Difunctional polymer nano-carrier system as well as preparation method and application thereof

Abstract

The invention provides a difunctional polymer nano-carrier system as well as a preparation method and application thereof, and belongs to the technical field of pharmaceutical chemicals. The nano carrier system is prepared from the following raw materials: DSPE-PEG-S2P, DSPE-PEG-beta-CD, DSPE-G2K and PLGA (poly (lactic-co-glycolic acid)) through a nano precipitation method. The siTRPM2 is complexed with a cationic lipid carrier DSPE-G2K through electrostatic interaction, the periphery of the cationic lipid is coated with the PLGA based on the hydrophobic interaction between the PLGA and the DSPE-G2K, and the modified targeting peptide S2P and beta-CD are jointly connected to the surface of the carrier. The cationic lipid is stably complexed with siRNA due to positive charges, S2P can rapidly target pro-inflammatory macrophages, modified cyclodextrin (beta-CD) can include, dissolve and remove cholesterol in atherosclerotic plaques and promote cholesterol excretion, and the carrier system has a good effect in silencing TRPM2 and CD36 proteins, and can be used for preparing an anti-inflammatory drug for treating atherosclerotic plaques. And intervention treatment can be performed on formation and development of plaques in a lesion area at the same time.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemical industry, and specifically relates to a dual-functional polymer nano-carrier system, a preparation method and application thereof. Background Art

[0002] Atherosclerosis (AS) is a chronic inflammatory vascular disease characterized by the accumulation of cholesterol and cholesterol-rich macrophage cell fragments on the vascular wall. After endothelial cell damage, low-density lipoprotein (LDL) accumulates in the intima layer and undergoes oxidation to form oxidized low-density lipoprotein (oxLDL). Strong pro-inflammatory signals induce monocyte-derived cells to aggregate under the endothelium, differentiate into mononuclear macrophages, phagocytize modified lipoproteins mainly composed of oxLDL, and finally transform into cholesterol-rich foam cells. Lipid accumulation caused by increased foam cells is a key feature of early and mid-stage atherosclerosis, which will further lead to an increase in the volume of lipid streaks, narrowing of the arterial lumen, obstruction of blood flow, and subsequently various cardiovascular diseases such as coronary heart disease, myocardial infarction and stroke. At the same time, the various mediators secreted by foam cells, such as cytokines (TNF-α, IL-1β) and chemokines (MCP-1), will further recruit more immune cells into the arterial wall, enhance local inflammatory response, accelerate the progression of atherosclerosis, and increase the instability of plaques, ultimately leading to the formation of necrotic cores and increasing the risk of myocardial infarction. Therefore, inhibiting the formation of foam cells, reducing their lipid load or preventing the inflammatory response they trigger is an important strategy for treating atherosclerosis.

[0003] Cyclodextrin (CD) is a cyclic oligosaccharide, which is called α-, β- and γ-CD. Among them, β-CD has good binding properties for hydrophobic drug molecules or amphiphilic molecules with hydrophobic parts such as cholesterol because of its hydrophobic inner surface and hydrophilic outer surface. It can form inclusion complexes with cholesterol in the internal hydrophobic cavity, promote the dissolution of cholesterol, increase cholesterol efflux, and reduce the accumulation of cholesterol in cells. It has similar effects to cholesterol ester transfer protein (CETP) inhibitors, which can enhance the efflux of cholesterol by cells. Therefore, β-CD has been widely used to reduce cholesterol levels and remove insoluble cholesterol crystals. Related studies have shown that cholesterol can be transported to extracellular apolipoprotein AI (apoA-I) and high-density lipoprotein (HDL) through macrophage ATP-binding cassette (ABC) transporters A1 (ABCA1) and G1 (ABCG1), respectively. The expression of these membrane lipid translocases is induced by peroxisome proliferator-activated receptor-γ (PPARγ) and the sterol-regulated transcription factor liver X receptor-α (LXRα). β-CD promotes macrophage cholesterol efflux by increasing LXRα content and improves atherosclerosis in preclinical animal models. As a promising therapeutic agent, it provides a new drug delivery strategy and treatment method for atherosclerosis.

[0004] However, the interaction between the C2 / C3 secondary hydroxyl groups of adjacent glucose units of β-CD largely limits the water solubility of β-CD. The formation and accumulation of CD-cholesterol or CD-cholesterol crystalline complexes may further reduce the solubility and even precipitate in aqueous solution systems, which may be an important factor in acute nephrotoxicity caused by intravenous injection. In addition, β-CD may also easily and efficiently extract plasma membrane-bound cholesterol, which may greatly induce hemolysis and increase ototoxicity. Therefore, chemical modification of β-CD to improve its solubility and biocompatibility is a prerequisite for safe clinical application.

[0005] In the early stages of atherosclerosis, macrophages differentiated from monocytes recognize and ingest oxLDL through specific receptors expressed on their surfaces (such as scavenger receptors, SR-A, and CD36). Related studies have shown that oxLDL in atherosclerotic lesions increases the expression of CD36 by driving the expression of lipoprotein receptors, thereby promoting the formation of foam cells, of which 70% of oxLDL is dominated by CD36. Transient receptor potential cation channel subfamily M member 2 (TRPM2) is a non-selective cation channel. Activation of the TRPM2 channel leads to the increase of intracellular Ca 2+The concentration of oxLDL increases, which in turn triggers a series of physiological and pathological reactions. Inhibiting the TRPM2 channel can reduce the production of reactive oxygen species (ROS), thereby reducing the damage to cells caused by oxidative stress. In addition, the activation of the TRPM2 channel also promotes the expression of monocyte chemoattractant protein-1 (MCP-1) and macrophage migration inhibitory factor (MIF), which play an important role in the inflammatory response. Therefore, inhibiting the TRPM2 channel can reduce the production of these inflammatory factors, thereby alleviating the inflammatory response and inhibiting the uptake of oxLDL by foam cells and the infiltration of macrophages. Therefore, achieving effective delivery of siTRPM2 is expected to become a new and efficient strategy to improve atherosclerotic plaques by regulating oxLDL uptake.

[0006] However, there is no existing solution for combining β-CD with siTRPM2 to effectively treat atherosclerosis, and due to the immature delivery technology, the safe and effective delivery of nucleic acid drugs (such as antisense oligonucleotides (ASO), small interfering RNA (siRNA), microRNA (miRNA) and messenger RNA (mRNA)) in vivo still faces many challenges. For AS, the formation and development of plaques are important events in the progression of the disease, so targeted delivery of plaques can improve the therapeutic effect of drugs. Viral vectors can be used for nucleic acid delivery because of their strong transfection ability, but there are a series of potential risks that cannot be ignored in clinical applications, such as immunogenicity and possible gene mutation. In contrast, non-viral nanocarriers have attracted much attention in nucleic acid drug delivery research because of their low price, easy synthesis, easy purification, and high nucleic acid transfection efficiency and low immunogenicity. For example, a dendritic nanocarrier system targeting atherosclerosis macrophages disclosed in Chinese patent CN110420198A, in which PAMAM G5.0 dendritic polymer is used as the basic carrier, and β-CD and targeting molecule mannose (Mannose) are used to modify the three effectively. The targeted nanocarrier system targeting atherosclerosis-related macrophages is constructed, and the average particle size range of the nanocarrier finally prepared is 100-150nm. The nanocarrier system has the effect of targeting atherosclerosis-related macrophages, can encapsulate, dissolve and remove cholesterol in atherosclerotic plaques, effectively reduce cholesterol crystals in plaques, and can inhibit inflammation, and finally reduce plaques. However, it is important to note that although the above-mentioned nanocarrier system shows good targeting and the effect of inhibiting inflammation to reduce plaques, the action pathway is relatively single. It only uses the dissolution-promoting effect of β-CD on cholesterol to treat AS, and cannot inhibit the TRPM2 channel to further reduce the production of inflammatory factors, that is, it cannot effectively intervene in the formation of plaques in the lesion area.

[0007] Cationic lipids, as a new type of biomolecule that combines the characteristics of dendritic molecules and lipopeptides, have brought new hope for the efficient delivery of siRNA with their unique structural and functional properties. Dendritic lipopeptides combine the branching structure of dendritic molecules with the biological activity of lipopeptides to form a stable three-dimensional structure. Its amino-containing head and hydrophobic tail together constitute a cationic lipid with a positive charge, which enables it to bind to siRNA through electrostatic interaction, thereby achieving the purpose of delivering siRNA. There are few reports in the prior art on the use of cationic lipids to deliver siRNA for the treatment of atherosclerosis, and there is still a lot of room for research on the corresponding technology. Summary of the invention

[0008] The purpose of the present invention is to solve the deficiencies in the prior art and provide a bifunctional polymer nanocarrier system and a preparation method thereof. The present application adopts a nanoprecipitation method to prepare a target polymer nanocarrier system, utilizes the positively charged property of cationic lipids to efficiently deliver siRNA, and innovatively combines β-CD with siTRPM2 to simultaneously intervene in the formation and development of plaques in the lesion area. Compared with the prior art, the siRNA transfection effect is better, and it also has a good promoting effect on the efflux of intracellular cholesterol.

[0009] The specific technical scheme of the present invention is: a dual-functional polymer nanocarrier system, which is composed of DSPE-PEG-S2P, DSPE-PEG-β-CD, DSPE-G 2 K and PLGA were used as raw materials to prepare the carrier system by nanoprecipitation method; siTRPM2 was complexed with the cationic lipid carrier DSPE-G2K through electrostatic interaction, and PLGA was based on DSPE-G 2 The hydrophobic interaction between K is coated on the periphery of the cationic lipid, and the modified targeting peptide S2P and β-CD are jointly connected to the carrier surface.

[0010] The preparation method of the above-mentioned bifunctional polymer nanocarrier system is as follows:

[0011] 1) β-CD-NH 2 DSPE-PEG-β-CD was prepared by coupling to DSPE-PEG-NHS via amidation reaction;

[0012] 2) Dissolve DSPE-PEG-MAL and S2P in DMF, add Et3N, stir to react, evaporate and concentrate, dialyze, and freeze-dry to obtain DSPE-PEG-S2P;

[0013] 3) Under an inert atmosphere, H-Lys-OMe·2HCl, Boc-Lys(Boc)-OH, HOBT, HBTU and DIPEA were mixed, and DMF was added to dissolve. After reacting in an ice bath, the reaction was stirred at room temperature, extracted, washed, salted out, and dried to obtain a crude product solution, which was concentrated and purified to obtain G 2 K(MeO);

[0014] 4) Dissolve G2K(MeO) in NaOH / MeOH, react in an ice bath, adjust the pH of the solution, remove MeOH by rotary evaporation, add ethyl acetate, wash, salt out, and dry to obtain a crude product solution, and concentrate the filtrate to obtain G 2 K(COOH);

[0015] 5) Under inert atmosphere, DSPE, G 2 Mix K(COOH), HOBT, HBTU and DIPEA, add anhydrous CHCl 3 / DMF dissolution, ice bath reaction, heating and stirring reaction, extraction, washing, salting out, drying, to obtain a crude product solution, concentration, purification, to obtain DSPE-G 2 K(Boc) 4 ;

[0016] 6) Weigh DSPE-G2K(Boc) 4 In the reactor, add a magnetic particle and seal it. Under the protection of inert gas, add DCM to dissolve, add TFA, stir and react in an ice bath, stir and react at room temperature. After the reaction is completed, evaporate and concentrate under reduced pressure, wash, and dry to obtain DSPE-G. 2 K is a white solid;

[0017] 7) Configure DSPE-G 2 K tetrahydrofuran solution, PLGA acetone solution, DSPE-PEG-S2P aqueous solution and DSPE-PEG-β-CD aqueous solution, siTRPM2 was first mixed with DSPE-G 2 The tetrahydrofuran solution of K was mixed and vortexed to complex siTRPM2 with the cationic lipid, and then the acetone solution of PLGA was added and vortexed to mix, and then the DSPE-PEG-S2P aqueous solution and the DSPE-PEG-β-CD aqueous solution were added, stirred in an ice bath, and centrifuged to obtain a bifunctional polymer nanocarrier system containing siTRPM2.

[0018] Furthermore, in step 2), the molar ratio of DSPE-PEG-MAL, S2P and Et3N is 1:1:1-3.

[0019] Furthermore, in step 3), the molar ratio of H-Lys-OMe·2HCl, Boc-Lys(Boc)-OH, HOBT, HBTU and DIPEA is 1:1-2:2-5:2-5:4-10, the reaction is carried out in an ice bath for 0.5-1 h, and the reaction is stirred at room temperature for 18-36 h.

[0020] Further, in step 5), DSPE, G 2 The molar ratio of K(COOH), HOBT, HBTU and DIPEA is 1:1-2:2-4:2-4:8-14. After reacting in an ice bath for 20-60 min, the mixture is heated to 45-50° C. and stirred for reaction for 18-24 h.

[0021] Furthermore, in step 7), the molar ratio of DSPE-PEG-S2P, DSPE-PEG-β-CD, DSPE-G2K and PLGA is 8-28:8-28:12-40:10-14, and the preferred molar ratio is 18:18:26:10; the time of the two vortex processes is 20-60 seconds, and the ice bath stirring time is 0.5-1.5 hours.

[0022] The above-mentioned bifunctional polymer nanocarrier system has good effects in rapidly targeting pro-inflammatory macrophages, enhancing the ability of intracellular cholesterol efflux, and silencing TRPM2 and CD36 proteins. It innovatively combines the treatment of atherosclerosis by cyclodextrin with siTRPM2, and can simultaneously intervene in the formation and development of plaques in the diseased area. Therefore, it can be effectively used in the preparation of targeted drugs for atherosclerosis.

[0023] The present application also claims protection for an atherosclerosis targeted drug, which comprises the above-mentioned bifunctional polymer nanocarrier system for achieving accurate and efficient drug delivery.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] 1. The present application uses DSPE-PEG-S2P, DSPE-PEG-β-CD, DSPE-G2K and PLGA as raw materials, and obtains a bifunctional polymer nanoparticle by nanoprecipitation method, and uses cationic lipid DSPE-G2K as a carrier. Based on the positively charged property of cationic lipids, it can be electrostatically complexed with siRNA to help achieve siRNA delivery, and the peripherally coated PLGA can effectively protect nucleic acids to prevent them from being degraded by nucleases. The targeting peptide S2P connected to its surface acts on proinflammatory macrophages as a targeting molecule, which can increase the drug concentration in the lesion site, and the modified cyclodextrin (β-CD) can encapsulate, dissolve and remove cholesterol in atherosclerotic plaques, promote the efflux of cholesterol in cells, inhibit inflammation, promote the regression of plaques, and have a protective effect on cardiovascular system;

[0026] 2. In the atherosclerotic lesion area, pro-inflammatory macrophages will continuously ingest oxLDL and become foam cells. After the foam cells continue to accumulate, plaques will form in the blood vessels, thereby causing a series of diseases. The present application can identify the highly expressed stable protein-2 on the surface of the pro-inflammatory macrophage membrane through the targeting peptide S2P, and can quickly target the lesion area; after the drug-loaded nanoparticles escape through the endosome, siTRPM2 is released, and the expression of the main receptor CD36 protein of oxLDL is reduced by reducing the expression of TRPM2 protein, thereby reducing the uptake of macrophages, thereby achieving the effect of reducing the formation of foam cells;

[0027] 3. The bifunctional polymer nanoparticles prepared in this application innovatively combine the treatment of atherosclerosis by cyclodextrin with siTRPM2, and simultaneously intervene in the formation and development of plaques in the lesion area. Compared with the existing technology, the siRNA transfection effect is better, and it also has a good effect of promoting the excretion of intracellular cholesterol;

[0028] 4. Both peptides S2P and β-CD are modified with polyethylene glycol (PEG), which can improve the water solubility, biocompatibility and biodegradability of the targeting factors S2P and β-CD for clinical applications, achieving the purpose of efficient and safe targeted delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the NMR spectrum of DSPE-PEG-β-CD;

[0030] Figure 2 is the NMR spectrum of DSPE-PEG-S2P;

[0031] Figure 3 G 2 Mass spectrum of K(COOH);

[0032] Figure 4 For DSPE-G 2 Mass spectrum of K;

[0033] Figure 5 The fluorescence intensity of FAM probe in cells after different nanoparticles targeted macrophages, where *** indicates p < 0.001, ** indicates p < 0.01, ns indicates p ≥ 0.05, n = 3;

[0034] Figure 6 The fluorescence intensity of NBD probe in cells after different nanoparticles promoted cholesterol efflux, where *** indicates p < 0.001, ** indicates p < 0.01, ns indicates p ≥ 0.05, n = 3;

[0035] Figure 7The expression levels of TRPM2 and CD36 in foam cells after treatment with different nanoparticles (a) and semi-quantitative analysis of TRPM2 (b) and CD36 (c), where *** indicates p < 0.001, ** indicates p < 0.01, ns indicates p ≥ 0.05, n = 3;

[0036] Figure 8 The targeting effects of nanoparticles ATCC-DLNPs and CC-DLNPs in vivo (a) and fluorescence semi-quantitative graph (b), where *** indicates p < 0.001, ** indicates p < 0.01, ns indicates p ≥ 0.05, n = 3;

[0037] Fig. 9 Figure 2 shows the aortic blood vessel staining after treatment with different nanoparticles (a) and the quantitative analysis of aortic lesion area (b), where *** indicates p<0.001, ** indicates p<0.01, ns indicates p≥0.05, n=3. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further described below in conjunction with the accompanying drawings, but is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be included in the protection scope of the present invention.

[0039] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0040] Among them, L-lysine methyl ester dihydrochloride (H-Lys-OMe·2HCl) was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.; (S)-2,6-di-tert-butyloxycarbonylaminocaproic acid (Boc-Lys(Boc)-OH) was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.; 1-hydroxybenzotriazole (HOBt) was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.; benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.; lipopolysaccharide (LPS) (derived from Escherichia coli 055:B5) was purchased from Sigma-Aldrich; oxidized low-density lipoprotein (oxLDL) was purchased from Guangzhou Yiyuan Biotechnology Co., Ltd.; distearoylphosphatidylethanolamine-polyethylene glycol-maleimide (DSPE-PEG-MAL) and distearoylphosphatidylacetamide-polyethylene glycol-succinimide (DSPE-PEG-NHS) were purchased from Chongqing Yusi Pharmaceutical Technology Co., Ltd.; S2P peptide (CRTLLTVRKC) was purchased from Gill Biochemical Co., Ltd.; mono-(6-amino-6-deoxy)-β-cyclodextrin (β-CD-NH 2) and poly(lactic acid-co-glycolic acid) (PLGA, Mw 38000-54000) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) was purchased from Shanghai Huawei Pharmaceutical Technology Co., Ltd.

[0041] Example 1

[0042] This example uses nanoprecipitation method to prepare polymer nanocarrier system ATCC-DLNPs, and the specific steps are as follows:

[0043] 1) Synthesis of DSPE-PEG-β-CD

[0044] β-CD-NH 2 The 6-amino-β-cyclodextrin (β-CD-NH 2 , 0.04mmoL) was dissolved in N,N-dimethylformamide (DMF, 4mL), phospholipid-polyethylene glycol-active ester (DSPE-PEG-NHS, 0.04mmoL) was dissolved in dichloromethane (DCM, 1mL), the above two solutions were mixed, triethylamine (Et3N, 0.15mmoL) was added, stirred at room temperature for 24h, concentrated by rotary evaporator, dialyzed for 24h, and freeze-dried for 48h to obtain DSPE-PEG-β-CD powder with a yield of 80%. Its H NMR spectrum is shown as follows: Figure 1 shown.

[0045] The synthetic route is as follows:

[0046]

[0047] 2) Synthesis of DSPE-PEG-S2P

[0048] Phospholipid-polyethylene glycol-maleimide (DSPE-PEG-MAL, 0.04mmoL) and S2P polypeptide (0.04mmoL) were dissolved in DMF (5mL), and Et3N (0.07mmoL) was added. The mixture was stirred at 40°C for 24h, then concentrated by rotary evaporator, dialyzed for 24h, and freeze-dried for 48h to obtain phospholipid-polyethylene glycol-polypeptide S2P (DSPE-PEG-S2P) powder with a yield of 83%. Its H NMR spectrum is shown in the following figure. Figure 2 The specific synthetic route is as follows:

[0049]

[0050] 3) G 2 Synthesis of K(COOH)

[0051] Weigh L-lysine methyl ester hydrochloride (H-Lys-OMe·2HCl, 10mmol), (S)-2,6-di-tert-butyloxycarbonylaminocaproic acid (Boc-lys(Boc)-OH, 20mmol), 1-hydroxybenzotriazole (HOBT, 30mmol), benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate (HBTU, 30mmol), N,N-diisopropylethylamine (DIPEA, 60mmol). HOBT and HBTU were mixed in equal moles as condensing agents, H-Lys-OMe·2HCl, Boc-Lys(Boc)-OH, condensing agents and DIPEA were added in a molar ratio of 1:2:6:6, and the reaction solvent was 50mL DMF. The raw materials were added to a branch-mouth bottle under ice bath and nitrogen protection, and DMF was added to dissolve the solid drug. The reaction was first ice bathed for 30 minutes, and then stirred at room temperature for 24 hours. After the reaction was completed, the reaction solution was extracted with ethyl acetate (EA) and saturated with NaHCO 3 , dilute hydrochloric acid, saturated NaCl, anhydrous Na 2 SO 4 Washing, salting out and drying were performed to obtain a crude product solution. The crude reaction solution was then concentrated and purified by thin layer chromatography, with the mobile phase being petroleum ether and ethyl acetate in a volume ratio (V:V) of 1:1 to obtain G 2 K(MeO) with a yield of 75%.

[0052] G 2 K(MeO) was dissolved in NaOH / MeOH (1 mol / L, 6 mL) and stirred in an ice bath for 4 h. The pH of the solution was adjusted to neutral with HCl (1 mol / L). Methanol (MeOH) was removed by rotary evaporation and ethyl acetate was added to the solution for extraction. 3 , dilute hydrochloric acid, saturated NaCl, anhydrous Na 2 SO 4 Washing, salting out and drying are performed to obtain a crude product solution. The filtrate is concentrated by rotary evaporation to obtain G 2 K(COOH) has a yield of 85% and its mass spectrum is shown in Figure 3 The synthetic route is as follows:

[0053]

[0054] 4) DSPE-G 2 Synthesis of K

[0055] Weigh polyethylene glycol phospholipids (DSPE, 0.5 mmol), G 2K(COOH) (0.7 mmol), HOBT (1.5 mmol), HBTU (1.5 mmol), DIPEA (0.8 mL, 5 mmol). DSPE, G2K(COOH), condensation agent [equimolar mixture of HOBT and HBTU] and DIPEA were added in a molar ratio of 1:1.2:6:10, and the reaction solvent was 60 mL of anhydrous CHCl 3 / DMF (10:1, V / V).

[0056] Add the above raw materials into a bottle under ice bath and nitrogen protection, then add anhydrous CHCl 3 / DMF to dissolve the solid drug, react in an ice bath for 30 min, and stir at 50°C for 24 h. After the reaction, the reaction solution was extracted with chloroform and washed with saturated NaHCO 3 , dilute hydrochloric acid, saturated NaCl, anhydrous Na 2 SO 4 Washing, salting out and drying were performed to obtain a crude product solution. The crude reaction solution was then concentrated and purified by thin layer chromatography, with the mobile phase being dichloromethane / methanol in a volume ratio (V:V) of 20:1, to obtain DSPE-G 2 K(Boc) 4 , with a yield of 70%.

[0057] Weigh 50 mg DSPE-G 2 K(Boc) 4 In a three-necked flask, add a magnetic particle and seal it with a rubber stopper and place it in an ice bath. Under nitrogen protection, slowly add 5 mL of DCM to dissolve it, then slowly add 5 mL of trifluoroacetic acid (TFA), stir the reaction in an ice bath for 30 minutes, and continue to stir the reaction at room temperature for 6 hours. After the reaction is completed, evaporate the reaction solution under reduced pressure and concentrate it to dryness, add pre-cooled anhydrous ether to wash it until it turns white, and vacuum dry it for 24 hours to obtain DSPE-G. 2 K is a white solid with a yield of 80%. Its mass spectrum is as follows Figure 4 The synthetic route is as follows:

[0058]

[0059] 5) Preparation of polymer nanoparticles by nanoprecipitation method

[0060] Formulating DSPE-G 2 The samples were prepared from tetrahydrofuran solution of DSPE-PEG-S2P (2.0 mg / mL), acetone solution of poly(lactic-co-glycolic acid) (PLGA) (5.0 mg / mL), aqueous solution of DSPE-PEG-S2P (1.0 mg / mL), and aqueous solution of DSPE-PEG-β-CD (1.0 mg / mL).

[0061] siRNA (2 nM) was rapidly mixed with DSPE-G 2 The tetrahydrofuran solution of K was mixed and vortexed for 30 seconds to allow the siRNA to complex with the cationic lipids through electrostatic interaction. Subsequently, the acetone solution of PLGA was added and vortexed rapidly for 30 seconds. 2 Hydrophobic interaction between K and siRNA / DSPE-G 2 K / PLGA mixed solution. The above mixture was added to DSPE-PEG solution (DSPE-PEG-S2P: DSPE-PEG-β-CD = 1:1, V / V), stirred at 4°C for 1 h, centrifuged at 5800 rpm for 40 min at 4°C, centrifuged with pure water for 20 min, and the process was repeated three times to remove the remaining organic solution to prepare nanoparticles containing siRNA.

[0062] The sequence information of the siRNA used in this example is as follows, and each siRNA sequence is suffixed with a TT base overhang (dTdT):

[0063] Non-specific siRNA (siNC): 5'-UUC UCC GAA CGU GUC ACG UdTdT-3' (sense strand, SEQ ID NO. 1) and 5'-ACG UGA CAC GUU CGG AGA AdTdT-3' (antisense strand, SEQ ID NO. 2);

[0064] Luc gene targeting (siLuc): 5'-GGA CGA GGA CGA GCA CUU CUU dTdT-3' (sense strand, SEQ ID NO. 3) and 5'-AAG AAG UGC UCG UCC UCG UCC dTdT-3' (antisense strand, SEQ ID NO. 4);

[0065] siRNA targeting TRPM2 (siTRPM2): 5'-GUC UCG GAC AUC ACU AUC UdTdT-3' (sense strand, SEQ ID NO. 5) and 5'-AGA UAG UGA UGU CCG AGA CdTdT-3' (antisense strand, SEQ ID NO. 6).

[0066] Orthogonal Formulation Screening of Nanoparticles:

[0067] Use the software "Orthogonal Design Assistant" according to L 9 (3 4 ) Design orthogonal experiment. Select DSPE-PEG-S2P, DSPE-PEG-β-CD, DSPE-G 2K and PLGA were four influencing factors, as shown in Table 1. Three different molar levels were set for each factor, and orthogonal screening was performed using the transfection efficiency of siLuc in 4T1-Luc cells as an indicator. The specific process of transfecting 4T1-Luc cells was as follows: 4T1-Luc cells were transfected at 8×10 3 Cells were seeded in a 96-well plate at a density of 1.50 μg / well and incubated at 37°C for 24 h in 15% RPMI-1640. Different formulations containing 150 ng of siLuc were added to each well and incubated at 37°C for 24 h. Protein concentration and Luc fluorescence intensity were determined by BCA method, and the wavelength at 562 nm was detected by microplate reader.

[0068] Table 1. Dosage design of different influencing factors

[0069]

[0070] The specific screening scheme is shown in Table 2.

[0071] Table 2. Orthogonal screening table for nanoparticles

[0072]

[0073]

[0074] The optimal ratios finally screened out are: DSPE-PEG-S2P, DSPE-PEG-β-CD, DSPE-G 2 K and PLGA were compounded in a molar ratio of 18:18:26:10.

[0075] Comparative Example 1

[0076] Preparation of the nanocarrier system CC-DLNPs without targeting peptide S2P: The difference from Example 1 is that in this comparative example, DSPE-PEG without targeting peptide S2P is selected as a raw material to replace DSPE-PEG-S2P, and DSPE-PEG is prepared. 2 K tetrahydrofuran solution (2.0 mg / mL), PLGA acetone solution (5.0 mg / mL), DSPE-PEG aqueous solution (1.0 mg / mL) and DSPE-PEG-β-CD aqueous solution (1.0 mg / mL). Nanoparticles CC-DLNPs, DSPE-PEG, DSPE-PEG-β-CD, DSPE-G 2 The molar ratio of K and PLGA was also selected as 18:18:26:10.

[0077] Comparative Example 2

[0078] Preparation of cyclodextrin-free nanocarrier system AT-DLNPs: The difference from Example 1 is that in this comparative example, DSPE-PEG without cyclodextrin is selected as a raw material to replace DSPE-PEG-β-CD, and DSPE-PEG-β-CD is prepared. 2 The tetrahydrofuran solution of K (2.0 mg / mL), the acetone solution of PLGA (5.0 mg / mL), the aqueous solution of DSPE-PEG (1.0 mg / mL) and the aqueous solution of DSPE-PEG-S2P (1.0 mg / mL) were prepared according to the same steps as step 5) in Example 1. Nanoparticles AT-DLNPs, DSPE-PEG-S2P, DSPE-PEG, DSPE-G 2 The molar ratio of K and PLGA was also selected as 18:18:26:10.

[0079] Comparative Example 3

[0080] Preparation of DLNPs without targeting peptide S2P and cyclodextrin: The difference from Example 1 is that DSPE-PEG is used as a raw material to replace DSPE-PEG-β-CD and DSPE-PEG-S2P to prepare DSPE-PEG- 2 K tetrahydrofuran solution (2.0 mg / mL), PLGA acetone solution (5.0 mg / mL), DSPE-PEG aqueous solution (1.0 mg / mL). Nanoparticles DLNPs, DSPE-PEG, DSPE-G 2 The molar ratio of K and PLGA was 36:26:10.

[0081] Related performance tests:

[0082] 1. Investigating the targeting ability of the nanocarrier systems prepared in Example 1 and Comparative Examples 1-3

[0083] Take RAW 264.7 cells in the logarithmic growth phase, collect them by centrifugation and discard the supernatant, add 5 mL of fresh complete medium, and gently pipette to resuspend the cells. Load the pipetted cell suspension onto a hemocytometer for counting and adjust the cell concentration to 1×10 5 / mL. The cell suspension was inoculated into a 12-well plate, and 700ng / mL lipopolysaccharide (LPS) was added and cultured for 24 hours to allow the cells to completely adhere to the wall. A blank control group and a drug-treated group were set up. The blank group Control was added with basal culture medium; the drug-treated groups were added with 1mL of DLNPs, CC-DLNPs, AT-DLNPs, and ATCC-DLNPs solutions loaded with siRNA (labeled with 0.18nM fluorescein FAM). After culturing for 4h and 8h respectively, each well was washed 3 times with PBS buffer. 400μL PBS solution was added to each well, and the cells were gently blown with a pipette until the cells fell off. The cell suspension was collected in a centrifuge tube, and the cell suspension was continued to be gently blown to disperse the cells evenly. The fluorescence intensity of FAM (EX: 488nm, EM: 525nm) was measured by flow cytometry. Two replicate wells were set for each sample (n=3). Results reference Figure 5 As can be seen from the figure, compared with the CC-DLNPs group, the fluorescence intensity of the AT-DLNPs group and the ATCC-DLNPs group was enhanced and the difference was significant. This proves that the nanocarrier systems AT-DLNPs and ATCC-DLNPs containing the targeting peptide S2P have obvious targeting ability to pro-inflammatory macrophages.

[0084] 2. Investigating the ability of the nanocarrier system prepared in Example 1 and Comparative Examples 1-3 to promote cholesterol efflux from cells

[0085] Take RAW264.7 cells in the logarithmic growth phase, collect them by centrifugation and discard the supernatant, add 5 mL of fresh complete medium, and gently pipette to resuspend the cells. Load the pipetted cell suspension onto a hemocytometer for counting and adjust the cell concentration to 1×10 5 / mL. The cell suspension was inoculated into a 12-well plate. After the cells were completely attached, 700ng / mL lipopolysaccharide (LPS) and culture medium containing 1% NBD cholesterol (10μL) were added and cultured for 12h. A blank control group and a drug-treated group were set up. The blank group Control was added with basal culture medium; the drug-treated groups were added with 1mL of DLNPs, CC-DLNPs, AT-DLNPs, and ATCC-DLNPs solutions containing 0.12nM siNC, respectively. After culturing for 2h and 4h, each well was washed 3 times with PBS buffer. 400μL PBS solution was added to each well, and the cells were gently blown with a pipette until the cells fell off. The cell suspension was collected in a centrifuge tube, and the cell suspension was continued to be gently blown to disperse the cells evenly. The fluorescence intensity of NBD (EX: 488nm, EM: 525nm) was measured by flow cytometry. Two replicate wells were set for each sample (n=3). For results, see Figure 6The results showed that compared with the AT-DLNPs group, the fluorescence intensity in the cells of the CC-DLNPs group and the ATCC-DLNPs group decreased and there was a significant difference. This proved that the cyclodextrin-containing nanocarrier systems CC-DLNPs and ATCC-DLNPs can promote the efflux of cholesterol from cells.

[0086] 3. Investigating the expression levels of TRPM2 and CD36 proteins by the nanocarrier systems prepared in Example 1 and Comparative Examples 1-3

[0087] The Western blot method was used to study the changes in the expression of TRPM2 and CD36, proteins related to the siTRPM2 pathway in cells, to verify whether the nanocarrier system was successfully transfected into the cells and played a role in the siTRPM2 pathway. Take RAW 264.7 cells in the logarithmic growth phase, collect the cells by centrifugation, discard the supernatant, add 5 mL of fresh complete medium, and gently blow to resuspend the cells. Load the evenly blown cell suspension onto a hemocytometer for counting, and adjust the cell concentration to 3×10 5 oxLDL (25 mg / mL) and LPS (0.7 mg / mL) were added. The cell suspension was inoculated into a 60 mm cell culture dish (5 mL of cell suspension per dish). The culture dish with cells was placed at 37°C and 5% CO. 2 Cultured in an incubator with appropriate conditions for 24 hours until the cells adhered to the wall. The experiment set up a blank control group Control, a drug-treated blank control group Control+ATCC-DLNPs (C+ATCC-DLNPs), a control group oxLDL+LPS (LO), drug-treated groups DLNPs, CC-DLNPs, AT-DLNPs, ATCC-DLNPs / siNC, and ATCC-DLNPs after the addition of oxLDL and LPS stimulation (except for the group marked with siNC, all other drug-treated groups not marked separately were complexed with siTRPM2). Placed in an incubator and cultured for 48 hours. Proteins were extracted and lysed with RIPA lysis buffer, and the protein concentration was determined by the BCA method. The content of each sample was calculated based on the measured absorbance. Proteins of each group were diluted to the same concentration with RIPA lysis buffer, and 5×SDS protein loading buffer was added at a mass ratio of 1:4, and heated for denaturation. Gel preparation, sample loading, electrophoresis, membrane transfer, blocking, incubation with primary and secondary antibodies, and development were performed. For results, see Figure 7The results in the figure show that in terms of TRPM2 protein expression, the TRPM2 expression level of the LO group was significantly higher than that of the Control group, and the TRPM2 expression level of the ATCC-DLNPs group was significantly lower than that of the LO group, while the TRPM2 expression levels of the DLNPs, CC-DLNPs, and AT-DLNPs groups did not change significantly. This proves that the ATCC-DLNPs group may have a stronger effect of silencing TRPM2. The trend of CD36 protein expression is the same as that of TRPM2 protein expression. This proves that the ATCC-DLNPs group has a stronger silencing effect on both TRPM2 and CD36 proteins.

[0088] 4. Investigation of the in vivo targeting ability of the nanocarrier systems ATCC-DLNPs and CC-DLNPs:

[0089] Cy5.5-CC-DLNPs and Cy5.5-ATCC-DLNPs containing 1.6 mg / kg Cy5.5 were injected into the tail vein of 8-week-old male ApoE- / - mice (after 2 weeks of high-fat diet modeling). After 4h and 12h, respectively, the mouse aorta was stripped and imaged and quantified under the conditions of excitation wavelength of 670±10nm and emission wavelength of 700±10nm. The results are shown in Figure 8 The results showed that the fluorescence intensity in the blood vessels of the ATCC-DLNPs group was significantly stronger than that of the CC-DLNPs group without targeting effect 4 hours after injection, and the ATCC-DLNPs group still had strong fluorescence after 12 hours. This proves that the ATCC-DLNPs group can quickly target the diseased aortic vascular area in the modeling mice within 4 hours and has a longer retention effect.

[0090] 5. Application of Nanocarrier Systems in the Treatment of Atherosclerosis

[0091] Eight-week-old male ApoE- / - mice were randomly divided into five groups (n=5): control group (Saline), DLNPs group, CC-DLNPs group, AT-DLNPs group, and ATCC-DLNPs group. The mice were fed a high-fat diet for 2 months. Subsequently, the five groups of mice were intravenously injected with normal saline, DLNPs, CC-DLNPs, AT-DLNPs, and ATCC-DLNPs, each containing 3 mg / kg siTRPM2, for 3 consecutive weeks. After the experiment, all mice were euthanized, the aorta of the mice was dissected under a microscope, and the plaques in the aorta were stained with oil red (ORO). For results, see Fig. 9The results in the figure show that the Saline group had the highest content of plaque in the aorta, while the ATCC-DLNPs group had significantly lower content of plaque in the aorta than the other three groups; compared with the Saline group, the differences in the content of plaque in the blood vessels of the DLNPs, AT-DLNPs and CC-DLNPs groups were smaller, proving that the treatment effects of the DLNPs, AT-DLNPs and CC-DLNPs groups were not ideal, and the ATCC-DLNPs group could significantly slow down the development of atherosclerotic disease and had great application potential in helping to treat atherosclerosis.

[0092] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A dual-functional polymer nanocarrier system, characterized in that: The carrier system is prepared by nanoprecipitation method with DSPE-PEG-S2P, DSPE-PEG-β-CD, DSPE-G2K and PLGA as raw materials; siTRPM2 is complexed with the cationic lipid carrier DSPE-G2K through electrostatic interaction, PLGA is coated on the periphery of the cationic lipid based on the hydrophobic interaction between PLGA and DSPE-G2K, and the modified targeting peptide S2P and β-CD are jointly connected to the carrier surface.

2. The method for preparing a bifunctional polymer nanocarrier system according to claim 1, characterized in that: The steps include: 1) coupling β-CD-NH2 to DSPE-PEG-NHS through an amidation reaction to prepare DSPE-PEG-β-CD; 2) Dissolve DSPE-PEG-MAL and S2P in DMF, add Et3N, stir to react, evaporate and concentrate, dialyze, and freeze-dry to obtain DSPE-PEG-S2P; 3) Under an inert atmosphere, H-Lys-OMe·2HCl, Boc-Lys(Boc)-OH, HOBT, HBTU and DIPEA were mixed, and DMF was added to dissolve. After reacting in an ice bath, the mixture was stirred at room temperature for reaction, extracted, washed, salted out, and dried to obtain a crude product solution, which was concentrated and purified to obtain G2K(MeO); 4) Dissolve G2K(MeO) in NaOH / MeOH, react in an ice bath, adjust the pH of the solution, remove MeOH by rotary evaporation, add ethyl acetate, wash, salt out, and dry to obtain a crude product solution, and concentrate the filtrate to obtain G2K(COOH); 5) Under an inert atmosphere, DSPE, G2K(COOH), HOBT, HBTU and DIPEA were mixed, anhydrous CHCl3 / DMF was added to dissolve, reacted in an ice bath, heated and stirred to react, extracted, washed, salted out, and dried to obtain a crude product solution, which was concentrated and purified to obtain DSPE-G2K(Boc)4; 6) Weigh DSPE-G2K(Boc)4 into a reactor, add a magnetic particle and seal it, add DCM to dissolve under inert gas protection, add TFA, stir and react in an ice bath, stir and react at room temperature, and after the reaction is completed, evaporate and concentrate under reduced pressure, wash, and dry to obtain DSPE-G2K as a white solid; 7) preparing a tetrahydrofuran solution of DSPE-G2K, an acetone solution of PLGA, an aqueous solution of DSPE-PEG-S2P and an aqueous solution of DSPE-PEG-β-CD, first mixing siTRPM2 with the tetrahydrofuran solution of DSPE-G2K, vortexing to complex siTRPM2 with the cationic lipid, then adding the acetone solution of PLGA, vortexing to mix, then adding the aqueous solution of DSPE-PEG-S2P and the aqueous solution of DSPE-PEG-β-CD, stirring in an ice bath, and centrifuging to obtain a bifunctional polymer nanocarrier system containing siTRPM2.

3. The method for preparing a bifunctional polymer nanocarrier system according to claim 2, characterized in that: In step 2), the molar ratio of DSPE-PEG-MAL, S2P and Et3N is 1:1:1-3.

4. The method for preparing a bifunctional polymer nanocarrier system according to claim 2, characterized in that: In step 3), the molar ratio of H-Lys-OMe·2HCl, Boc-Lys(Boc)-OH, HOBT, HBTU and DIPEA is 1:1-2:2-5:2-5:4-10, the reaction is carried out in an ice bath for 0.5-1 h, and the reaction is stirred at room temperature for 18-36 h.

5. The method for preparing a bifunctional polymer nanocarrier system according to claim 2, characterized in that: In step 5), the molar ratio of DSPE, G2K(COOH), HOBT, HBTU and DIPEA is 1:1-2:2-4:2-4:8-14. After reacting in an ice bath for 20-60 min, the mixture is heated to 45-50° C. and stirred for reaction for 18-24 h.

6. The method for preparing a dual-functional polymer nanocarrier system according to claim 2, characterized in that: In step 7), the molar ratio of DSPE-PEG-S2P, DSPE-PEG-β-CD, DSPE-G2K and PLGA is 8-28:8-28:12-40:10-14; the time of the two vortex processes is 20-60 seconds, and the ice bath stirring time is 0.5-1.5 hours.

7. The method for preparing a bifunctional polymer nanocarrier system according to claim 6, characterized in that: In step 7), the molar ratio of DSPE-PEG-S2P, DSPE-PEG-β-CD, DSPE-G2K and PLGA is 18:18:26:

10.

8. Use of a bifunctional polymer nanocarrier system as claimed in claim 1 in the preparation of targeted drugs for atherosclerosis.

9. An atherosclerosis targeted drug, characterized in that: The drug comprises the bifunctional polymer nanocarrier system according to claim 1.

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