3-6 h, preferably 4 h; precipitate the obtained solution with ice ethyl ether, centrifuge to obtain white solid hexadecylamine-PLL 90 , and dry under vacuum overnight.

[0060] (3) Synthesis of PLL 90 -Arg

[0061] Take 0.2-40 g of Nα-Boc-Nω-(2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl)-L-arginine, preferably 1 g; 0.3-30 g of EDCI, preferably 0.75 g; and 0.2-20 g of HOBT, preferably 0.5 g; dissolve in 5-800 mL of DMF, preferably 20 mL. Then take 0.1-20 g of hexadecylamine-PLL90, preferably 0.5 g; and 0.2-20 g of N,N-diisopropylethylamine, preferably 0.5 g; dissolve in 4-400 mL of deionized water, preferably 10 mL, then add and react at room temperature for 12-96 h, preferably 72 h; dialyze the reaction solution with deionized water (dialysis bag molecular weight cut-off 3500 Da) for 24-96 h, preferably 72 h; and lyophilize to obtain a white solid. Then the white solid is subjected to trifluoroacetic acid to obtain PLL 90 -Arg.

[0062] (4) PLL 90 Synthesis of PLL-Arg-DMA (abbreviation: LAD)

[0063] Take 0.1-10 g of PLL-Arg 90 -Arg, preferably 0.5 g; dissolved in 2-200 mL of NaOH solution with pH = 8.0, preferably 10 mL; then take out more than 0 and less than or equal to 30 g of DMA, preferably 1.5 g; dissolved in 1-20 mL of 2,4-dioxane, preferably 5 mL, and reacted for 4-6 h, preferably 5 h. Take out the dialysis (dialysis bag with a molecular weight cutoff of 3500 Da) for 24-96 h, preferably 72 h; and freeze-drying to obtain PLL 90 -Arg-DMA white solid (polyamino acid carrier).

[0064] By a similar method, maleic anhydride (MA) or succinic anhydride (SSA) is replaced by DMA to prepare maleic anhydride or succinic anhydride modified polymer PLL 90 -Arg-MA (abbreviation: LAM) and PLL 90 -Arg-SSA (abbreviation: LAS).

[0065] The siCamk2g in the application is from Guangzhou Rui Bo Biological Technology Co., Ltd.; the sequence (5'-3') is as follows: 5'-AAC GUG GUA CAU AAU GCU ACA-3'.

[0066] Example 1 Preparation of polyamino acid carrier

[0067] The embodiment provides a preparation method of a polyamino acid carrier, which comprises the following steps:

[0068] (1) Synthesis of ZLL (NCA)

[0069] Take 8 g of Nε-benzylcarbonyl-L-lysine and dissolve it in 40 mL of anhydrous tetrahydrofuran to obtain a reaction bottom liquid. Then take 4 g of triphosgene and dissolve it in 20 mL of anhydrous tetrahydrofuran, and drop it into the reaction bottom liquid at a constant pressure through a dropping funnel at a constant speed, and condense it back to flow at 50°C. When the reaction solution becomes clear and transparent, precipitate it with n-hexane, filter the obtained solid, and recrystallize it in THF / n-hexane = 1:2 (v / v). Dry the recrystallized solid at room temperature under reduced pressure to obtain ZLL (NCA), the Chinese full name of which is Nε-benzyl-benzylcarbonyl-L-lysine-N-carboxylic cyclic anhydride.

[0070] (2) Synthesis of hexadecylamine-PLL

[0071] Take 7 mg of solid hexadecylamine into a round bottom flask; then add 1 g of ZLL (NCA) solid and 5 mL of anhydrous DMF into it, and react at 37°C for 72 h; then precipitate the reaction solution with ice-ethyl ether twice to obtain a solid, which is vacuum dried overnight to obtain hexadecylamine-PLL 90 . Then take the obtained hexadecylamine-PLL 90 , add 5 mL of trifluoroacetic acid to it to dissolve it thoroughly. Then add 3 mL of 33% HBr / acetic acid solution into it, and hydrolyze in an ice-water bath for 4 h; then centrifuge the obtained solution with ice-ethyl ether to obtain white solid hexadecylamine-PLL 90 , which is vacuum dried overnight.

[0072] (3) Synthesis of PLL 90 -Arg

[0073] Take 1 g of Nα-Boc-Nω-(2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl)-L-arginine, 0.75 g of EDCI (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride), and 0.5 g of HOBT (1-hydroxybenzotriazole) to be dissolved in 20 mL of DMF to obtain a reaction solution at room temperature for 30 min. Then take 0.5 g of hexadecylamine-PLL 90 and 0.5 g of N,N-diisopropylethylamine to be dissolved in 10 mL of deionized water, and then added into the above reaction solution and reacted at room temperature for 72 h. The reaction solution is dialyzed (dialysis bag with a molecular weight cut-off of 3500 Da) with deionized water for 72 h; and then freeze-dried to obtain a white solid. Then the white solid is added into 6 mL of trifluoroacetic acid; and stirred at room temperature for 4 h. Then the mixture is precipitated with anhydrous ethyl ether and centrifuged, and the obtained solid is redissolved with DMSO and dialyzed (dialysis bag with a molecular weight cut-off of 3500 Da) for 72 h; then 2 mL of mercaptoethanol is added into it; and stirred at room temperature overnight, and then dialyzed (dialysis bag with a molecular weight cut-off of 3500 Da) for 72 h; and then freeze-dried to obtain PLL 90 -Arg.

[0074] (4) Synthesis of PLL 90 -Arg-DMA (abbreviation: LAD)

[0075] Take 0.5 g of PLL 90 -Arg to be dissolved in 10 mL of a NaOH solution with pH = 8.0 to obtain a reaction solution, and then take 1.5 g of dimethyl maleic anhydride (DMA) to be dissolved in 5 mL of 2,4-dioxane and added into the above reaction solution to react for 5 h. Take out and dialyze (dialysis bag with a molecular weight cut-off of 3500 Da) for 72 h; and then freeze-dried to obtain PLL 90 -Arg-DMA white solid (polyamino acid carrier, abbreviated as “LAD carrier”).

[0076] Example 2 Preparation of polyamino acid carrier

[0077] The present example provides a method for preparing a polyamino acid carrier, which is basically the same as the method of Example 1, except that in step (4), 1.5 g of maleic anhydride (MA) is used instead of dimethyl maleic anhydride (DMA), and finally PLL 90 - Arg-MA white solid (abbreviation: LAM carrier).

[0078] Example 3 Preparation of polyamino acid carrier

[0079] The present example provides a method for preparing a polyamino acid carrier, which is basically the same as the method of Example 1, except that in step (4), 1.5 g of maleic anhydride (MA) is used instead of dimethyl maleic anhydride (DMA), and finally PLL 90 - Arg-MA white solid (abbreviation: LAM carrier).

[0080] Example 4 Preparation of drug-loaded nanoparticles

[0081] The present example provides a method for preparing drug-loaded nanoparticles, comprising the following steps:

[0082] The LAD carrier prepared in Example 1 is dissolved in water to prepare a dispersion liquid with a LAD concentration of 1 mg / mL. 20 μL of the above LAD carrier dispersion liquid is added to 460 pmol of nucleic acid drug siCamk2g, mixed for 1 min to obtain drug-loaded nanoparticles, which are then diluted into 1 mL of water to prepare a suspension containing drug-loaded nanoparticles LAD@siCamk2g (hereinafter referred to as "LAD@siCamk2g").

[0083] The particle size distribution and morphology of the drug-loaded nanoparticles LAD@siCamk2g are characterized by a nanoflow instrument and a transmission electron microscope, as shown in Figures 2-3 The results show that the average particle size of the drug-loaded nanoparticles is 54 nm and the PDI is 0.12.

[0084] The ultraviolet spectra of the polyamino acid carrier, the nucleic acid drug, and the drug-loaded nanoparticles are detected by an ultraviolet-visible spectrophotometer, respectively. As can be seen from Figure 4 the ultraviolet spectrum of LAD@siCamk2g, there are both characteristic absorptions of LAD carrier alone and characteristic absorptions of siCamk2g nucleic acid drug alone. This indicates that the drug-loaded nanoparticles can successfully load the nucleic acid drug.

[0085] Example 5 Preparation of drug-loaded nanoparticles

[0086] The present example provides a preparation method of drug-loaded nanoparticles, which is basically the same as that of Example 4, except that the LAM carrier of Example 2 is used instead of the polyamino acid carrier prepared in Example 1, to prepare drug-loaded nanoparticles and a suspension containing drug-loaded nanoparticles LAM@siCamk2g (hereinafter referred to as "LAM@siCamk2g").

[0087] Example 6 Preparation of drug-loaded nanoparticles

[0088] The present example provides a preparation method of drug-loaded nanoparticles, which is basically the same as that of Example 4, except that the LAM carrier of Example 2 is used instead of the polyamino acid carrier prepared in Example 1, to prepare drug-loaded nanoparticles and a suspension containing drug-loaded nanoparticles LAM@siCamk2g (hereinafter referred to as "LAM@siCamk2g").

[0089] Comparative Example 1

[0090] The siCamk2g was loaded with a commercial liposome carrier (specific name: Lipofectamine 2000, purchased from Thermo Fisher Company, item number 11668019). The specific method is as follows: 8 μL of liposome was mixed with 250 μL of serum-free medium for 5 min to prepare a dispersion of the liposome carrier. 460 pmol of nucleic acid drug siCamk2g was mixed with 250 μL of serum-free medium for 5 min to prepare a dispersion of the nucleic acid drug siCamk2g. The dispersion of the liposome carrier and the dispersion of the nucleic acid drug siCamk2g were mixed for 10 min, and then diluted into 1 mL of water to prepare a suspension containing drug-loaded liposome particles (hereinafter referred to as "Lipo@siCamk2g").

[0091] The particle size distribution of Lipo@siCamk2g was measured by a particle size analyzer. The results showed that the average particle size of Lipo@siCamk2g was 110 nm, and the PDI was 0.24.

[0092] Experimental Example 1 Synthesis and characterization of polyamino acid carriers with different grafting ratios

[0093] 1. Preparation of polyamino acid carrier

[0094] The PLL was prepared according to the method of Example 1. 90 -Arg. The polyamino acid carrier does not graft DMA, and the carrier is denoted as LAD0.

[0095] 0.5 g of PLL 90 -Arg was dissolved in 10 mL of NaOH solution with pH = 8.0 to obtain a reaction solution.

[0096] 0.1g, 0.2g, 0.5g, and 1.5g of dimethylmaleic anhydride (DMA) were taken out respectively and dissolved in 5mL of 2,4-dioxane, added to the above reaction solution, and reacted for 5h. The solution was taken out and dialyzed (dialysis bag molecular weight cutoff 3500Da) for 72h; freeze-dried to obtain LAD carriers with different grafting ratios (named in descending order according to grafting ratio: LAD 10 , LAD 20 , LAD 50 , LAD 100 ).

[0097] 2. Characterization

[0098] The polyamino acid carrier synthesized above was characterized by NMR spectroscopy. Figure 1 As shown by Figure 1 It can be seen that compared with the polyamino acid carrier LAD0, LAD 10 , LAD 20 , LAD 50 , LAD 100 The peak area at the chemical shift of 1.6-2.0 ppm gradually increases. The characteristic peak at the chemical shift of 1.6-2.0 ppm is the characteristic peak of the dimethylmaleic acid group in the polyamino acid carrier, denoted as peak a.

[0099] The modification ratio of DMA in each group of polyamino acid carriers was calculated according to the following formula: modification ratio = 1 / 6 * (integrated area of ​​peak a - area of ​​peak a0) / integrated area of ​​peak at chemical shift of 4.2 ppm × 100%, where: the area of ​​peak a0 is the integrated area of ​​LAD0 at peak a. 10 , LAD 20 , LAD 50 , LAD 100 The modification ratios of the polyamino acid carriers were 0%, 10%, 20%, 50% and 100%, respectively. It was confirmed that the polyamino acid carriers with different modification ratios were successfully synthesized.

[0100] Experimental Example 2 Nucleic acid loading capacity of polyamino acid carriers with different grafting ratios

[0101] 1. Preparation of drug-loaded nanoparticles

[0102] The polyamino acid carrier and the nucleic acid drug were mixed for 1 min according to the molar ratio of nitrogen element in the polyamino acid carrier to phosphorus element in the nucleic acid drug (nitrogen-phosphorus ratio, i.e., N / P) of 0:1, 0.5:1; 1:1, 2:1, 4:1, 8:1, 16:1 (specifically, LAD0 and LAD 10 , LAD 20 , LAD 50 , LAD 100The carrier is LAD carrier, 0 g, 0.5 μg, 0.9 μg, 1.9 μg, 3.7 μg, 7.4 μg, 14.8 μg of LAD carrier and 92 pmol of nucleic acid drug siCamk2g are mixed for 1 min to obtain drug-loaded nanoparticles with different N / P.

[0103] 2. Preparation of agarose gel

[0104] 2 g of agarose is dissolved in 100 mL of 1 × TAE solution; then it is heated in a microwave oven for 1 min at medium-high fire; it is completely dissolved, then after it cools slightly, 1 / 10000 of the total volume of the solution of nucleic acid dye (Yixing Biotech (Shanghai) Co., Ltd., 10204ES76) is added, and then it is poured into a mold and allowed to cool completely to form.

[0105] 3. Electrophoresis

[0106] 5 μL of loading buffer is added to the drug-loaded nanoparticles prepared above with different N / P, and mixed for 1 min to obtain the loading system.

[0107] 2 L of 1 × TAE solution is added to the electrophoresis tank, the gel is immersed in the TAE solution, the prepared loading system is added to the gel well, and electrophoresis is started with a voltage of 140 V; the electrophoresis time is selected for 30 min. After electrophoresis, it is placed in an imager for imaging, and the results are shown in Figure 5 .

[0108] As can be seen from Figure 5 , when N / P is 4:1, the nucleic acid drug has already completely combined with the polyamino acid carrier, resulting in weaker negative charge and unable to move downward; it shows that the polyamino acid carrier can efficiently load the nucleic acid drug, achieving 100% full loading of the nucleic acid drug.

[0109] Experimental Example 3 Evaluation of the efficiency of the production of nitric oxide by drug-loaded nanoparticles in macrophages

[0110] 1. Preparation of test drugs

[0111] The LAD carrier prepared in Example 1, the LAM carrier prepared in Example 2, and the LAS carrier prepared in Example 3 were respectively assembled with control siRNA (abbreviated as "scr", purchased from Guangzhou Ribobio Biotechnology Co., Ltd., sequence (5'-3') as follows: 5'-UUGGGAAAAAGUUGAGUGGUU-3') into polyamino acid carrier-based nucleic acid drug delivery systems LAD@scr, LAM@scr, and LAS@scr according to the following methods. Specifically, the polyamino acid carrier was dissolved in water to prepare a polyamino acid carrier aqueous solution with a concentration of 1 mg / mL. 20 μL of the polyamino acid carrier aqueous solution was added to 460 pmol of scr, and mixed for 1 min to obtain the polyamino acid carrier-based nucleic acid drug delivery systems LAD@scr, LAM@scr, and LAS@scr, respectively.

[0112] 2. Cell culture and administration

[0113] Raw 264.7 cells were seeded in an 8-well cell culture chamber at a density of 5 x 10 4 Raw 264.7 cells were seeded in an 8-well cell culture chamber at a density of 5 x 10

[0114] PBS group: 2 μL of PBS buffer was added to the cells;

[0115] LPS group: 0.2 μL of 1 mg / mL LPS (lipopolysaccharide) was added to the cells;

[0116] LPS+Arg group: 2 μL of arginine solution (solvent: PBS, concentration 0.7 mg / mL) was added to the cells, followed by the addition of 0.2 μL of 1 mg / mL LPS;

[0117] LPS+LAS@scr group: 4 μL of the polyamino acid carrier-based nucleic acid drug delivery system LAS@scr was added to the cells, followed by the addition of 0.2 μL of 1 mg / mL LPS.

[0118] LPS+LAM@scr group: 4 μL of the polyamino acid carrier-based nucleic acid drug delivery system LAM@scr was added to the cells, followed by the addition of 0.2 μL of 1 mg / mL LPS.

[0119] LPS+LAD@scr group: 4 μL of the polyamino acid carrier-based nucleic acid drug delivery system LAD@scr was added to the cells, followed by the addition of 0.2 μL of 1 mg / mL LPS.

[0120] 3. Detect the efficiency of polyamino acid-based nucleic acid drug delivery system in producing nitric oxide in macrophages

[0121] All the above groups were incubated at 37°C for 24 h. 2 μL of DAF-FM DA (green fluorescence, nitric oxide indicator, Beyotime, S0019S) was added to each well and incubated at 37°C for 0.5 h. The cells were washed with PBS and stained with 1 μg of DAPI (nuclear dye) for 10 min. The cells were washed with PBS and then observed under a confocal microscope.

[0122] 4. Test results

[0123] The results are as follows Figure 6 As shown, the fluorescence intensity of the PBS group was (1.52±0.15)×10 3 The fluorescence intensity of the LPS group was (1.47±0.14)×10 3 The fluorescence intensity of the LPS+Arg group was (6.01±0.54)×10 3 The fluorescence intensity of the LPS+LAS@scr group was (1.53±0.01)×10 3 The fluorescence intensity of the LPS+LAM@scr group was (2.5±0.58)×10 3 The fluorescence intensity of the LPS+LAD@scr group was (6.16±1.35)×10 3 Compared with the PBS and LPS groups, the fluorescence intensity of the LPS+LAD@scr group was significantly increased (p<0.0001). However, compared with the LPS group, the fluorescence intensity of the LPS+LAM@scr and LPS+LAS@scr groups was slightly increased, but the differences were not significant. These results indicate that the LAD@scr-treated group is more effective in interacting with inducible nitric oxide synthase (iNOS) in macrophages to produce nitric oxide.

[0124] Experimental Example 4 Gene Silencing Efficiency

[0125] 1. Cell culture and drug administration

[0126] 7 × 10 cells were seeded in a 6-well plate. 5 Raw 264.7 cells were plated. After the cells adhered, the complete medium was replaced with serum-free medium to resuspend the cells. The cell suspension was randomly divided into two groups, with three parallel wells per group and 2 mL per well. 40 μL of LAD@siCamk2g and Lipo@siCamk2g prepared in Example 4 and Comparative Example 1 were added to each group, respectively. The cells were incubated at 37°C for 8 h. After incubation, the supernatant was removed and replaced with 2 mL / well of complete medium. The cells were cultured at 37°C for another 40 h. The medium was then discarded and the cells were harvested.

[0127] 2. Western-blot experiment detection

[0128] In the hole, add the appropriate amount of lysis solution, and blow the cells down with a gun several times. Centrifuge at 12000 rpm for 10 min at 4°C. After centrifugation, the protein is aspirated and placed in an EP tube, and the protein is quantified using a BCA kit (protein loading amount is 30 μg). Add Loading buffer (loading buffer), heat in a metal bath at 100°C for 10 min to denature the protein, and further separate and degrade the DNA bound to the protein. After cooking, it should no longer be sticky.

[0129] Prepare an 8% polyacrylamide gel, place the gel in the built electrophoresis tank, then add the samples to the gel holes in turn, pour the electrophoresis liquid, and start electrophoresis. 80V constant voltage, electrophoresis for 30 min, then 120V constant voltage, electrophoresis for 1.5h. After electrophoresis, take out the gel and place it in the built membrane transfer system for membrane transfer. Start membrane transfer. Constant current 200mA, membrane transfer for 40min. After membrane transfer, block with 5% defatted milk for 2h. After blocking, wash with TBST, and after washing, add the primary antibody of GAPDH and CaMKIIγ protein and incubate at 4°C overnight. After incubation of the primary antibody, wash with TBST, and after washing, add the secondary antibody and incubate at room temperature for 2h. After incubation of the secondary antibody, develop with the developer.

[0130] 3. Test results

[0131] The results are shown in Figure 7 From the results, it can be seen that both Lipo@siCamk2g and LAD@siCamk2g drug delivery systems can efficiently silence the expression of CaMKIIγ protein in macrophages.

[0132] Experimental Example 5 Cell migration

[0133] 1. Test method

[0134] Draw lines on the back of the 6-well plate with a marker pen for positioning, then inoculate 6×10 5 cells / well of HUVEC cells in the well plate; inoculate 1×10 5 cells / well of Raw 264.7 cells; after the cells adhere, use a 200 μL pipette gun to scratch the well plate, then wash the fallen cells with PBS, and replace them with fresh complete medium to resuspend the cells. Randomly divide the cell suspension into PBS group, LPS group, LPS+Arg group and LPS+LAD@siCamk2g group, 3 wells in each group, and 2 mL per well.

[0135] PBS group: add 20 μL of PBS buffer to the cells;

[0136] LPS group: 2 μL of 1 mg / mL LPS was added to the cells;

[0137] LPS+Arg group: 20 μL of arginine solution (solvent: PBS, concentration 0.7 mg / mL) was added to the cells, followed by 2 μL of 1 mg / mL LPS;

[0138] LPS+LAD@siCamk2g group: 40 μL of LAD@siCamk2g prepared in Example 4 was added to the cells, followed by 2 μL of 1 mg / mL LPS.

[0139] Each group was incubated at 37°C for 24 h, during which the same position was photographed at 0 h, 8 h, and 24 h.

[0140] 2. Test results

[0141] The results are shown in Table 1. Figure 8 As shown in Table 1, the cell migration area of the PBS group was (6.7 ± 0.39) x 10 4 pixels, the cell migration area of the LPS group was (8.42 ± 0.33) x 10 4 pixels, the cell migration area of the LPS+Arg group was (11.45 ± 1.35) x 10 4 pixels, and the cell migration area of the LPS+LAD@siCamk2g group was (12.36 ± 1.85) x 10 4 pixels. The p value between the PBS group and the LPS+LAD@siCamk2g group was <0.0001. It can be seen from the results that the cell migration area of the LPS+Arg group and the LPS+LAD@siCamk2g group was significantly increased (p <0.0001) compared with the PBS group, indicating that the cells in the two groups migrated significantly. This indicates that the drug-loaded nanoparticles can efficiently produce nitric oxide gas in the cells to promote cell migration.

[0142] Example 6: Investigation of the Targeted Accumulation Ability of Drug-Loaded Nanoparticles in Mice

[0143] 1. Preparation of the sample to be tested

[0144] ①The polyamino acid carrier prepared in Example 1 was dissolved in water to prepare a polyamino acid carrier aqueous solution with a polyamino acid carrier concentration of 1 mg / mL. 62.5 μL of the polyamino acid carrier aqueous solution was mixed with 62.5 μL of Cyanine 5-labeled siCamk2g (purchased from Guangzhou Ribo Biotechnology Co., Ltd., solvent: DEPC water, molar concentration: 230 pmol / μL) for 30 s-2 min (1 min in this experimental example) to obtain Cyanine 5-labeled LAD@siCamk2g.

[0145] ②31.25 μL of liposomes (specific name: Lipofectamine 2000, purchased from Thermo Fisher, model number Lipo2000) was mixed with 31.25 μL of PBS solution at room temperature for 5 minutes, and then mixed with 62.5 μL of Cyanine 5-labeled siCamk2g (purchased from Guangzhou Ribo Biotechnology Co., Ltd., solvent: DEPC water, molar concentration: 230 pmol / μL) for 10 minutes to obtain Cyanine 5-labeled Lipo@siCamk2g.

[0146] 2. Animal grouping, administration and testing

[0147] 12-week-old Apoe - / - Mice (model group) 6. At the same time, 3 nine-week-old wild-type C57BL / 6J mice were used as a blank treatment group (WT). The model group was randomly divided into two groups, 3 in each group, and the two model groups: 125 μL of Cyanine 5-labeled LAD@siCamk2g and Cyanine 5-labeled Lipo@siCamk2g were injected into the mice through the tail vein. Blank treatment group: 125 μL of Cyanine 5-labeled LAD@siCamk2g was injected into the mice through the tail vein, and the three groups were continued to be fed for 24 h. The aorta of the mice was taken out and imaged by a small animal live imaging instrument.

[0148] 3. Test results

[0149] The results are shown in Figure 9 The fluorescence intensity of the WT group was (1.21 ± 0.86) x 10 6 , the fluorescence intensity of the Lipo@siCamk2g group was (7.63 ± 3.21) x 10 6 , and the fluorescence intensity of the LAD@siCamk2g group was (18.07 ± 4.88) x 10 6Lipo@siCamk2g group and LAD@siCamk2g group, p<0.01. Compared with the WT group and the model group injected with Lipo@siCamk2g, the fluorescence intensity of the mouse aorta of the model group injected with LAD@siCamk2g provided by the application was significantly improved (p<0.01), indicating that the drug-loaded nanoparticles provided by the application can accumulate more accurately at the lesion plaque, without affecting the normal tissue cells, and showing excellent targeting ability for atherosclerotic plaques.

[0150] Experimental Example 7: Treatment effect of drug-loaded nanoparticles on atherosclerotic mice

[0151] 1. Animal grouping and administration

[0152] 12-week-old Apoe - / - The model group was randomly divided into three groups, each group of 3, namely: PBS group, LAD@siCamk2g treatment group and Lipo@siCamk2g treatment group.

[0153] LAD@siCamk2g treatment group: 125 μL of LAD@siCamk2g prepared in Example 4 was injected into the mouse body through the tail vein. Lipo@siCamk2g treatment group: 125 μL of Lipo@siCamk2g prepared in Comparative Example 1 was injected into the mouse body through the tail vein. Each injection was performed twice a week, and a total of 4 weeks. The PBS group was injected with the same volume of PBS solution.

[0154] 2. Test method

[0155] After treatment, the aortas of the mice in each group were removed, washed with PBS twice, and carefully cut longitudinally along the vessel wall with dissecting scissors. The cut blood vessels were immersed in oil red O staining solution at 37°C for 60 min; then taken out, differentiated with 75% ethanol until the fat plaques in the lumen were orange or bright red, and other parts were nearly colorless, and then washed with distilled water twice. The staining results were photographed with a camera.

[0156] 3. Test results

[0157] The results are shown in Figure 10As shown, the percentage of Oil Red O area to total area in the PBS group was 61.79±18.73%, in the Lipo@siCamk2g group was 27.73±11.46%, and in the LAD@siCamk2g group was 8.47±3.17%. P < 0.05 was found between the PBS and LAD@siCamk2g groups. Compared with the PBS and Lipo@siCamk2g groups, the LAD@siCamk2g group had significantly smaller intraluminal fatty plaque area (red area) after LAD@siCamk2g injection (P < 0.05), demonstrating a significantly greater therapeutic effect than the other two groups, demonstrating that in situ gene-edited macrophages combined with nitric oxide gas therapy can achieve a significant therapeutic effect on atherosclerosis.

[0158] Experimental Example 8: Study on the in situ editing effect of drug-loaded nanoparticles on macrophages in vivo

[0159] 1. Animal grouping and drug administration

[0160] 12-week-old Apoe - / - 9 mice (model group) were randomly divided into three groups, 3 mice in each group: PBS group, LAD@siCamk2g treatment group and Lipo@siCamk2g treatment group.

[0161] In the LAD@siCamk2g treatment group, mice were injected with 125 μL of LAD@siCamk2g prepared in Example 4 via the tail vein. In the Lipo@siCamk2g treatment group, mice were injected with 125 μL of Lipo@siCamk2g prepared in Comparative Example 1 via the tail vein. Injections were administered twice weekly for a total of four weeks. The PBS group was injected with the same volume of PBS solution in parallel.

[0162] 2. Test methods

[0163] After treatment, the aorta of each group of mice was removed, embedded with OTC, and then frozen sectioned. The frozen sections were stained with TUNEL (red) and F8 / 40 (green) immunofluorescence. The staining results were observed using a slice scanner. The fluorescence staining imaging results are shown in Figure 2. Figure 11 White arrows indicate the co-localization of macrophages (green) and apoptotic cells (red).

[0164] 3. Test results

[0165] The results are as follows Figure 11 As shown, the colocalization fluorescence intensity of the PBS group was (3.95±0.68)×10 5, the co-localization fluorescence intensity of Lipo@siCamk2g group was (6.44±0.7) x 10 5 , the co-localization fluorescence intensity of LAD@siCamk2g group was (14.65±2.85) x 10 5 . The p value between Lipo@siCamk2g group and LAD@siCamk2g group was less than 0.001. Compared with PBS group and Lipo@siCamk2g treatment group, the LAD@siCamk2g treatment group had a significantly higher degree of co-localization (yellow) between macrophages (green) and apoptotic cells (red) in vivo by injecting LAD@siCamk2g (p<0.001). This indicates that it can significantly genetically modify macrophages, restore their phagocytic ability, clear apoptotic cells, and its therapeutic effect is significantly higher than that of the other two groups.

[0166] Experiment Example 9: Investigation of the Repair Effect of Drug-loaded Nanoparticles on the Endothelial Barrier in vivo

[0167] Evans blue (EB) dye is commonly used for vascular endothelial cell barrier permeability testing. Under physiological conditions, the endothelium cannot permeate albumin; under pathological conditions, the dysfunction of intercellular junction proteins in endothelial cells leads to increased barrier permeability, at which time, the leakage of Evans blue-albumin can be detected in a monolayer endothelial cell model.

[0168] 1. Animal grouping and administration

[0169] Twelve-week-old Apoe - / - mice (model group) 9. The model group was randomly divided into three groups, each group of 3, respectively: PBS group, LAD@siCamk2g treatment group and Lipo@siCamk2g treatment group.

[0170] LAD@siCamk2g treatment group: 125 μL of LAD@siCamk2g prepared in Example 4 was injected into the mouse body through the tail vein. Lipo@siCamk2g treatment group: 125 μL of Lipo@siCamk2g prepared in Comparative Example 1 was injected into the mouse body through the tail vein. Each injection was 2 times a week, a total of 4 weeks. The PBS group was injected with the same volume of PBS solution.

[0171] 2. Test method

[0172] After treatment, 2% Evans blue dye was injected into each group of mice through the tail vein. After 30 min of injection, perfusion was performed with 4% paraformaldehyde solution, then the aorta was collected and opened longitudinally for photography and staining intensity statistics. The heart was then embedded with OTC, and the aortic root section was observed for staining results using a frozen sectioning technique.

[0173] 3. Test results

[0174] Results as shown in Figure 12 Figure 6, the blood vessels of the PBS-treated atherosclerosis model mice were significantly colored by Evans blue, the color of the Lipo@siCamk2g-treated group was lighter, and the color of the LAD@siCamk2g-treated group was the lightest. Therefore, compared with the Lipo@siCamk2g group without nitric oxide gas treatment, the LAD@siCamk2g group treated by gene editing combined with nitric oxide gas treatment can significantly promote the proliferation and migration of vascular endothelial cells, and ultimately achieve the repair of the vascular endothelial barrier.

[0175] Experimental Example 10: Reprogramming macrophage gene silencing efficiency test

[0176] (1) Test method

[0177] Samples with different DMA modification rates and L-lysine grafting rates and L-lysine polymerization degrees were prepared.

[0178] Samples 1-3: Polyamino acid carriers corresponding to Examples 1-3, respectively, as shown in Table 2.

[0179] Samples 4-5: The preparation method is basically the same as Example 1, the only difference is that the amount of dimethyl maleic anhydride (DMA) in step (4) is adjusted from 1.5 g to 0.1 g, 0.2 g and 0.5 g, respectively.

[0180] Comparative samples 2-5: The preparation method is basically the same as Example 1, the only difference is that step (4) is not performed, and in step (3), the amount of Nα-Boc-Nω-(2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl)-L-arginine is adjusted from 1 g to 0.5 g, no adjustment, 1.4 g and 2 g, respectively.

[0181] Comparative samples 6-8: The preparation method is basically the same as Example 1, the only difference is that steps (3) and (4) are not performed, and in step (2), the amount of ZLL(NCA) solid is adjusted from 1 g to 0.67 g, no adjustment, 1.34 g, respectively.

[0182] The number average polymerization degree of L-lysine, the grafting rate of arginine, and the modification rate of DMA of the above polyamino acid carrier samples are shown in Table 1. In the present application, the grafting rate or modification rate of DMA is tested and calculated according to item (1) of Experimental Example 1; the grafting rate of arginine is measured by nuclear magnetic resonance hydrogen spectrum, and the number average polymerization degree is measured by nuclear magnetic resonance hydrogen spectrum.

[0183] ① Preparation of the sample to be tested: each group of polyamino acid carrier was dissolved in PBS solution to prepare 1 mg / mL polyamino acid carrier PBS solution. 20 μL of 1 mg / mL polyamino acid carrier PBS solution was added to 460 pmol of nucleic acid drug siRNA (i.e. siCamk2g), mixed for 1 min to obtain a nucleic acid drug delivery system based on polyamino acid carrier.

[0184] ② Cell treatment: log phase growing Raw 264.7 cells were seeded in a cell 6-well plate at a density of 7 x 10 5 cells / well, and the cells were allowed to adhere; the complete culture medium was replaced with serum-free culture medium to resuspend the cells, and the cell suspension was randomly divided into 14 groups, namely test groups 1-13 and a PBS group, each group having 3 parallel wells, and 2 mL of cell suspension was added to each well. 40 μL of each group of sample to be tested was added to the cell suspension in the test groups 1-13, and the same volume of PBS solution was added to the PBS group; incubation was performed for 8 h. After incubation, the supernatant was removed and replaced with complete culture medium, and incubation was continued for 16 h; the cells were collected.

[0185] ③ Testing: before the sample of the cultured adherent cells was collected, the culture medium was removed, 1 mL of TRIzol was added to lyse the cells, and the lysate was repeatedly blown with a pipette gun until no obvious precipitate was present in the lysate, and the lysate was placed on ice for 5 min. 200 μL of chloroform was added to 1 mL of the sample; oscillation was performed for 15 s, and the sample was placed on ice for 3 min. Centrifugation was performed at 12000 g at 4°C for 15 min, and the sample was carefully taken out and placed on a test tube rack. 500 μL of the supernatant was taken to a new centrifuge tube, and attention was paid to make a mark on the tube cap, 500 μL of isopropanol was added; gentle mixing was performed, and the sample was placed on ice for 10 min; centrifugation was performed at 12000 g at 4°C for 10 min; the supernatant was slowly discarded, 1 mL of 75% ethanol was added; the precipitate was gently popped up with the hand, centrifugation was performed at 7500 g at 4°C for 5 min, and the supernatant was slowly discarded; then, short-term centrifugation was performed at 7500 g for 30 s; residual liquid was removed as much as possible; the centrifuge tube was inverted on a clean PE glove, and the sample was cooled until the precipitate edge became transparent. 40 μL of RNase-Free H2O was added; the sample was placed at room temperature for 10 min; the RNA precipitate was dissolved, and finally the sample was repeatedly blown with a pipette gun several times to mix it well; agarose gel electrophoresis was performed to detect the quality of the extracted RNA, and three ribosomal RNA bands (28S, 18S, 5S) were used to evaluate the quality of the RNA. 1.5 μL of total RNA was taken, and the RNA concentration of the sample was determined with a microspectrophotometer. Then, a mark was made on the tube wall and cap, and the sample was stored at -80°C for standby use. Reverse transcription reaction was performed according to the operation of the RT-qPCR kit, and cDNA was synthesized. Finally, qPCR experiment was performed to detect the content of the target mRNA in the cells according to the operation of the qPCR kit. The siRNA silencing efficiency was calculated using the ΔΔCT method, and the calculation formula was as follows:

[0186] ΔCT 样本 = CT靶基因 -CT 内参基因

[0187] DeltaCT 对照 = CT 靶基因 -CT 内参基因

[0188] DeltaDeltaCT = DeltaCT 样本 - DeltaCT 对照

[0189] Target gene expression = 2-DeltaDeltaCT

[0190] (2) Test results

[0191] The results are shown in the following Table 1:

[0192] Table 1: Effect of different lysine polymerization degrees, arginine grafting rates, and DMA modification ratios on siRNA silencing efficiency

[0193]

[0194]

[0195] From the above table, it can be seen that the silencing efficiency of the polyamino acid carrier prepared by grafting DMA is significantly improved compared to not grafting DMA, especially when the L-lysine polymerization degree is 90, the arginine grafting rate is 50%, and the DMA modification ratio is 100%.

[0196] Table 2: Effect of different small molecule acid anhydrides on siRNA silencing efficiency

[0197]

[0198] From the above table, it can be seen that the silencing efficiency of the polyamino acid carrier prepared by grafting DMA is significantly improved compared to grafting other small molecule acid anhydrides.

[0199] Obviously, the above examples are merely examples for clarity and do not limit the embodiments. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. All embodiments do not need to be exhausted here. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. Use of drug-loaded nanoparticles in the manufacture of a medicament for the prevention or treatment of atherosclerosis, characterized in that, The drug-loaded nanoparticles comprise: (1) a polyamino acid carrier grafted with a nitric oxide donor, the polyamino acid carrier having a structural formula as shown in formula (I): wherein m and n are the number-average polymerization degree, and y is an integer selected from 2-16; n is 10-500, m is 10-200, and n≥m; R1has the structural formula as shown below: (2) a nucleic acid drug for silencing Camk2g gene expression, which is encapsulated in the polyamino acid carrier; the nucleic acid drug is siCamk2g, and its sequence (5'-3') is as follows: 5'-AAC GUG GUA CAU AAU GCU ACA-3', and the nitric oxide donor is arginine.

2. Use according to claim 1, characterized in that, The preparation method of the polyamino acid carrier comprises the following steps: (1) reacting lysine with a protecting group with triphosgene or phosgene in the presence of a solvent to obtain L-lysine-N-carboxyl cyclic anhydride with a protecting group; (2) ring-opening polymerization of L-lysine-N-carboxyl cyclic anhydride with a protecting group with a monomer as shown in formula (II) in the presence of a solvent, and then removing the protecting group of the amino acid to obtain polylysine with an alkyl chain; (3) reacting arginine with a protecting group with an amide catalyst in the presence of a solvent, and then grafting polymerization of the reaction product with polylysine with an alkyl chain in the presence of an acid-binding agent, and then removing the protecting group of the amino acid; (4) modifying the polymeric product obtained in step (3) after removing the protecting group of the amino acid with a small molecule anhydride to obtain a polyamino acid carrier; wherein y is an integer selected from 2-16; and the small molecule anhydride is dimethyl maleic anhydride (DMA).

3. Use according to claim 2, characterized in that, n is 60-120, and the grafting rate of arginine is greater than 0 and less than or equal to 100%.

4. Use according to claim 3, characterized in that, The grafting rate of arginine is 25-100%, and the modification ratio of DMA is greater than 0 and less than or equal to 100%.

5. Use according to claim 4, characterized in that, The modification ratio of DMA is 10-100%.

6. Use according to claim 5, characterized in that, n is 90, the grafting rate of arginine is 50%, and the modification ratio of DMA is 100%.

7. Use according to any one of claims 1 to 6, characterized in that, The molar ratio between nitrogen elements in the polyamino acid carrier and phosphorus elements in the nucleic acid drug is 0.25-16:1; and / or, the average particle size of the drug-loaded nanoparticles is 40-80 nm.

8. Use according to any one of claims 1 to 6, characterized in that, The preparation method of the drug-loaded nanoparticles comprises mixing the polyamino acid carrier and the nucleic acid drug in the presence of an aqueous solution.

9. Use according to claim 8, characterized in that, The mass of the polyamino acid carrier and the molar ratio of the nucleic acid drug are 0.25 μg-0.03 mg:80-500 pmol.

10. Use according to claim 8, characterized in that, The mixing time is 30 s-5 min.

11. Use according to claim 1, characterized in that, The drug also comprises other pharmaceutically acceptable carriers.

12. Use according to claim 11, characterized in that, The pharmaceutically acceptable carrier is selected from at least one of a pharmaceutically acceptable solvent, a solubilizer, a cosolvent, an emulsifier, a coloring agent, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure adjusting agent, a stabilizer, a glidant, a corrigent, a preservative, a suspending agent, a coating material, an aroma, an antiadherent, an integrating agent, a penetration enhancer, a pH adjusting agent, a buffer, a plasticizer, a surfactant, a thickening agent, an inclusion agent, a humectant, an absorbent, a flocculating agent, an anti-flocculating agent, a filtration aid, a release retarder, a high molecular backbone material, and a film forming material.

13. The use according to claim 1, characterized in that, The dosage form of the drug is selected from an injection or an oral preparation.

14. Use according to claim 13, characterized in that, The injection is selected from an injection solution or a powder injection; the oral preparation is selected from a tablet, a solution, a capsule, a powder, a pill, a granule, a syrup, a suspension, or an oral sustained / controlled release preparation.

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