ROS-responsive siRNA nano-micelle as well as preparation method and application thereof

Abstract

The invention relates to the field of compounds and biological medicines, and discloses ROS (reactive oxygen species) responsive siRNA (small interfering Ribonucleic Acid) nano-micelles as well as a preparation method and application thereof. According to the invention, a polymer synthesized by adopting a TK-GUA monomer with guanidyl is tightly combined with siRNA through electrostatic interaction, a hydrogen bond and a stable salt bridge structure. Under the action of high-concentration ROS, the ROS response type siRNA nano-micelle disclosed by the invention can be subjected to responsive splitting, so that efficient release of siRNA is realized. Wherein the polymer B has remarkable ROS responsiveness and can trigger release of siRNA under the action of 5 mM hydrogen peroxide, and the regulation and control capability of the polymer B is superior to that of the prior art. The siRNA polymer nano-micelle disclosed by the invention can be combined with sorafenib to realize a synergistic anti-tumor effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of compounds and biological medicine, in particular, to ROS-responsive siRNA nanomicelles and preparation method and application thereof. BACKGROUND

[0002] Primary liver cancer mainly includes hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC) and combined hepatocellular-cholangiocarcinoma (cHCC-CCA) three different pathological types, the three in pathogenesis, biological behavior, histopathology, treatment method and prognosis etc. have big difference, among them HCC accounts for 75% ~ 85%, ICC accounts for 10% ~ 15%.

[0003] The occurrence and development of HCC are closely related to multiple signal pathways, including Wnt, MAPK, mTOR, STAT, Hippo and Hedgehog, etc. The existing liver cancer treatment methods have the defects of large trauma, difficult to cure, easy to relapse, high drug resistance and large systemic toxicity. Therefore, it is urgent to develop new technology to provide more high-quality options for patients.

[0004] RNA interference (RNAi) is a biological process that specifically degrades target gene mRNA by small molecule RNA (such as small interfering RNA, siRNA), thereby inhibiting gene expression. RNA interference technology has the characteristics of high efficiency and strong specificity, which can target to silence cancer genes or regulate tumor-related signal pathways, thereby inhibiting the proliferation, migration and invasion of tumor cells. Although RNA interference technology has good prospects in liver cancer, RNA interference technology in drug delivery faces many challenges. First, siRNA molecules are negatively charged and have poor stability, and are easily degraded by nucleases, making it difficult to penetrate the cell membrane. Second, siRNA is easily cleared in the body, difficult to circulate for a long time, and lacks tissue targeting. Therefore, it is particularly important to design a delivery carrier to protect siRNA and achieve targeted delivery.

[0005] CN109880021A discloses a polymer and a preparation method thereof, a ROS-responsive siRNA nanomicelle and an application thereof. The polymer is a strong brain-targeting ROS-responsive siRNA nanomicelle, and is not suitable for tumors other than brain tumors. The polymer is formed by polymerization of specific monomers, and can bind with siRNA to form stable nanomicelles. The nanomicelle has the characteristics of no cytotoxicity and effective release of siRNA, and can be used for the preparation of drugs for inhibiting tumor cell growth and / or inhibiting neovascularization in tumor sites. However, the polymer nanomicelle of the patent realizes the active transmembrane ability of the blood-brain barrier through the Angiopep-2 ligand, and is mainly used for targeting central nervous system tumors such as brain glioma (GBM). This brain penetration effect may not have significant therapeutic effect on other non-brain metastatic tumor diseases, and may increase the distribution of drugs in brain tissue, thereby bringing unnecessary side effect risks. Therefore, its brain penetration characteristics do not have advantages in non-brain tumor treatment, and may even limit its wide application in other tumor types. SUMMARY

[0006] The purpose of the present application is to provide a ROS-responsive siRNA nanomicelle and a preparation method and application thereof.

[0007] To achieve the purpose of the present application, in a first aspect, the present application provides a polymer B, which is Meo-PEG 2k -P(TK-GUA), and the structure is as follows: Wherein, n is 45; x is an integer between 10-20.

[0008] Preferably, the polymer B is Meo-PEG 2k -P(TK-GUA) 6.2k , wherein n is 45, and x is 16.

[0009] In a second aspect, the present application provides a polymer C, which is GA-PEG 3.4k -P(TK-GUA), and the structure is as follows: Wherein, n is 77; x is an integer between 10-20.

[0010] Preferably, the polymer C is GA-PEG 3.4k -P(TK-GUA) 5.5k , wherein n is 77, and x is 14.

[0011] In the present application, PEG 2k refers to PEG2000, and PEG 3.4k refers to PEG3400.

[0012] In a third aspect, the present application provides a composition comprising the polymers B and C.

[0013] In a fourth aspect, the present application provides a preparation method of the polymer B, which is obtained by polymerization of TK-GUA and Meo-PEG 2k -CPADN, according to the following reaction equation: wherein, Meo-PEG 2k -CPADN is obtained by reaction of 4-cyano-4-(thiobenzoyl) valeric acid and Meo-PEG 2k -NH2. Refer to CN109880021A. The synthesis route is as follows: In a fifth aspect, the present application provides a preparation method of the polymer C, which is obtained by polymerization of TK-GUA and GA-PEG 3.4K -CPADN, according to the following reaction equation: wherein, GA-PEG 3.4k -CPADN is obtained by reaction of glycyrrhetic acid and Fmoc-PEG 3.4k -NH2. The synthesis route is as follows: In the present application, the synthesis route of TK-GUA is as follows: In a sixth aspect, the present application provides a ROS-responsive siRNA nanomicelle, which comprises a polymer and siRNA (molecular weight of about 13 kDa); The polymer is selected from at least one of the polymers B and C.

[0014] In a seventh aspect, the present application provides a preparation method of the nanomicelle, comprising the following steps: 1) Dissolve the polymer in HEPES buffer (10 mM, pH 7.4), and dissolve the siRNA powder in DEPC-treated water to prepare a polymer solution and an siRNA solution, respectively; 2) Mix the siRNA solution and the polymer solution at a molar ratio of 1:2.5-1:40, and incubate at room temperature (for example, for 30 minutes) to form siRNA polymer nanomicelles by self-assembly of siRNA and the polymer.

[0015] Preferably, the siRNA solution and the polymer solution in 2) are mixed at a molar ratio of 1:8.

[0016] More preferably, the polymer is formed by mixing polymers B and C in a molar ratio of 9:1.

[0017] In an eighth aspect, the present application provides any of the following applications of the nanomicelles: i) as siRNA delivery systems (including non-disease diagnosis and treatment purposes); ii) for preparing anti-tumor (including but not limited to hepatocellular carcinoma) drugs.

[0018] In a ninth aspect, the present application provides an anti-tumor drug comprising sorafenib and the nanomicelles.

[0019] In a tenth aspect, the present application provides the use of the anti-tumor drug in the treatment of cancer (including but not limited to hepatocellular carcinoma).

[0020] By means of the above technical solutions, the present application has at least the following advantages and beneficial effects: (1) Enhanced siRNA loading capacity. The polymer synthesized by the TK-GUA monomer with a guanidyl (Gu + ) group can be tightly combined with siRNA through electrostatic interaction, hydrogen bonding and stable salt bridge structure. Gel retardation experiments show that the polymer B / C can completely load siRNA when the molar ratio of polymer to siRNA is 8:1.

[0021] (2) Significant improvement in serum stability. Serum stability tests show that the polymer B-siRNA complex can still observe clear siRNA bands after incubation in 50% fetal bovine serum for 24 hours, while free siRNA and commercial carrier Lip3000-siRNA complex are significantly degraded after 6 hours. This improvement significantly improves the serum stability of siRNA, ensuring its stability in the in vivo environment.

[0022] (3) Significant ROS response. Polymer B has significant ROS responsiveness and can trigger the release of siRNA under the action of 5 mM hydrogen peroxide, while polymer A does not show H2O2 responsiveness. Therefore, the regulation ability of polymer B on siRNA release under ROS environment is superior to that of polymer A.

[0023] (4) Good biological safety. Through CCK-8 research, it is verified that the carriers do not show obvious cytotoxicity at a drug concentration of 200 nM siNC.

[0024] (Five) Significant synergy with sorafenib. The present application creates an intelligent platform that can synergize with sorafenib and "self-activate", sorafenib, while exerting anti-tumor effects, will induce tumor cells to produce exogenous ROS. This drug-induced ROS, combined with the inherent endogenous ROS in the tumor microenvironment, can greatly accelerate the cleavage of the thioether bond, thereby significantly increasing the release rate of siRNA. The released siRNA down-regulates the expression of β-Catenin protein, enhances the anti-tumor activity of sorafenib, and forms a virtuous cycle with sorafenib treatment: the degradation of β-Catenin enhances the efficacy of sorafenib, and the ROS induced by sorafenib promotes the release of more siRNA. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 For Meo-PEG in the preferred embodiment of the present application 2k H-NMR of -P(GUA).

[0026] Figure 2 For GUA H-NMR in the preferred embodiment of the present application

[0027] Figure 3 For TK-GUA H-NMR in the preferred embodiment of the present application

[0028] Figure 4 For Meo-PEG in the preferred embodiment of the present application 2k H-NMR of -P(TK-GUA).

[0029] Figure 5 For GA-PEG in the preferred embodiment of the present application 3.4k H-NMR of -P(TK-GUA).

[0030] Figure 6 For the characterization of siRNA polymer nanomicelles in the preferred embodiment of the present application

[0031] Figure 7 For the release of siRNA in A-siRNA and B-siRNA after H2O2 action determined by gel electrophoresis in the preferred embodiment of the present application

[0032] Figure 8 For the results of agarose experiments after siRNA micelles were treated with FBS or inactivated FBS for different times in the preferred embodiment of the present application

[0033] Figure 9 For the results of carrier cell toxicity CCK-8 assay in the preferred embodiment of the present application

[0034] Figure 10Hep-G2 cell uptake of siRNA polymer nanomicelles observed by confocal microscope in the preferred embodiment of the present application.

[0035] Figure 11 Hep-G2 cell uptake of siRNA polymer nanomicelles detected by flow cytometry in the preferred embodiment of the present application.

[0036] Figure 12 Lysosomal escape of siRNA polymer nanomicelles observed by confocal microscope in the preferred embodiment of the present application.

[0037] Figure 13 Effect of siRNA micelles on expression of β-Catenin protein in Hep-G2 analyzed by Western-Blot in the preferred embodiment of the present application.

[0038] Figure 14 Cytotoxicity CCK-8 assay results of siRNA and Sorafenib combination in the preferred embodiment of the present application.

[0039] Figure 15 Effect of Sorafenib on ROS in Hep-G2 cells analyzed by flow cytometry in the preferred embodiment of the present application.

[0040] Figure 16 Effect of Sorafenib on lysosomal co-localization of siRNA analyzed by confocal microscope in the preferred embodiment of the present application.

[0041] Figure 17 Results of ex vivo imaging of main organs of mice after tail vein injection of B-siRNA and B / C-siRNA for 24 hours in the preferred embodiment of the present application. DETAILED DESCRIPTION

[0042] The present application provides polymers B and C and a preparation method thereof.

[0043] The present application also provides a ROS-responsive self-activated targeted siRNA nanomicelle and an application thereof.

[0044] The present application provides a ROS-responsive siRNA nanomicelle, which can be responsively cleaved under the action of high-concentration ROS to realize efficient release of siRNA. The polymer B has significant ROS responsiveness and can trigger release of siRNA under the action of 5 mM hydrogen peroxide, and has better regulation capacity than the prior art. The polymer of the present application can be combined with Sorafenib to realize synergistic anti-tumor effect.

[0045] The present application adopts the following technical solutions: The application provides a ROS-responsive siRNA nanomicelle, which comprises a polymer and siRNA. wherein n is 45, and x is an integer between 10 and 20.

[0046] Preferably, the polymer B is Meo-PEG 2k -P(TK-GUA) 6.2k wherein n is 45, and x is 16.

[0047] The polymer C is: wherein n is 77, which is the monomer repeat number of PEG3400, and x is an integer between 10 and 20.

[0048] Preferably, the polymer C is GA-PEG 3.4k -P(TK-GUA) 5.5k wherein n is 77, and x is 14.

[0049] The preparation of the polymer carrier is as follows: TK-GUA and Meo-PEG 2k -CPADN is polymerized to obtain the polymer B; TK-GUA and GA-PEG 3.4K -CPADN is polymerized to obtain the polymer C.

[0050] wherein Meo-PEG 2k -CPADN is obtained by the reaction of 4-cyano-4-(thiobenzoyl) valeric acid and Meo-PEG 2k -NH2; GA-PEG 3.4k -CPADN is obtained by the reaction of glycyrrhetic acid and Fmoc-PEG 3.4k -NH2.

[0051] The application also provides a ROS-responsive siRNA nanomicelle, which comprises a polymer and siRNA, and the polymer is a combination of the polymer B and the polymer C.

[0052] The ROS-responsive siRNA nanomicelle is mainly prepared by the following steps: The high molecular polymer is dissolved in 10 mM HEPES buffer solution (pH 7.4) respectively, and the siRNA powder is dissolved in DEPC-treated water to prepare a solution for use.

[0053] 1) Mix siRNA solution with each polymer solution at pre-set molar ratio (e.g. 1:2.5, 1:5, 1:8, 1:10, 1:20, 1:40), make sure the total volume of each sample is consistent, and the final concentration of siRNA is kept constant.

[0054] 2) Mix the solution well by blowing, then incubate at room temperature for 30 minutes, so that siRNA and polymer form siRNA polymer nanomicelles through self-assembly.

[0055] The application further provides the ROS-responsive siRNA nanomicelles in the field of cancer.

[0056] Preferably, the cancer is hepatocellular carcinoma.

[0057] The following examples are used to illustrate the present application, but not to limit the scope of the present application. If not specifically indicated, the technical means used in the examples are the conventional means known to those skilled in the art, and the raw materials used are commercially available.

[0058] Comparative Example 1 Polymer A (reference CN109880021A) Dissolve Meo-PEG 2k -CPADN, GUA and azobisisobutyronitrile in 2 mL of anhydrous N,N-dimethylformamide (DMF), under nitrogen protection, stir the reaction mixture at 70°C for 24 hours. After the reaction is completed, the light yellow solid product Meo-PEG 2k -P(GUA) 3.3k , wherein n is 45 and x is 18. The hydrogen nuclear magnetic resonance spectrum is as shown in Figure 1 .

[0059] Synthesis of key intermediates in Example 1 1. Synthesis of GUA N-(3-aminopropyl)methacrylate hydrochloride, 2.80 mmol of pyrazole amidine and 6.70 mmol of triethylamine (TEA) were dissolved in 8 mL of N,N-dimethylformamide (DMF), followed by the addition of 0.045 mmol of hydroquinone. The reaction mixture was stirred at room temperature for 24 hours under nitrogen protection. After the reaction was completed, the reaction solution was slowly added dropwise into 50 mL of pre-cooled diethyl ether to induce the precipitation of the product. The supernatant was removed by centrifugation, and the obtained organic phase precipitate was washed with acetonitrile (10 mL) and triethylamine (0.5 mL) twice, respectively, and then washed with dichloromethane (15 mL) once. Finally, the obtained pale yellow precipitate was dried under vacuum to obtain the target product GUA, whose proton nuclear magnetic resonance spectrum is shown in Figure 2

[0060] 2. Synthesis of TK-GUA hydrochloride was dissolved in 60 mL of dichloromethane (DCM), followed by the addition of 425.66 mmol (59.3 mL) of triethylamine (TEA). Under nitrogen protection and 0°C ice bath conditions, 210.03 mmol (30 mL) of trifluoroacetic anhydride (TFAA) was slowly added dropwise through a constant pressure dropping funnel. After the addition was completed, the reaction temperature was maintained and stirring was continued for 2 hours. After the reaction was completed, 20 mL of deionized water was slowly added to the system to quench the reaction. The reaction solution was transferred to a separatory funnel and extracted with 60 mL of dichloromethane and saturated sodium chloride solution three times. The organic phase was combined, dried with an appropriate amount of anhydrous sodium sulfate, filtered, and the filtrate was collected. The solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. Finally, the target product S1 in the form of yellow oil was obtained by purification using a 200-mesh alumina column chromatography with dichloromethane as the eluent.

[0061] hydrochloride was dissolved in 60 mL of dichloromethane (DCM), followed by the addition of 425.66 mmol (59.3 mL) of triethylamine (TEA). Under nitrogen protection and 0°C ice bath conditions, 210.03 mmol (30 mL) of trifluoroacetic anhydride (TFAA) was slowly added dropwise through a constant pressure dropping funnel. After the addition was completed, the reaction temperature was maintained and stirring was continued for 2 hours. After the reaction was completed, 20 mL of deionized water was slowly added to the system to quench the reaction. The reaction solution was transferred to a separatory funnel and extracted with 60 mL of dichloromethane and saturated sodium chloride solution three times. The organic phase was combined, dried with an appropriate amount of anhydrous sodium sulfate, filtered, and the filtrate was collected. The solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. Finally, the target product S1 in the form of yellow oil was obtained by purification using a 200-mesh alumina column chromatography with dichloromethane as the eluent.​

[0062] Dissolve 16.08 mmol S2 in 10 mL of methanol (CH3OH) and slowly add 10 mL of 4 M aqueous sodium hydroxide (NaOH) solution through a constant pressure dropping funnel under stirring at room temperature. After the addition is completed, continue stirring the reaction at room temperature for 4 hours. After the reaction is completed, remove the methanol by rotary evaporation under reduced pressure. Transfer the remaining reaction solution to a separatory funnel and extract three times with 20 mL of ethyl acetate (EtOAc) and saturated sodium chloride solution. Combine the organic phases, dry over an appropriate amount of anhydrous sodium sulfate, filter, and collect the filtrate. Remove the solvent by rotary evaporation under reduced pressure to obtain the product S3 as a yellow oil.

[0063] Dissolve 0.71 mmol (60.8 mg) of methacrylic acid, 0.71 mmol (269.8 mg) of O-(7-azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and 1.42 mmol (247.3 μL) of N,N-diisopropylethylamine (DIPEA) in 5 mL of anhydrous N,N-dimethylformamide (DMF) and stir at room temperature (25 °C) for 5 minutes to activate. Then add 0.59 mmol of S3 and continue stirring the reaction at room temperature for 2 hours. After the reaction is completed, quench the reaction by adding 10 mL of 1 M aqueous sodium hydroxide (NaOH) solution to the reaction system. Transfer the reaction solution to a separatory funnel and extract three times with 20 mL of dichloromethane and saturated sodium chloride solution. Combine the organic phases, dry over an appropriate amount of anhydrous sodium sulfate, filter, and collect the filtrate. Remove the solvent by rotary evaporation under reduced pressure to obtain the crude product. Finally, purify the product using reverse phase chromatography with water and acetonitrile as eluents and an octadecylsilane-bonded silica gel (C18) column to obtain the product S4 as a yellow oil.

[0064] Dissolve 0.63 mmol (204.20 mg) of 4-(fluorenylmethoxycarbonylamino) butyric acid, 0.63 mmol (239.00 mg) of HATU and 1.25 mmol (219.00 μL) of N,N-diisopropylethylamine (DIPEA) in 5 mL of anhydrous N,N-dimethylformamide (DMF) and stir for activation at room temperature (25 °C) for 5 minutes. Then add 0.52 mmol of S4 and continue stirring the reaction at room temperature for 2 hours. After the reaction is completed, quench the reaction by adding 10 mL of 1M aqueous sodium hydroxide (NaOH) solution to the reaction system. Transfer the reaction solution to a separatory funnel and extract three times with 20 mL of dichloromethane and saturated sodium chloride solution. Combine the organic phases, dry over an appropriate amount of anhydrous sodium sulfate, filter, and collect the filtrate. Concentrate the filtrate by rotary evaporation under reduced pressure to remove the solvent and obtain the crude product. Finally, purify the product using reverse phase chromatography with water and acetonitrile as eluents and an octadecylsilane-bonded silica gel (C18) column to obtain the product S5 as a yellow oil.

[0065] Dissolve 0.35 mmol (200.00 mg) of compound S5 in 4 mL of anhydrous N,N-dimethylformamide (DMF) and slowly add 1 mL of piperidine while stirring at room temperature (25 °C) for 4 hours. After the reaction is completed, add 20 mL of deionized water to the reaction system and observe the precipitation of white solids. Isolate the white solids by filtration, retain the filtrate, and rotary evaporate the filtrate under reduced pressure to remove the solvent and finally obtain the product S6 as a yellow oil.

[0066] Dissolve 0.33 mmol of compound S6, 0.33 mmol of Praxadine and 0.79 mmol of triethylamine (TEA) in 4 mL of anhydrous N,N-dimethylformamide (DMF) and then add 0.005 mmol of hydroquinone as a catalyst. Stir the reaction mixture at room temperature (25 °C) for 24 hours under nitrogen protection. After the reaction is completed, dissolve in a small amount of water and freeze-dry to remove the solvent. Finally, separate the product using an octadecylsilane-bonded silica gel (C18) column with water and acetonitrile as gradient eluents to obtain the product TK-GUA as a pale yellow solid. The nuclear magnetic resonance hydrogen spectrum is shown in Figure 3

[0067] The synthesis route is as follows: 3、Meo-PEG 2k Synthesis of CPADN ​Dissolve 1.19 mmol of N-hydroxysuccinimide and 1.19 mmol of 4-cyano-4-(thiobenzoyl) valeric acid in 3.5 mL of anhydrous chloroform (CHCl3). Dissolve another 1.19 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in 1.5 mL of anhydrous chloroform, and slowly add it dropwise to the reaction system under stirring, and continue the reaction at room temperature for 24 hours. After the reaction is completed, filter the reaction solution, and reserve the filtrate. Add 5 mL of water to the filtrate, extract it with dichloromethane three times, combine the organic phases, and wash them with saturated brine. Dry the organic phase over anhydrous sodium sulfate, and then spin dry the solvent. Purify the crude product by silica gel column chromatography, using dichloromethane:methanol (800:1, v / v) as the eluent, to obtain the target product S1 as a pink solid.

[0068] Dissolve 0.11 mmol of compound S1 and 0.08 mmol of Meo-PEG 2k -NH2 in anhydrous dichloromethane, and stir the reaction at room temperature for 24 hours. After the reaction is completed, slowly add the reaction solution dropwise to 80 mL of ice ethyl ether, and observe the generation of a large amount of pink suspension. Then, centrifuge the precipitated product at 4°C. To obtain a high-purity product, redissolve the precipitate in 4 mL of dichloromethane, add it dropwise to ice ethyl ether again for precipitation, centrifuge and separate, and repeat this purification step three times. Finally, place the collected pink precipitate in a vacuum freeze dryer to obtain the target product Meo-PEG 2k -CPADN.

[0069] 4、GA-PEG 3.4k Synthesis of GA-PEG Dissolve 6.00 mmol of glycyrrhetinic acid, 6.00 mmol of HATU, and 12.00 mmol of N,N-diisopropylethylamine (DIPEA) in 5 mL of anhydrous N,N-dimethylformamide (DMF), and stir the mixture at room temperature (25°C) for 5 minutes. Then, add 5.00 mmol of Fmoc-PEG 3.4k -NH2 to the reaction system, and continue to stir the reaction at room temperature for 4 hours. After the reaction is completed, transfer the reaction solution to a 4000 Da dialysis bag, and first dialyze it in a mixture of methanol and water (1:1 by volume) for 24 hours, and then dialyze it in pure water for 24 hours. After the dialysis is completed, take out the sample, and dry it using a freeze dryer to obtain the product S1 as a white powder.

[0070] S1 was dissolved in 4 mL of anhydrous N,N-dimethylformamide (DMF), 1 mL of piperidine was slowly added under stirring at room temperature (25 °C) and the reaction was continued for 6 hours. After the reaction was completed, the reaction solution was transferred to a 4000 Da dialysis bag, first dialyzed in a mixture of methanol and water (volume ratio 1:1) for 24 hours, and then dialyzed in pure water for 24 hours. After dialysis was completed, the sample was taken out and dried using a freeze dryer to obtain a white powdery product S2.

[0071] CPADN, 0.62 μmol HATU and 1.23 μmol N,N-diisopropylethylamine (DIPEA) were dissolved in 2 mL of anhydrous N,N-dimethylformamide (DMF), and stirred at room temperature (25 °C) for 5 minutes. Then, 0.51 μmol S2 was added to the reaction system, and the reaction was continued under stirring at room temperature for 4 hours. After the reaction was completed, the reaction solution was transferred to a 4000 Da dialysis bag, first dialyzed in a mixture of methanol and water (volume ratio 1:1) for 24 hours, and then dialyzed in pure water for 24 hours. After dialysis was completed, the sample was taken out and dried using a freeze dryer to obtain a light pink solid product GA-PEG 3.4k -CPADN.

[0072] Example 2 Preparation of polymer B Meo-PEG 2k -CPADN, 260.00 μmol TK-GUA and 4.14 μmol azobisisobutyronitrile were dissolved in 2 mL of anhydrous N,N-dimethylformamide (DMF), and the reaction mixture was stirred at 70 °C for 24 hours under nitrogen protection. After the reaction was completed, the reaction solution was transferred to a 2000 Da dialysis bag, first dialyzed in a mixture of methanol and water (volume ratio 1:1) for 24 hours, and then dialyzed in pure water for 24 hours. After dialysis was completed, the sample was taken out and dried using a freeze dryer to obtain a light yellow solid product Meo-PEG 2k -P(TK-GUA) 6.2k , and the nuclear magnetic resonance hydrogen spectrum is as shown in Figure 4 .

[0073] wherein n is 45 and x is 16.

[0074] Example 3 Preparation of polymer C GA-PEG 3.4kCPADN, 173.00 μmol TK-GUA and 2.16 μmol azobisisobutyronitrile were dissolved in 2 mL of anhydrous N,N-dimethylformamide (DMF) under nitrogen protection. The reaction mixture was stirred at 70 °C for 24 hours. After the reaction was completed, the reaction solution was transferred to a 4000 Da dialysis bag and dialyzed in a mixture of methanol and water (1:1, by volume) for 24 hours, and then dialyzed in pure water for 24 hours. After dialysis was completed, the sample was taken out and dried using a freeze dryer to obtain a light yellow solid product GA-PEG 3.4k P(TK-GUA) 5.5k , whose 1H nuclear magnetic resonance spectrum is shown in Figure 5

[0075] wherein n is 77 and x is 14.

[0076] Example 4 Preparation and characterization of siRNA polymer nanomicelles The high molecular polymer Meo-PEG 2k P(GUA) 3.3k , Meo-PEG 2k P(TK-GUA) 6.2k and GA-PEG 3.4k P(TK-GUA) 5.5k were respectively dissolved in 10 mM HEPES buffer solution (pH 7.4) to prepare a solution with a concentration of 1 mg / mL for standby. At the same time, siRNA (siRNA target sequence: GCCACAAGATTACAAGAAA) powder was dissolved in DEPC-treated water to prepare a stock solution with a concentration of 20 μM for standby.

[0077] In the preparation of siRNA-polymer nanomicelles, the specific molar ratio between siRNA and polymer was calculated. The specific operation steps are as follows: (1) The siRNA solution and each polymer solution were mixed according to the preset molar ratio (for example, 1:2.5, 1:5, 1:8, 1:10, 1:20, 1:40) to ensure that the total volume of each sample was consistent and the final concentration of siRNA was constant.

[0078] (2) The mixed solution was thoroughly mixed by blowing and then incubated at room temperature for 30 minutes to allow siRNA and polymer to form siRNA polymer nanomicelles through self-assembly.

[0079] Through the above method, siRNA polymer nanomicelles with stable structure can be successfully prepared.

[0080] The high molecular polymer Meo-PEG​2k -P(GUA) 3.3k , Meo-PEG 2k -P(TK-GUA) 6.2k , GA-PEG 3.4k -P(TK-GUA) 5.5k , Meo-PEG 2k -P(TK-GUA) 6.2k + GA-PEG 3.4k -P(TK-GUA) 5.5k (molar ratio 9:1) and siRNA were A-siRNA, B-siRNA, C-siRNA and B / C-siRNA, respectively. Then, the binding ability of polymers and siRNA was evaluated by agarose gel electrophoresis, and the morphology and particle size of siRNA polymer nanomicelles were characterized by dynamic light scattering (Malvern Panalytical) and transmission electron microscopy (Hitachi HT-7800) as shown in Figure 6 When the molar ratio of polymer A and siRNA was 6, siRNA could be completely complexed with polymer A; when the molar ratio of polymer B and siRNA was 8, siRNA could be completely complexed with polymer B; when the molar ratio of polymer C and siRNA was 10, siRNA could be completely complexed with polymer C; when the molar ratio of polymer B / C (molar ratio B:C = 9:1) and siRNA was 8, siRNA could be completely complexed with polymer B / C. TEM results showed that each group of polymers and siRNA formed nearly circular nanomicelles with a size of about 30 nm. Considering the loading capacity and particle size, the molar ratio of polymer and siRNA was 8:1 in the subsequent experiments.

[0081] Example 5 ROS-responsive test of siRNA polymer nanomicelles With the A group of polymers without thio ketone bond as a control, A-siRNA and B-siRNA polymer nanomicelles were prepared according to the molar ratio of siRNA (siRNA target sequence: GCCACAAGATTACAAGAAA) and polymer was 1:8. Subsequently, different concentrations of hydrogen peroxide were added to each group, and after 12 hours, the release of siRNA was detected by agarose gel electrophoresis experiment to evaluate the responsiveness of the polymer in the ROS environment and its effect on the release of siRNA.

[0082] As shown in Figure 7As shown in the ROS response experiment results of the ROS response experiment results, A-siRNA does not exhibit obvious H2O2 responsiveness, while B-siRNA has the ability of ROS-responsive release of siRNA, and this property is derived from the fact that the TK-GUA monomer contained therein is broken under ROS conditions, resulting in the loose structure of the nanomicelle, thereby releasing siRNA.

[0083] Example 6 Serum stability test of siRNA polymer nanomicelles After incubating each group of carriers in 50% (v / v) fetal bovine serum for different times, 10 μL aliquots were taken, and EDTA was added to terminate the degradation reaction. Subsequently, siRNA (siRNA target sequence: GCCACAAGATTACAAGAAA) in the carriers was replaced out using sodium heparin, and the aliquots were analyzed by agarose gel electrophoresis.

[0084] As shown in the serum stability results of the serum stability results, Figure 8 the protection effect of carrier B on siRNA is significantly better than that of commercial carrier Lip3000, which can effectively protect siRNA from degradation by nucleases in serum, thereby significantly improving the serum stability of siRNA.

[0085] Example 7 Cytotoxicity test of carriers The cytotoxicity of Blank (culture medium containing the same volume of HEPES), B-siNC, C-siNC and B / C-siNC at concentrations of 400 nM, 200 nM and 100 nM on Hep-G2 was detected by CCK-8 method. siNC was purchased from Guangzhou Ribo Bioscience Co., Ltd.

[0086] As shown in the serum stability results of the serum stability results, Figure 9 It can be seen that at a drug concentration of 200 nM, each carrier is essentially non-toxic.

[0087] Example 8 Cell uptake test of siRNA polymer nanomicelles The cell uptake behavior was analyzed by laser confocal microscopy (Cy3-labeled siRNA, showing green fluorescence) and flow cytometry (Cy5-labeled siRNA). Four groups of Free-siRNA (naked siRNA), B-siRNA, C-siRNA and B / C-siRNA were subjected to drug administration.

[0088] As shown in the serum stability results of the serum stability results, Figure 10 , 11 As shown in the serum stability results of the serum stability results,

[0089] Example 9 Lysosomal co-localization test of siRNA polymer nanomicelles The co-localization of Cy5-labeled siRNA (red fluorescence) and lysosome probe Lyso-tracker (green fluorescence) was observed by laser confocal microscope.

[0090] As shown in Figure 12 Figure 9, the overlapping area of red fluorescence (Cy5-siRNA) and green fluorescence (Lyso) decreased compared with 1 h after administration, and the Pearson coefficient decreased, indicating that part of the siRNA had escaped from the lysosome.

[0091] Example 10 Effect of siRNA polymer nanomicelles on β-Catenin protein expression in Hep-G2 cells Western blot was used to detect the expression of β-Catenin at the protein level. Over Western blot was used to detect the expression of β-Catenin at the protein level.

[0092] As shown in Figure 13 Figure 10, each group of vectors can significantly inhibit the expression of β-Catenin protein in Hep-G2 cells.

[0093] Example 11 Anti-tumor effect test of siRNA polymer nanomicelles combined with sorafenib Inhibition of β-Catenin protein can enhance the anti-tumor effect of sorafenib (SF) on hepatocellular carcinoma, so this experiment used CCK-8 method to evaluate the cell survival rate when sorafenib was used alone and when sorafenib and siRNA polymer nanomicelles targeting CTNNB1 gene (CTNNB1 gene is the encoding gene of β-Catenin protein) (siRNA target sequence: GCCACAAGATTACAAGAAA) were used in combination.

[0094] As shown in Figure 14 Figure 11, at sorafenib concentrations of 0.5 μM and 1 μM, siRNA polymer nanomicelles combined with sorafenib can significantly reduce cell viability compared with sorafenib alone.

[0095] Example 12 Analysis of the effect of sorafenib on ROS level in HepG2 cells and lysosomal co-localization of siRNA micelles Flow cytometry and confocal microscope were used to detect the changes in intracellular ROS level and the effect of sorafenib on lysosomal co-localization of siRNA.

[0096] The flow cytometry analysis results showed that Figure 15), the ROS content in HepG2 cells was significantly increased after sorafenib treatment. The lysosome escape behavior of siRNA was observed by confocal microscopy (Fig. 6B) and it was found that (Fig. 6B) the overlapping area of red fluorescence (Cy5-siRNA) and green fluorescence (Lyso-tracker) was significantly reduced in sorafenib treatment group and the Pearson correlation coefficient decreased from 0.43 in control group to 0.19, indicating that sorafenib effectively promoted the release of siRNA by increasing the ROS level in tumor cells. Figure 16

[0097] Example 13 In vivo biodistribution of siRNA polymer nanomicelles The ROS-responsive B-siRNA or the targeted B / C-siRNA containing glycyrrhetinic acid was injected into the tail vein of mice (6-8 weeks old, 18-22 g female Balb / c nude mice, the dosage of drug was 3 mg / kg (calculated by the mass of siRNA)), and the heart, liver, spleen, lung, kidney and tumor tissues were collected for ex vivo imaging after 24 h.

[0098] As shown in Fig. 9B, compared with the ROS-responsive B-siRNA nanomedicine, the targeted B / C-siRNA group showed better liver targeting ability. Figure 17

[0099] Although the present application has been described in detail with general description and specific embodiments, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of the present application.​​

Claims

1. Polymer B, characterized in that, It is Meo-PEG 2k -P(TK-GUA), the structure is as follows: Where n is 45; x is an integer between 10 and 20.

2. Polymer C, characterized in that, It is GA-PEG 3.4k -P(TK-GUA), structure as follows: Where n is 77; x is an integer between 10 and 20.

3. The composition, characterized in that, The composition comprises polymer B as described in claim 1 and polymer C as described in claim 2.

4. The method for preparing polymer B according to claim 1, characterized in that, Composed of TK-GUA and Meo-PEG 2k -CPADN is obtained through polymerization, and the reaction equation is as follows: 。 5. The method for preparing polymer C according to claim 2, characterized in that, Composed of TK-GUA and GA-PEG 3.4K -CPADN is obtained through polymerization, and the reaction equation is as follows: 。 6. ROS-responsive siRNA nanomicelles, characterized in that, The nanomicelles comprise polymers and siRNA; The polymer is selected from at least one of polymer B of claim 1 and polymer C of claim 2.

7. The method for preparing nanomicelles according to claim 6, characterized in that, Includes the following steps: 1) Dissolve the polymer in HEPES buffer and dissolve the siRNA powder in DEPC-treated water to prepare polymer solutions and siRNA solutions respectively; 2) Mix the siRNA solution and polymer solution at a molar ratio of 1:2.5 to 1:40, and incubate at room temperature to allow the siRNA and polymer to self-assemble into siRNA polymer nanomicelles. Preferably, in step 2), the siRNA solution and the polymer solution are mixed at a molar ratio of 1:8; More preferably, the polymer is composed of polymers B and C mixed in a molar ratio of 9:

1.

8. Any of the following applications of the nanomicelles of claim 6: i) Used as a siRNA delivery system; ii) Used in the preparation of antitumor drugs; i) is used for purposes other than disease diagnosis and treatment.

9. The application according to claim 8, characterized in that, (ii) Tumors include hepatocellular carcinoma.

10. An antitumor drug, characterized in that, Including sorafenib and the nanomicelles of claim 6.

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