Redox-responsive nucleolin aptamer-paclitaxel conjugate, preparation method and application thereof

Abstract

The present invention discloses a series of novel redox-responsive nucleolin aptamer-paclitaxel conjugates as shown in formula I, a preparation method thereof and an application thereof: #imgabs0#R is -(CH2) m1 S(CH2) n1 ‑,‑(CH2) m2 SS(CH2) n2 ‑、‑(CH2) m3 SC(CH3)2S(CH2) n3 ‑, m1, n1, m2, n2, m3, n3 are 0 or positive integers. The conjugate is designed to respond to the redox heterogeneity of triple-negative breast cancer, has good water solubility and tumor specificity, and exhibits redox dual responsiveness, rapid release of PTX, and the highest anti-triple-negative breast cancer ability.

Description

Technical field:

[0001] The invention relates to a redox-responsive nucleolin aptamer-paclitaxel conjugate, a preparation method and application thereof. Background technology:

[0002] Paclitaxel (PTX) is a tetracyclic diterpene compound that blocks cell mitosis by inhibiting tubulin. Despite decades of discovery, scientists still focus on PTX-based chemotherapy because of its broad-spectrum anticancer activity and high efficiency. However, PTX has adverse reactions, such as bone marrow suppression, gastrointestinal irritation, etc. In addition, PTX has poor solubility in water, which greatly limits its dosage in clinical applications. To overcome these obstacles, various marketed drugs have been developed, such as solvent-based paclitaxel, liposome (Lipso), albumin-based paclitaxel (Abraxane), etc. However, these drugs can reduce rather than eliminate side effects, and it is increasingly difficult to meet the exact clinical criteria.

[0003] In recent years, attention in the field of cancer treatment is shifting from traditional drugs to targeted drugs. Triple-negative breast cancer (TNBC) is a highly malignant and poorly prognostic form of breast cancer (BC). Targeted therapies for BC are generally ineffective due to the lack of receptors (ER / PR / HER2). Currently, the main focus is on improving the specificity of PTX by attaching tumor-targeting ligands. A large number of ligands have been involved in PTX-specific strategies, including small molecules, peptides, antibodies, nanoparticles, magnetic iron oxides, and biofilms. Few developed approaches have proven to be perfect and are far from ready for use in commercial systems. Antibody-drug conjugates (ADCs) have been positioned at the forefront of chemotherapy, improving clinical outcomes for patients with specific DNA mutations. However, heavily modified antibodies have the risks of difficult transmembrane crossing, strong immunogenicity, storage rigidity, reduced target affinity, altered pharmacokinetics, and increased heterogeneity. Aptamers, as targeting components, have the advantages of fast screening, fast cell penetration, low immunogenicity, and easy synthesis, modification, and industrialization, making them a promising cancer treatment.

[0004] Unlike normal cells, nucleolin is abundantly expressed on the surface of various cancer cells and reportedly is an attractive target for triple-negative breast cancer (TNBC) therapy. ACT-GRO-777 (also known as AS1411) is a nucleolin-targeted aptamer (NCT00740441) that has entered clinical phase II studies (see Rosenberg JE, Bambury RM, VanAllen EM, et al. A phase II trial of AS1411 (a novel nucleolin-targeted DNA aptamer) in metastatic renal cell carcinoma. Invest New Drugs 2014; 32(1): 178-187). Notably, AS1411 enhances cell permeability through the macrocystin pathway, which is beneficial for the development of novel aptamer-drug conjugates (ApDCs), such as doxorubicin and melittin. Increasing evidence suggests that tumor microenvironment (TME)-responsive prodrugs have considerable therapeutic value because cancer tissues and adjacent normal tissues are clearly distinguished. In our previous work, we designed a highly water-soluble aptamer-paclitaxel conjugate that is sensitive to cathepsin B and can selectively deliver paclitaxel (PTX) to ovarian tumors (Xue F, Lin X, Cai Z, et al. Doxifluridine-based pharmacosomes delivering miR-122 as tumor microenvironments-activated nanoplatforms for synergistic treatment of hepatocellular carcinoma. Colloids Surf B Biointerfaces 2021; 197: 111367). In addition, due to abnormal tumor metabolism, not only is the concentration of glutathione (GSH) in tumor cells significantly higher than that in normal cells (500-1.0×10 4 μM vs. 2-20μM), and the level of reactive oxygen species (ROS, especially H2O2) was significantly higher than that of normal cells (5-1000μM vs. 0.001-0.7μM). However, current studies have not yet determined whether oxidative or alternative reductive conditions are the optimal conditions for the tumor microenvironment (TME) to stimulate ApDCs. Summary of the invention:

[0005] The purpose of the present invention is to provide a series of novel redox-responsive nucleolin aptamer-paclitaxel conjugates, preparation methods and applications thereof, wherein the conjugates are designed to respond to the redox heterogeneity of triple-negative breast cancer (TNBC), have good water solubility and tumor specificity, exhibit redox dual responsiveness (oxidation + reduction microenvironment), rapid release of PTX and the highest anti-triple-negative breast cancer (TNBC) ability.

[0006] The present invention is achieved through the following technical solutions:

[0007] The redox-responsive nucleolin aptamer-paclitaxel conjugate shown in Formula I:

[0008]

[0009] Where R is -(CH2) m1 S(CH2) n1 -、-(CH2) m2 SS(CH2) n2 -、-(CH2) m3 SC(CH3)2S(CH2) n3 -Any one of which m1, n1, m2, n2, m3, n3 are 0 or positive integers.

[0010] Preferably, m1, n1, m2, n2, m3, n3 are positive integers of 0 or 1-3.

[0011] The 5' and 3' ends of any of the unmodified or modified DNA / RNA such as aptamers, antisense nucleic acids, siRNAs, and CpGs.

[0012] The preparation method of the redox-responsive nucleolin aptamer-paclitaxel conjugate shown in formula I comprises the following steps: firstly, a redox-responsive diacid is constructed; then, the redox-responsive diacid is coupled with paclitaxel (PTX) to obtain a carboxylic acid derivative; finally, the carboxylic acid derivative is further coupled with amino DNA to obtain a redox-responsive nucleolin aptamer-paclitaxel conjugate ApDC.

[0013] In particular, when R in Formula I is -(CH2) m1 S(CH2) n1 -When monosulfur, m1=0, n1=0, the synthesis route is as follows:

[0014]

[0015] Reagent description: (i) 1,4-oxythiazane-2,6-dione, Py, THF; (ii) NHS, DCC, THF; (iii) amino-DNA, NaHCO3, H2O, DMF

[0016] The synthesis method comprises the following steps:

[0017] 1) Using equivalent amounts of 1,4-sulfane-2,6-dione and pyridine (Py) as raw materials, then adding paclitaxel (PTX) in anhydrous tetrahydrofuran (THF), the molar ratio of 1,4-sulfane-2,6-dione to paclitaxel is 1:(0.5-0.9), stirring at room temperature for 2-4 days, concentrating the mixture under reduced pressure, and then co-evaporating with toluene, and chromatography to obtain compound 2;

[0018] 2) Compound 2 is mixed with N-hydroxysuccinimide (NHS) in anhydrous tetrahydrofuran (THF), dicyclohexylcarbodiimide (DCC) is added and stirred at room temperature, the mixture is filtered, and the filtrate is concentrated in vacuo to obtain a crude solid, which is finally cooled to 4° C. with ether for recrystallization, and evaporated to dryness to obtain a white solid compound 3; wherein the molar ratio of compound 2, NHS and DCC is 1:(1-1.5):(1-1.5);

[0019] 3) Add 5' or 3'-amino DNA solution to NaHCO3 buffer, add compound 3 to DMF, the molar ratio of compound 3 to 5' or 3'-amino DNA is (30-100):1, stir at room temperature for 2h-12h, purify and desalt on a column to obtain compound 4 or compound 5.

[0020] When R in Formula I is -(CH2) m2 SS(CH2) n2 - disulfide, m2 = 1, n2 = 1, the synthesis route is as follows:

[0021]

[0022] Reagents and conditions: (i) I2, MeOH; (ii) AcCl, 60-80°C reflux; (iii) PTX, Py; (iv) NHS, DCC, THF; (v) Amino-DNA, PB buffer (pH = 8.0), DMF

[0023] The synthesis method comprises the following steps:

[0024] 1) Cooling the solution of compound 6 to 0°C, adding iodine dropwise, the molar ratio of compound 6 to iodine being 1:(0.2-0.45), quenching the reaction mixture by adding saturated Na2S2O3 aqueous solution after complete reaction, adjusting the pH to about 4 by gradually adding solid NaHCO3, and then filtering and concentrating the mixture, passing through a silica gel column to obtain compound 7;

[0025] 2) Add acetyl chloride to compound 7, heat to 60-80°C and reflux for 2-12 hours under nitrogen to obtain crude compound 8, dissolve crude compound 8 and paclitaxel (PTX) in a solvent, then add pyridine (Py) dropwise and stir at room temperature for 2 days, remove the solvent in vacuo, and obtain compound 9 by silica gel chromatography on the solid residue; wherein the molar ratio of crude compound 8, paclitaxel (PTX) and Py is 1:1:(0.3-0.5);

[0026] 3) Compound 9 is mixed with N-hydroxysuccinimide (NHS) in a molar ratio of 1:(1-2), dicyclohexylcarbodiimide (DCC) is added, and the mixture is stirred at room temperature for 1-4 days, a solid is precipitated, and the solid is filtered and the filtrate is evaporated to obtain a crude product of compound 10;

[0027] 4) Add 5' or 3'-amino DNA solution to PB buffer, add compound 10 to DMF, the molar ratio of compound 10 to 5' or 3'-amino DNA is (30-100):1, stir at 30-50°C for 2h-12h, purify and desalt with a column to obtain compound 11 or compound 12.

[0028] When R in Formula I is -(CH2) m3 SC(CH3)2S(CH2) n3 -, m3 = 1, n3 = 1, the synthesis route is as follows:

[0029]

[0030] Description of reagents and conditions: (i) H2SO4, acetone; (ii) PTX, DCC, DMAP, DMF; (iii) amino-DNA, DCC, HBTU, DIPEA, H2O, DMF.

[0031] The synthesis method comprises the following steps:

[0032] 1) Compound 6 is cooled to 0°C in acetone, sulfuric acid is added dropwise, the molar ratio of compound 6 to sulfuric acid is 1:(0.5-0.8:), stirred for 2-12 hours, then diluted with water, and NaOH aqueous solution is gradually added to adjust the pH to 5, and then post-treated to obtain compound 13;

[0033] 2) Compound 13 and paclitaxel (PTX) were added in a molar ratio of 1:(1-2), DMF was added, and then dicyclohexylcarbodiimide (DCC) and a catalytic amount of DMAP were added to the solution, and the molar ratio of DCC to compound 13 was (1-2):1. The mixture was allowed to stand at room temperature overnight, filtered, evaporated, and subjected to column chromatography on silica gel to obtain compound 14;

[0034] 3) Compound 14, dicyclohexylcarbodiimide (DCC) and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) are added to DMF respectively and stirred. After cooling to 0°C, 5' or 3'-aminoDNA and N,N-diisopropylethylamine (DIPEA) aqueous solution are dropped into the mixture and stirred at 30-50°C for 4-12h, wherein the molar ratio of compound 14, dicyclohexylcarbodiimide (DCC), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), N,N-diisopropylethylamine (DIPEA) and 5' / 3'-aminoDNA is (30-100):(30-100):(30-100):(30-100):1, and triethylamine acetate (TEAA) is added to quench the reaction mixture, and then purified by post-treatment to obtain compound 15 or compound 16.

[0035] The redox-responsive nucleolin aptamer-paclitaxel conjugate obtained by the present invention has good water solubility and tumor specificity. The present invention also protects the use of the redox-responsive nucleolin aptamer-paclitaxel conjugate in preparing anti-triple-negative breast cancer (TNBC) drugs.

[0036] The beneficial effects of the present invention are as follows: The present invention provides a series of novel redox-responsive nucleolin aptamer-paclitaxel conjugates, preparation methods and applications thereof, wherein the conjugates are designed to respond to the redox heterogeneity of triple-negative breast cancer (TNBC), have good water solubility and tumor specificity, exhibit redox dual responsiveness (oxidation + reduction microenvironment), rapid release of PTX and the highest anti-triple-negative breast cancer (TNBC) ability. Description of the drawings:

[0037] Figure 1 is the drug release and serum stability of ApDCs in response to different TMEs;

[0038] Among them, A is the drug release of ApDCs in different TME responses in phosphate buffered saline (PBS, 0.1x), reducing microenvironment (DTT, 10mM), and oxidizing microenvironment (H2O2, 1mM); ApDCs (0.15nmol) of aptamer-drug conjugates were incubated in different buffers at 37°C and sampled every 3 hours. The intact ApDCs normalized at 0h were considered as 100%. The data are expressed as the mean ± standard deviation of three replicates.

[0039] B is the serum stability of ApDCs with different TME stimulation connections; ApDCs (0.15 nmol) were incubated in 90% fetal bovine serum at 37°C, samples were taken at different time points, and images were visualized with 0.01% GelGreen;

[0040] In the figure, PSA is the abbreviation of compound 4, ASP is the abbreviation of compound 5, PSSA is the abbreviation of compound 11, ASSP is the abbreviation of compound 12, PTKA is the abbreviation of compound 15, and ATKP is the abbreviation of compound 16

[0041] Figure 2 The cytotoxicity and apoptosis of ApDC-treated cells in response to different TMEs.

[0042] Wherein, A is the cytotoxicity of ApDCs to cancer cells and normal cells in different TME responses. Wherein, ad is the cell viability of mouse triple-negative breast cancer cell line (4T1), human triple-negative breast cancer cell line (MDA-MB-231), human ovarian cancer cell line (SKOV3) and human normal liver cell line (MIHA) after treatment with free AS1411, free PTX and ApDCs at 250nM for 72h. Data are presented as mean ± standard deviation of at least three replicates. Statistical significance was determined by the Holm-Sidak method, alpha = 0.05. ns indicates P>0.05; * indicates P<0.05; ** indicates P<0.01; *** indicates P<0.001; **** indicates P<0.0001. GraphPad Prism 8.0.1 software was used for statistical analysis.

[0043] B shows the apoptosis of MDA-MB-231 cells treated with ApDC in different TME responses. Annexin V-FITC / PI apoptosis detection kit, flow cytometry analysis of the percentage of normal cells, early apoptosis, late apoptosis and dead cells. The concentrations of ApDC and free PTX were both 250 nM. Data are presented as the mean ± SD of three replicates. Specific implementation method:

[0044] The following is a further description of the present invention, rather than a limitation of the present invention.

[0045] Example 1: Synthesis of compounds

[0046] The reaction equation is as follows:

[0047]

[0048] Wherein R is -S- in compound 4 and compound 5. Compound 4 is referred to as PSA, and compound 5 is referred to as ASP. Compound 11 is referred to as PSSA, compound 12 is referred to as ASSP, compound 15 is referred to as PTKA, and compound 16 is referred to as ATKP.

[0049] Synthesis of compound 2:

[0050] Py (242 μL, 3.006 mmol) was added to a suspension of 1,4-sulfane-2,6-dione (397 mg, 3.006 mmol) in THF (20 mL) and stirred for 5 min. Then, PTX (2.176 g, 2.548 mmol) was added to anhydrous THF (20 mL) and stirred at room temperature for 4 d. The mixture was concentrated under reduced pressure and then co-evaporated with toluene three times to remove Py. The residue was then chromatographed on silica gel (PE / EA=1 / 1) to give compound 2 (1.807 g, 72%) as a white solid.

[0051] MS (ESI) m / z for C 51 H 54 NO 17 S - [MH] - ,Calculated 984.3118,Found 984.3153; 1 HNMR(400MHz,MeOD)δ8.02(dd,J=8.2,6.8Hz,2H),7.74(dd,J=5.3,3.2Hz,2H),7.60–7.55(m,1H),7.49(t,J=7.5Hz,2H),7.44–7.38(m ,3H),7.37–7.31(m,4H),7.17(t,J=7.3Hz,1H),6.35(d,J=4.5Hz,1H),5.99(t,J=8.6Hz,1H),5.77(d,J=6.1Hz,1H),5.54(d,J=7.3Hz, 1H),5.41(d,J=6.1Hz,1H),4.93–4.87(m,1H),4.25(dd,J=11.0,6.7Hz,1H),4.09(s,2H),3.72(d,J=7.2Hz,1H),3.56–3.35(m,3H),3. 30–3.24(m,1H),3.16(d,J=15.0Hz,1H),2.43–2.24(m,4H),2.15–2.03(m,4H),1.85–1.65(m,5H),1.55(s,3H),1.04(d,J=3.5Hz,6H); 13C NMR(101MHz,MeOD)δ205.24,171.33,170.24,167.71,142.40,138.37,135.48, 134.90,134.66,132.93,131.34,130.14,129.97–129.47,128.87–128.4,85.9 3,82.28,79.04,77.49,76.85,76.51,76.28,73.07,72.33,59.24,55.24,47.9 2,44.62,37.55,36.45,33.68,26.97,23.35,22.43,20.84,14.99,10.50.Peak overlapping wasobserved.

[0052] Synthesis of compound 3: Compound 2 (1.2 g, 1.218 mmol) was mixed with NHS (147 mg, 1.279 mmol) in anhydrous tetrahydrofuran (30 mL), and DCC (276 mg, 1.34 mmol) was added and stirred at room temperature for 3 days. The mixture was filtered and the filtrate was concentrated in vacuo to obtain a crude solid. Finally, it was cooled to 4 ° C with ether (100 mL) for recrystallization and evaporated to dryness to obtain a white solid. MS (ESI) m / z for C 55 H 57 N2O 19 S - [MH] - ,Calculated 1081.3282,Found 1081.3203.

[0053] Synthesis of compounds 4-5: Add 5' / 3'-amino DNA (100nmol) solution to NaHCO3 buffer (50mM, 0.1mL), add compound 3 (3μmol) to DMF (0.2mL), stir at 30°C (800rpm) for 2h, and add TEAA (2M, 0.1mL) to quench the reaction mixture. The solvent was removed in vacuo, and the solid residue was redissolved in ddH2O (1ml). Then, the mixture was purified using HPLC (Agilent 1260). Phase A is ACN, and phase B is TEAA (50mM). Oligonucleotide BEH C18OBDTM Prep column (2.5 m, 10 mm × 50 mm), flow rate 1.2 mL min -1The column temperature was ambient temperature, and the gradient was run from 5% to 60% of phase A in 30 minutes. The purified aptamer-drug conjugate (ApDC) was desalted on a Sephadex G25 column to obtain compound 4 or 5. The sample was further lyophilized to dryness for storage and confirmed by ESI mass spectrometry analysis.

[0054] Synthesis of compound 7: A solution of compound 6 (2.3 g, 21.7 mmol) in methanol (30 mL) was cooled to 0 ° C, and iodine (5.634 g, 11.1 mmol) in methanol (30 mL) was dropped into the stirred solution for 2 hours. Thin layer chromatography analysis showed that compound 6 was completely consumed. The reaction mixture was quenched by adding saturated aqueous Na2S2O3 solution, and the pH was adjusted to about 4 by gradually adding solid NaHCO3. The mixture was then filtered and concentrated, and chromatographed on silica gel (DCM / MeOH=30 / 1) to obtain compound 7 (2.012 g, 87%) as a white solid.

[0055] MS (ESI) m / z for C6H9O4S2 - [MH] - ,Calculated 208.9948,Found 208.9964; 1 HNMR (400MHz, MeOD) δ2.92 (t, J = 7.0 Hz, 4H), 2.71 (t, J = 7.0 Hz, 4H). 13 C NMR (101MHz, MeOD) δ175.36, 34.76, 34.30.

[0056] Synthesis of compounds 8 and 9:

[0057] Compound 7 ((0.211 g, 1.005 mmol)) and acetyl chloride (5 mL) were added to a round-bottom flask. Then, the mixture was heated to 65°C under nitrogen and refluxed for 2 hours to obtain crude compound 8. MS (ESI) m / z for C6H9O3S2 + [M+H] + ,Calculated192.9988,Found 192.9990.

[0058] All crude compound 8 and PTX (854 mg, 1 mmol) were dissolved in anhydrous DCM (20 mL). Then, Py (0.282 mL, 3.5 mmol) was added dropwise to the mixture and stirred at room temperature for 2 days. The solvent was removed in vacuo and the solid residue was redissolved in DCM (20 mL). Compound 9 was obtained by chromatography on silica gel (DCM / MeOH = 20 / 1 to 3 / 1) as a white solid. MS (ESI) m / z for C 53 H58 NO 17 S2 - [M-H] - ,Calculated 1044.3152,Found 1044.3238; 1 H NMR(400MHz,MeOD)δ8.02(dd,J=8.2,6.8Hz,2H),7.74(dd,J=5.3,3.2Hz,2H),7.60–7.55(m,1H),7.49(t,J=7.5Hz,2H),7.44–7.38(m,3H),7.37–7.31(m,4H),7.17(t,J=7.3Hz,1H),6.35(d,J=4.5Hz,1H),5.99(t,J=8.6Hz,1H),5.77(d,J=6.1Hz,1H),5.54(d,J=7.3Hz,1H),5.41(d,J=6.1Hz,1H),4.93–4.87(m,1H),4.25(dd,J=11.0,6.7Hz,1H),4.09(s,2H),3.72(d,J=7.2Hz,1H),3.56–3.35(m,3H),3.30–3.24(m,1H),3.16(d,J=15.0Hz,1H),2.43–2.24(m,4H),2.15–2.03(m,4H),1.85–1.65(m,5H),1.55(s,3H),1.04(d,J=3.5Hz,6H); 13 C NMR(101MHz,MeOD)δ205.19,172.60,171.63,171.31,170.61,170.34,167.66,142.38,138.29,135.54,134.87,134.64,132.95,131.32,130.14,129.68,128.67,85.92,82.28,79.03,77.47,76.84,76.16,73.02,72.33,59.23,55.24,49.90,47.93,44.61,37.56,36.43,35.58,34.78,34.47,33.71,26.96,23.35,22.43,20.85,15.02,10.51.Peak overlappingwas observed.

[0059] Synthesis of compound 10: Compound 9 (648 mg, 0.62 mmol) and NHS (72.8 mg, 0.633 mmol) were mixed in anhydrous THF (20 mL), and DCC (133.3 mg, 0.646 mmol) was added and stirred at room temperature for 2 days. Solid precipitated. The mixture was filtered and the filtrate was evaporated to obtain crude product 10. MS (ESI) m / z for C 57 H 62 N2NaO 19 S2 + [M+Na] + ,Calculated1165.3280,Found 1165.3274.

[0060] Synthesis of compounds 11-12: 5' / 3'-amino DNA (100 nmol) solution was added to PB buffer (50 mM, 0.1 mL), compound 10 (3 μmol) in DMF (0.2 mL) was added to the solution, stirred at 30 °C for 2 h, and TEAA (2 M, 0.1 mL) was added to quench the reaction mixture. The solvent was removed in vacuo, and the solid residue was redissolved in ddH2O (1 ml). The mixture was then purified using HPLC (Agilent 1260). Phase A was ACN, and phase B was TEAA (50 mM). Oligonucleotide BEH C18OBDTM Prep column (2.5 m, 10 mm × 50 mm), flow rate 1.2 mL min -1 The column temperature was ambient temperature, and the gradient was run from 5% to 60% of phase A in 30 minutes. The purified aptamer-drug conjugate (ApDC) was desalted using a Sephadex G25 column to obtain compound 11 / 12. The sample was further lyophilized to dryness and confirmed by ESI mass spectrometry.

[0061] Synthesis of compound 13: Compound 6 (3.412 g, 32.2 mmol) was cooled to 0 °C in acetone (20 mL), sulfuric acid (1.882 g, 19.2 mmol) was added dropwise to the stirred mixture and stirred for 2 hours. Then it was diluted with ddH2O (200 mL), and the pH was adjusted to 5 by gradually adding aqueous NaOH solution. Then, the mixture was evaporated to remove acetone, and the aqueous layer was extracted with ethyl acetate (100 mL×3). Finally, the crude product was chromatographed on reduced pressure silica gel (n-hexane / EA=4 / 1) to obtain compound 13 (2.313 g, 57%) as a white solid. MS (ESI) m / z for C9H 15 O4S2 - [MH] -,Calculated 251.0417,Found251.0448; 1 H NMR (400MHz, MeOD) δ2.76 (dd, J=9.0, 5.4Hz, 4H), 2.49 (t, J=7.1Hz, 4H), 1.49 (s, 6H); 13 C NMR (101MHz, MeOD) δ56.97,35.33,31.31,26.33.

[0062] Synthesis of compound 14: Compound 13 (0.433 g, 1.718 mmol) and PTX (1.547 g, 1.812 mmol) were added to a round-bottom flask and added to DMF (20 mL). DCC (355 mg, 1.722 mmol) and a catalytic amount of DMAP were added to the solution. The mixture was left at room temperature overnight. The mixture was filtered, evaporated, and chromatographed on silica gel (DCM / MeOH=50 / 1-30 / 1) to obtain compound 14 (1.064 g, 57%) as a white solid. MS (ESI) m / z for C 56 H 64 NO 17 S2 - [MH] - ,Calculated1086.3621,Found 1086.3747; 1 H NMR (400MHz, MeOD) δ8.05–7.98(m,2H),7.71(dd,J=5.2,3.3Hz,2H),7.57(td,J=7.0,3.2Hz,1H),7.39(dddd,J=23.6,15.3,11.1,4.7Hz ,9H),7.17(t,J=7.3Hz,1H),6.35(s,1H),5.97(t,J=8.7Hz,1H),5.77(d,J=6.5Hz,1H),5.53(d,J=7.2Hz,1H),5.43(d,J=6.5Hz,1H),4.9 3–4.87(m,1H),4.24(dd,J=10.9,6.7Hz,1H),4.09(s,2H),3.71(d,J=7.2Hz,1H),2.71(t,J=6.8Hz,2H),2.61(t,J=6.6Hz,2H),2.41–2. 28(m,4H),2.13–2.00(m,8H),1.82(d,J=0.8Hz,3H),1.77–1.65(m,2H),1.55(s,3H),1.34(s,3H),1.24–1.07(m,3H),1.06–0.99(m,6H); 13C NMR(101MHz,MeOD)δ205.19,172.86,171.66,171.34,170.41,170.35,167. 68,142.36,138.36,135.51,134.94,132.95,131.44,131.25,130.16,129.7 6,129.60,128.75,128.72,85.91,82.29,79.07,76.82,76.28,76.03,73.02 ,31.19,30.70,26.15,23.38,22.41,20.84,15.08,10.48.Peakoverlapping was observed.

[0063] Synthesis of compounds 15-16: Compound 14 (3 μmol), DCC (3 μmol) and HBTU (3 μmol) were added to DMF (0.4 mL) respectively and stirred for 10 min. After cooling to 0°C, a solution of 5′ / 3′-amino DNA (100 nmol) and DIPEA (3 μmol) in ddH2O (0.1 mL) was added dropwise to the mixture, stirred at 30°C for 4 h, and TEAA (2 M, 0.1 mL) was added to quench the reaction mixture. The solvent was removed in vacuo, and the solid residue was redissolved in ddH2O (1 ml). The mixture was then purified using high performance liquid chromatography (Agilent 1260). Phase A was ACN and phase B was TEAA (50 mM). Oligonucleotide BEH C18 OBDTM Prep column (2.5m, 10mm×50mm), flow rate of 1.2mL·min-1, column temperature at ambient temperature, gradient from 5% to 60% of phase A in 30 minutes. The purified ApDC was desalted by Sephadex G25 column to obtain compound 15 / 16. The sample was further lyophilized to dryness and confirmed by ESI mass spectrometry.

[0064] Example 2: Drug release and stability of different tumor microenvironment (TME)-responsive aptamer-drug conjugates (ApDCs).

[0065] Drug release assay: ApDCs (0.15 nmol) were dissolved in PBS (0.1×, 60 μL), DTT (10 mM, 60 μL) and H2O2 (1 mM, 60 μL), respectively. Incubated at 37°C, the intact ApDCs were detected at regular intervals using an Agilent 1290 HPLC system and a 260 nm UV detector. Phase A was ACN and phase B was TEAA (50 mM). In a YMC-Triart C18 column (3 m, 150 mm × 4.6 mm), the flow rate was 0.5 mL min-1, the column temperature was ambient temperature, and the gradient was run from 2% to 55% of phase A in 30 min. The percentage of intact ApDCs was normalized, and the initial amount was 100%.

[0066] In order to compare the differences in drug release of the aptamer-drug conjugates (ApDCs) stimulated by different tumor microenvironments (TME), HPLC was used to evaluate the relatively intact aptamer-drug conjugates (ApDCs), including compound 4, compound 5, compound 11, compound 12, compound 15 and compound 16, after incubation in physiological buffer (PBS), reducing microenvironment (DTT) or oxidizing (H2O2) microenvironment.

[0067] like Figure 1 As shown in A, various aptamer-drug conjugates ApDCs maintained high stability after incubation in physiological buffer at 37°C for 24 hours. ApDCs with disulfide linkers (ASSP, PSSA) and thio linkers (ATKP, PTKA) were activated by reducing microenvironment and oxidizing microenvironment, respectively. ApDCs with thioether linkers (ASP, PSA) were co-activated simultaneously in reducing microenvironment and oxidizing microenvironment. The results showed that the hydrolysis efficiency of ApDCs in H2O2 solution (1mM) was: ASP≈PSA>ATKP≈PTKA>ASSP≈PSSA. In addition, the hydrolysis efficiency of ApDCs in DTT solution (10mM) was ASSP≈PSSA>ASP≈PSA>ATKP≈PTKA. The results showed that ApDCs containing thioether linkers were more sensitive to oxidative microenvironment than reducing microenvironment. Compared with thioether-linked ApDCs, thioether-linked ApDCs had higher drug release efficiency. The coupling end of ApDCs had little effect on drug release.

[0068] Degradation experiments were performed to evaluate the serum stability of ApDCs with different TME stimulation conjugates ( Figure 1B). These ApDCs (0.15 nmol) were incubated in 90% fetal bovine serum at 37°C, and samples were taken at different time points and visualized with 0.01% GelGreen. The study found that 3′-end ApDCs generally have higher stability than 5′-end ApDCs, which may be related to the function of the 3′-end exon. In addition, the serum stability of ASP and ASSP is lower than that of ATKP. This may be related to the steric hindrance of the dimethyl group in the thiol linker.

[0069] Example 3: In vitro anti-triple negative breast cancer (TNBC) ability of aptamer-drug conjugate ApDC in response to different tumor microenvironments TME

[0070] CCK-8 cell viability assay: To identify the most potent ApDCs in tumor cells, a cell viability assay will be performed using the Cell Counting Kit-8 (CCK8, Dojindo). MDA-MB-231, 4T1, SKOV-3, and MIHA cells will be seeded in 96-well plates at approximately 5,000 cells per well and incubated overnight to adhere according to the protocol provided. The solution of ApDCs and controls will be in the medium. Remove the cell culture medium, add the solution, and incubate at 37°C for 72h. At the end of the incubation, 100 μL of medium containing 10% CCK8 solution will be added to each well. The plates will be read after an additional 2 hours of incubation using a spectrometer at a wavelength of 450nm.

[0071] Apoptosis experiment: To analyze the different stages of apoptosis, cells were seeded in 6-well plates and treated with ApDC and control groups for 48 h. The cells were collected, rinsed with cold PBS, and resuspended with 100 μL 1× Annexin-V binding buffer. Then 5 μL Annexin-V-FITC and 5 μL PI were added, incubated in the dark at room temperature for 15 min, and quantitatively measured by flow cytometry.

[0072] The CCK-8 method was used to evaluate the in vitro antiproliferative effects of different TME stimulation conjugates on ApDCs. Figure 2The results shown in ab in A showed that various ApDCs could effectively inhibit the proliferation of cancer cells. Compared with free PTX, ASP was more effective against 4T1 cells (24.3% ± 2.1% vs 12.0% ± 2.1%, P < 0.01) and MDA-MB-231 cells (12.7% ± 0.5% vs 11.8% ± 0.4%, P < 0.05). In contrast, ASSP or ATKP had lower antiproliferative activity than ASP against 4T1 cells (27.3% ± 0.5% vs 35.5% ± 2.9% vs 12.0% ± 2.1%) and MDA-MB-231 cells (14.6% ± 0.8% vs 17.9% ± 0.3% vs 11.8% ± 0.4%), indicating the potential role of thioether linkers in TME-responsive chemotherapy. Importantly, ASP had a more stable anti-TNBC ability than PSA. Combined with serum stability data ( Figure 1 B), indicating that 3′-end coupling may be more conducive to the design of AS1411-PTX. In contrast, all ApDCs showed reduced cytotoxicity against SKOV3 cells and MIHA cells ( Figure 2 Ac-d).

[0073] Consistent with the antiproliferative results, ASP was more capable of inducing cell apoptosis in vitro than ASSP and ATKP. Figure 2 As shown in B, ASP increased the proportion of late apoptosis compared with ASSP and ATKP (32% ± 1% vs 22% ± 1% vs 19% ± 1%), while there was no significant difference between early apoptotic and dead cells. Overall, our in vitro data further confirmed the role of the thioether linker in AS1411-PTX conjugation. Compared with PTX, ASP has better antiproliferative ability against TNBC cells because ASP can effectively target TNBC cells and induce apoptosis, but with few biosafety issues.

Claims

1. Redox-responsive nucleolin aptamer-paclitaxel conjugate AS1411-PTX represented by Formula I: Ⅰ in, R is -(CH2) m1 S(CH2) n1 -, m1 and n1 are positive integers of 0 or 1-3, It is an AS1411 aptamer.

2. The method for preparing the redox-responsive nucleolin aptamer-paclitaxel conjugate AS1411-PTX represented by formula I according to claim 1, characterized in that: In formula Ⅰ, R is -(CH2) m1 S(CH2) n1 -When monosulfur, m1=0, n1=0, the synthesis route is as follows: ； The following steps are involved: 1) Using equivalent amounts of 1,4-sulfane-2,6-dione and pyridine as raw materials, paclitaxel was added, with a molar ratio of 1,4-sulfane-2,6-dione to paclitaxel of 1:(0.5-0.9), and stirred at room temperature for 2-4 days. The mixture was concentrated under reduced pressure, co-evaporated with toluene, and chromatographed to obtain compound 2; 2) Compound 2 was mixed with N-hydroxysuccinimide, dicyclohexylcarbodiimide was added and stirred at room temperature, the mixture was filtered, and the filtrate was concentrated in vacuo to obtain a crude solid, which was finally cooled to 4°C with ether for recrystallization and evaporated to dryness to obtain a white solid compound 3; wherein the molar ratio of compound 2, NHS, and DCC was 1:(1-1.5):(1-1.5); 3) Add 5' or 3'-amino DNA solution to NaHCO3 buffer, add compound 3 in DMF, the molar ratio of compound 3 to 5' or 3'-amino DNA is (30-100):1, stir at room temperature for 2 h-12 h, purify and desalt on a column to obtain compound 4 or compound 5.

3. Use of the redox-responsive nucleolin aptamer-paclitaxel conjugate AS1411-PTX according to claim 1 in the preparation of an anti-triple-negative breast cancer drug.

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