Preparation method of liver-targeted chemotherapeutic drug nano-micelle, chemotherapeutic drug and application

Abstract

The invention relates to the technical field of medical application, in particular to a liver-targeted chemotherapeutic drug nano-micelle preparation method, a chemotherapeutic drug and application, and the preparation method comprises the following steps: S100, preparing pluronic-succinimide formate; s200, preparing a pluronic-polyethyleneimine copolymer; s300, preparing divinyl sebacate; carrying out enzymatic synthesis on the Pluronic F127-SE; the preparation method comprises the following steps: preparing a Pluronic F127-C8-GalNAc nano micelle; and preparing the GalNAc (at) LFT-MMs / siCOP1 compound. The preparation method disclosed by the invention is beneficial to rapidly preparing the nano-micelle with multiple effects of active targeting, chemotherapy and gene therapy.

Description

Technical Field

[0001] The present invention relates to the field of medical application technology, and in particular to a method for preparing liver-targeted chemotherapy drug nano-micelles, the chemotherapy drug and applications. Background Art

[0003] Hepatocellular carcinoma is one of the most common malignant tumors in the world, with a poor prognosis and high recurrence rate. In recent years, the multikinase inhibitor lenvatinib has attracted much attention for its higher efficacy than sorafenib in improving patient prognosis. However, due to its low oral bioavailability, it needs to increase the dose to produce anti-tumor effects, which leads to side effects such as diarrhea and vomiting.

[0004] Small interfering RNA is used to silence cancer-related genes, thereby activating apoptotic pathways or inhibiting anti-apoptotic pathways in cancer cells. COP1 is a highly conserved E3-ubiquitin ligase. For the tumor suppressor protein P53, it is a key negative regulatory protease that can affect P53-dependent transcription, apoptosis, cell cycle regulation, and DNA repair. In recent years, combining anticancer drugs with siRNA has become an innovative therapeutic strategy. However, the challenge faced by free siRNA lies in its hydrophilicity, negative charge, and sensitivity to RNase, which make it difficult for siRNA to cross the negatively charged cell membrane and circulate stably in vivo. To this end, researchers focus on constructing co-delivery carriers that can efficiently encapsulate anticancer drugs and siRNA to ensure the precise localization and release of these therapeutic molecules at the tumor site. Polyethyleneimine (PEI) is a non-viral carrier that is widely used in nucleic acid delivery due to its unique proton sponge effect and superior transfection efficiency.

[0005] In order to improve the targeting of nanoparticles in tumor treatment, researchers use chemical methods to modify ligands that can bind to specific receptors on the tumor surface onto polymer micelle carriers to achieve active targeted delivery to tumors. In particular, in the field of liver treatment, the asialoglycoprotein receptor widely expressed on the membrane of hepatic parenchymal cells has become an ideal target for liver-specific drug delivery, which not only reduces the systemic toxicity of treatment, but also improves the efficacy of the drug. On this basis, N-acetylgalactosamine, as a ligand with high affinity for ASGPR on the surface of hepatocytes, can effectively deliver chemotherapy drugs specifically to hepatocytes expressing ASGPR. This precise targeted delivery method ensures that higher concentrations of therapeutic drugs can be concentrated at the tumor site, thereby improving the therapeutic effect while significantly reducing the risk of toxicity to healthy cells. However, at present, the preparation efficiency of nanocarriers for liver-targeted chemotherapy drugs is low, and the targeting is insufficient, making it difficult to apply on a large scale.

[0006] Therefore, those skilled in the art are committed to developing a method for preparing liver-targeted chemotherapy drug nanomicelles, chemotherapy drugs and applications, which are conducive to the rapid preparation of nanomicelles with multiple functions of active targeting, chemotherapy and gene therapy. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide a method for preparing liver-targeted chemotherapy drug nano-micelles, chemotherapy drugs and applications, which are conducive to the rapid preparation of nano-micelles with multiple functions of active targeting, chemotherapy and gene therapy. The technical solution of the present invention to solve the above technical problems is as follows: A method for preparing liver-targeted chemotherapy drug nano-micelles, characterized in that it comprises the following steps: S100. Preparation of Pluronic-formic acid succinimidyl ester (P123-NHS): S101. Pluronic P123 vacuum dried and dehydrated; S102. The vacuum-dried Pluronic P123 was dissolved in anhydrous toluene and anhydrous dichloromethane, triphosgene was added and magnetic stirring was performed until the reaction was completed, and then the mixed solution was rotary evaporated to evaporate the solvent, and then dissolved in a mixed solution of anhydrous toluene and anhydrous dichloromethane to prepare a mixed solution; S103. treating the mixed solution prepared in step S102 with N-hydroxysuccinimide, S104. After diluting anhydrous triethylamine with anhydrous dichloromethane, the mixture is added dropwise to the mixed solution treated with N-hydroxysuccinimide in step S103 to prepare a primary solution; S105. Stirring the primary solution prepared in step S104 until the reaction is complete, filtering the primary solution and evaporating it in a vacuum to dryness to form a residue; S106. The residue prepared in step S105 is dissolved in ethyl acetate, filtered, and the filtrate is subjected to rotary evaporation to remove the ethyl acetate, cooled and solidified to form P123-NHS, and then the P123-NHS is stored in a low temperature dry environment for standby use; S200. Preparation of Pluronic-polyethyleneimine copolymer (P123-PEI): S201. dissolving polyethyleneimine (PEI) in phosphate buffer; S202. Activating the P123-NHS prepared in step S106 and dissolving it in a phosphate buffer; S203. Add the P123-NHS mixed solution in step S202 dropwise to the polyethyleneimine (PEI) solution in step S201, and magnetically stir to form a solution after saturation with nitrogen; S204. The stirred solution in step S203 is added to the activated dialysis bag to remove unreacted activated P123, and the ionized water is continuously replaced during the dialysis process. Finally, the solution is freeze-dried to prepare a P123-PEI polymer, which is dried and stored at low temperature; S300. Preparation of divinyl sebacate: S301. Sebacic acid, vinyl acetate, mercuric acetate and copper acetate were added to the reaction flask and stirred in a constant temperature water bath to prepare a reaction solution; S302. Concentrated sulfuric acid is added dropwise to the reaction solution prepared in step S301, sodium acetate is added after the reaction is completed, and excess vinyl acetate is removed by vacuum distillation to prepare a secondary reaction solution; S303. The secondary reaction solution in step S302 is separated on a column using silica gel as the stationary phase and petroleum ether-ethyl acetate as the mobile phase to separate divinyl sebacate; S400. Enzymatic synthesis of Pluronic F127-SE: S401. Pluronic F127, divinyl sebacate prepared in step S303, lipase (CRL) and acetone are added to a reaction flask to prepare a mixed solution. S402. After the mixed solution in step S401 is shaken in an air bath for reaction, the reaction solution is filtered to remove lipase (CRL), the filtrate is vacuum dried and sterile water for injection is added, and then dialyzed in a dialysis bag, and freeze-dried to prepare Pluronic F127-SE powder; S500. Preparation of Pluronic F127-C 8 -GalNAc Nanomicelles: S501. Add GalNAc, Pluronic F127-SE prepared in step S401, Lipozyme TL IM lipase, and acetone to the reaction flask; S502. After the mixed solution in step S501 is shaken in an air bath for reaction, the reaction solution is filtered to remove the enzyme to prepare an enzyme-free solution; S503. The enzyme-free solution in step S502 is vacuum dried, sterile water for injection is added, and the solution is placed in a dialysis bag for dialysis, and freeze-dried to prepare Pluronic F127-C. 8 -GalNAc; S600. Preparation of GalNAc@LFT-MMs / siCOP1 complex: S601. P123-PEI 2KDa, Pluronic F127-C 8 -GalNAc and LFT are placed in a reaction vessel and methanol is added to dissolve to obtain an organic phase, which is rotary evaporated until the organic solvent is completely removed and then vacuum dried to obtain a drug film; S602. Add sterile water for injection to the drug film prepared in step S601, stir it magnetically and cool it to room temperature, then add siCOP1 to incubate to obtain a transparent micelle solution; S603. The transparent micelle solution prepared in step S602 is filtered through a microporous filter membrane, and freeze-dried to prepare a GAlNAc@LFT-MMs / siCOP1 nanocomposite.

[0009] The beneficial effects of adopting the above scheme are: the polymer micelles prepared by the above steps can simultaneously target the chemotherapy drug LFT and siRNA to liver cancer cells, and effectively release them in tumor cells to achieve stronger tumor inhibition and lower systemic toxicity; Taking advantage of the nano-micelles and combining them with the combined effects of siRNA and chemotherapy drugs, we can fully explore the optimal conditions for the combined use of the two drugs; At the same time, the preparation method of the present invention is simple, has important significance for developing new anti-liver cancer gene drugs and improving the therapeutic effect of liver cancer, and has broad application prospects.

[0010] Based on the above technical solution, the present invention can also be improved as follows.

[0011] Furthermore, in step S501, the ratio of GalNAc, Pluronic F127-SE, Lipozyme TL IM lipase, and acetone is: 0.4 mmol: 0.8 mmol: 100 mg: 50 ml.

[0012] The beneficial effect of adopting the above further scheme is that the appropriate ratio of ingredients is conducive to the subsequent preparation of Pluronic F127-C 8 -GalNAc.

[0013] Further, in step S401, the mixing ratio of Pluronic F127, divinyl sebacate, lipase (CRL) and acetone is: 1.3 mmol: 11.6 mmol: 200 mg: 50 ml.

[0014] The beneficial effect of adopting the above further scheme is that the appropriate ratio of ingredients is conducive to the subsequent preparation of Pluronic F127-SE.

[0015] Further, in step S402, the molecular weight cut-off of the dialysis bag is 10000 Da.

[0016] The beneficial effect of adopting the above further scheme is that the molecular weight cut-off of the dialysis bag is 10000Da, which is conducive to removing impurities and moisture in the solution.

[0017] Furthermore, in step S303, the ratio of petroleum ether and ethyl acetate in the mobile phase is 9:1.

[0018] The beneficial effect of adopting the above further scheme is that it is conducive to the preparation of pure divinyl sebacate.

[0019] Furthermore, in step S603, the microporous filter membrane is a 0.20 μm to 0.24 μm microporous filter membrane.

[0020] The beneficial effect of adopting the above further scheme is: further purifying the reaction solution.

[0021] Furthermore, in step S602, after adding siCOP1, the incubation time is 20 min to 40 min.

[0022] The beneficial effect of adopting the above further scheme is: improving the synthesis efficiency of the transparent micelle solution.

[0023] Further, in step 402, the dialysis time is 45 hours to 51 hours.

[0024] The beneficial effect of adopting the above further scheme is: removing impurities and moisture in the solution.

[0025] A liver-targeted chemotherapy drug comprises the liver-targeted chemotherapy drug nano-micelles as described above, and further comprises N-acetylgalactosamine, siRNA and lenvatinib.

[0026] Furthermore, liver-targeted chemotherapy drugs are used in products for the synergistic treatment of liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a flowchart of the steps of an embodiment of the present invention; Figure 2 The synthetic route of Pluronic P123-PEI of Example 1 of the present invention is shown in FIG. Figure 3 Fourier infrared spectrum of an embodiment of the present invention Figure 1 ; Figure 4 Fourier infrared spectrum of an embodiment of the present invention Figure 2 ; Figure 5 This is a diagram showing the particle size and Zeta potential distribution of the nanomicelles of Example 1 of the present invention; Figure 6 This is a graph showing the in vitro release results of nanomicelles in Example 1 of the present invention; Figure 7 This is a graph showing the relative survival rates of human liver cancer cells HepG2 and Huh7 cells, human cervical cancer cells Hela cells and normal liver cells MIHA after being cultured for 24, 48 and 72 hours in the presence of different concentrations of nano-preparations according to the present invention; Figure 8 This is a diagram showing the results of cell apoptosis after incubation of human liver cancer cells HepG2, Huh7 cells and LM3 cells with the drug preparation according to the embodiment of the present invention; Fig. 9 This is a flow cytometry result diagram of human liver cancer cells HepG2 and Huh7 cells, and human cervical cancer cells Hela cells according to the embodiments of the present invention; Fig.10 This is a diagram showing the cell cycle results of human liver cancer cells HepG2, Huh7 cells and LM3 cells after incubation with the drug preparation according to the embodiment of the present invention; Fig.11 This is a graph showing the results of a laser confocal microscopy experiment after incubation of human liver cancer cells HepG2 and Huh7 cells, and human cervical cancer cells Hela cells with a drug preparation according to an embodiment of the present invention; Fig.12 This is a WB result diagram of human liver cancer cells HepG2, Huh7 cells and LM3 cells after incubation with the drug preparation according to the embodiment of the present invention. DETAILED DESCRIPTION

[0029] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0030] In the description of the present invention, it is necessary to understand that the terms "center", "length", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "inside", "outside", "peripheral", "circumferential" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the referred system or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present invention.

[0031] In the description of the present invention, “plurality” means at least two, for example, two, three, etc., unless otherwise clearly and specifically defined.

[0032] In the present invention, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements, unless otherwise clearly defined. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0033] like Figure 1As shown, a method for preparing liver-targeted chemotherapy drug nano-micelles comprises the following steps: S100. Preparation of Pluronic acid succinimidyl ester (P123-NHS): S101. Pluronic P123 vacuum dried and dehydrated; S102. The vacuum-dried Pluronic P123 was dissolved in anhydrous toluene and anhydrous dichloromethane, triphosgene was added and magnetic stirring was performed until the reaction was completed, and then the mixed solution was rotary evaporated to evaporate the solvent, and then dissolved in a mixed solution of anhydrous toluene and anhydrous dichloromethane to prepare a mixed solution; S103. treating the mixed solution prepared in step S102 with N-hydroxysuccinimide, S104. After diluting anhydrous triethylamine with anhydrous dichloromethane, the mixture is added dropwise to the mixed solution treated with N-hydroxysuccinimide in step S103 to prepare a primary solution; S105. Stirring the primary solution prepared in step S104 until the reaction is complete, filtering the primary solution and evaporating it in a vacuum to dryness to form a residue; S106. The residue prepared in step S105 is dissolved in ethyl acetate, filtered, and the filtrate is subjected to rotary evaporation to remove the ethyl acetate, cooled and solidified to form P123-NHS, and then the P123-NHS is stored in a low temperature dry environment for standby use; S200. Preparation of Pluronic-polyethyleneimine copolymer (P123-PEI): S201. dissolving polyethyleneimine (PEI) in phosphate buffer; S202. Activating the P123-NHS prepared in step S106 and dissolving it in a phosphate buffer; S203. The P123-NHS mixed solution in step S202 is added dropwise to the polyethyleneimine (PEI) solution in step S201, and stirred to form a solution after saturation with nitrogen; S204. The stirred solution in step S203 is added to the activated dialysis bag to remove unreacted activated P123, and the ionized water is continuously replaced during the activation process. Finally, the solution is freeze-dried to prepare a P123-PEI polymer, which is then dried and stored at low temperature; S300. Preparation of divinyl sebacate: S301. Sebacic acid, vinyl acetate, mercuric acetate and copper acetate were added to the reaction flask and stirred in a constant temperature water bath to prepare a reaction solution; S302. Concentrated sulfuric acid is added dropwise to the reaction solution prepared in step S301, sodium acetate is added after the reaction is completed, and excess vinyl acetate is removed by vacuum distillation to prepare a secondary reaction solution; S303. The secondary reaction solution in step S302 is separated on a column, with silica gel as the stationary phase and petroleum ether-ethyl acetate as the mobile phase, the ratio of silica gel to petroleum ether-ethyl acetate is 9:1, and divinyl sebacate is separated; S400. Enzymatic synthesis of Pluronic F127-SE: S401. Add Pluronic F127, divinyl sebacate prepared in step S303, lipase (CRL) and acetone into a reaction bottle to prepare a mixed solution. Specifically, the mixing ratio of Pluronic F127, divinyl sebacate, lipase (CRL) and acetone is: 1.3 mmol: 11.6 mmol: 200 mg: 50 ml.

[0034] S402. After the mixed solution in step S401 is shaken in an air bath for reaction, the reaction solution is filtered to remove lipase (CRL), the filtrate is vacuum-dried and added with sterile water for injection, then placed in a dialysis bag for dialysis, and freeze-dried to prepare Pluronic F127-SE powder, wherein the molecular weight cutoff of the dialysis bag is 10,000 Da, and the dialysis time is 45 to 51 hours.

[0035] S500. Preparation of Pluronic F127-C 8 -GalNAc Nanomicelles: S501. Add GalNAc, Pluronic F127-SE prepared in step S401, Lipozyme TL IM lipase and acetone to the reaction flask. Specifically, the ratio of GalNAc, Pluronic F127-SE, Lipozyme TL IM lipase and acetone is: 0.4 mmol: 0.8 mmol: 100 mg: 50 ml.

[0036] S502. After the mixed solution in step S501 is shaken in an air bath for reaction, the reaction solution is filtered to remove the enzyme to prepare an enzyme-free solution; S503. The enzyme-free solution in step S502 is vacuum dried, sterile water for injection is added, and the solution is placed in a dialysis bag for dialysis, and freeze-dried to prepare Pluronic F127-C. 8 -GalNAc; S600. Preparation of GAlNAc@LFT-MMs / siCOP1 complex: S601. P123-PEI 2KDa, PluronicF127-C 8 -GalNAc and LFT are placed in a reaction vessel and methanol is added to dissolve to obtain an organic phase, which is rotary evaporated until the organic solvent is completely removed and then vacuum dried to obtain a drug film; S602. Add sterile water for injection to the drug film prepared in step S601, stir it magnetically and cool it to room temperature, add siCOP1 and incubate it to obtain a transparent micelle solution. The incubation time is 20 min to 40 min.

[0037] S603. The transparent micelle solution prepared in step S602 is filtered through a microporous filter membrane, wherein the microporous filter membrane is a 0.20 μm to 0.24 μm microporous filter membrane, and freeze-dried to prepare a GAlNAc@LFT-MMs / siCOP1 nanocomposite.

[0038] A liver-targeted chemotherapy drug comprises the liver-targeted chemotherapy drug nano-micelles as described above, and further comprises N-acetylgalactosamine, siRNA and lenvatinib.

[0039] Use of liver-targeted chemotherapy drugs in products for the synergistic treatment of liver cancer.

[0040] Embodiment 1 1. Synthesis of P123-PEI / siRNA Nanopolymers 1.1 Synthesis of Pluronic formic acid succinimidyl ester (P123-NHS): Pluronic P123 was dried under vacuum at 50 °C to remove moisture, and the dehydrated P123 (6.96 g, 1.2 mmol) was weighed and dissolved in a mixed solution of anhydrous toluene / anhydrous dichloromethane (3:1, 40 mL), and triphosgene (0.356 g, 1.2 mmol) was added to react with magnetic stirring at room temperature overnight. The reaction solution was rotary evaporated to remove the solvent, and the resulting residue was redissolved in a mixed solution of anhydrous toluene / anhydrous dichloromethane (2:1, 30 mL), then treated with N-hydroxysuccinimide (0.137 g, 1.2 mmol), and anhydrous triethylamine (0.17 mL, 1.2 mmol) was diluted with 3 ml of anhydrous dichloromethane and added dropwise to the reaction solution, and the reaction was continued with stirring for about 4 hours. After the reaction is complete, the reaction solution is filtered and evaporated to dryness in a vacuum. The residue is dissolved in 100 ml of ethyl acetate at 50°C. The filtrate is rotary evaporated to remove the ethyl acetate. The reactant is cooled to solidify and stored at -20°C under dry conditions.

[0041] 1.2. Synthesis of Pluronic-polyethyleneimine copolymer (P123-PEI): PEI (0.2 g, 0.1 mmol) was dissolved in 10 mL phosphate buffer (PBS, pH 7.4), and activated P123-NHS (0.58 g, 0.1 mmol) was dissolved in an appropriate amount of phosphate buffer (PBS, pH 7.4), and slowly added dropwise to the stirring PEI solution. After saturation with nitrogen, the solution was stirred at room temperature for 24 hours, and the reaction solution was added to the activated dialysis bag and dialyzed for 72 hours to remove the unreacted activated P123. The deionized water was replaced every 24 hours. Then, the PluronicP123-PEI polymer was obtained by freeze drying and stored at -20℃. The results were characterized by infrared spectrometer and nuclear magnetic resonance.

[0042] 2. Synthesis and characterization of Pluronic F127-GalNaC: 2.1. Preparation of divinyl sebacate: Take 72.8 g (0.36 mol) of sebacic acid, 150 mL of vinyl acetate (1.62 mol), 2.1 g of mercuric acetate and a trace of copper acetate and add them to a 250 mL three-necked flask. Heat the three-necked flask containing the above substances in a constant temperature water bath at 50 °C and stir. After a few minutes, add 0.25 mL of concentrated sulfuric acid and react for 8 h. After the reaction is completed, add about 2 g of sodium acetate and shake it thoroughly to neutralize the sulfuric acid. After removing the excess vinyl acetate by water pump vacuum distillation, the remaining blue solution is separated on the column. With silica gel as the stationary phase and petroleum ether-ethyl acetate (9:1) as the mobile phase, the pure product is separated.

[0043] 2.2. Enzymatic synthesis of Pluronic F127-SE: Accurately weigh Pluronic F127 1.3 mmol, divinyl sebacate 11.6 mmol, CRL (lipase) 200 mg, and acetone 50 mL, add them into a stoppered conical flask, place in an air bath shaker at 35 ° C, 250 r / min and react for 11.5 hours. After the reaction is completed, the reaction solution is filtered to remove the enzyme, the filtrate is vacuum-dried and added with an appropriate amount of sterile water for injection, and loaded into a pre-treated dialysis bag (molecular weight cutoff of 10000Da), dialyzed for 48 hours, and freeze-dried to obtain Pluronic F127-SE powder.

[0044] 2.3. Enzymatic synthesis of Pluronic F127-C 8 -GalNAc: Take 0.4 mmol of GalNAc, 0.8 mmol of Pluronic F127-SE, 100 mg of Lipozyme TL IM lipase, and 50 mL of acetone into a stoppered conical flask, place it in an air bath shaker at 45 °C and 250 r / min for 24 h. After the reaction is completed, the reaction solution is filtered to remove the enzyme, and the filtrate is vacuum-dried and added with an appropriate amount of sterile injection water, and then placed in a pre-treated dialysis bag (molecular weight cutoff is 10000Da), dialyzed for 48 h, and freeze-dried to obtain Pluronic F127-C. 8 -GalNAc. It was characterized by infrared spectrometer and nuclear magnetic resonance spectrometer respectively.

[0045] like Figure 2 As shown, Figure 2 The synthetic route of Pluronic P123-PEI is: The left A is Pluronic P123; Left B. is triphosgene; Left C is NHS; Left D is P123-NHS; Left E is Pluronic P123-PEI copolymer; A on the right is sebacic acid; Right B is vinyl acetate; Right C is divinyl sebacate; Right D is Puluronic F127; Right E is Puluronic F127-SE; Right F is N-acetylgalactosamine; Right G is Pluronic F127-C 8 -GalNAc.

[0046] 3. Preparation of GalNAc@LFT-MMs / siCOP1 complex 3.1. Weigh the prescription amount of P123-PEI 2KDa, PluronicF127-C 8-GalNAc, LFT in a round-bottom flask, add 10ml methanol and ultrasonically dissolve to obtain an organic phase. Rotate and evaporate at 50℃ for 30 min until the organic solvent is completely removed and a dry transparent film is obtained, which is then vacuum dried in a vacuum oven overnight. Heat in a 50℃ water bath and add an appropriate amount of sterile injection water at the same temperature to hydrate for 1h with magnetic stirring. After cooling to room temperature, add an appropriate amount of siCOP1 and incubate for 30min to obtain a transparent micelle solution. Filter with a 0.22 μm microporous membrane and freeze-dry overnight to obtain a nanocomplex loaded with GalNAc@LFT-MMs / siCOP1.

[0047] 3.2. LFT mixed micelles (LFT-MMs), N-acetylgalactosamine-modified LFT mixed micelles (GalNaC@LFT-MMs), LFT / siRNA mixed micelles (LFT / siRNA-MMs) and blank nanomicelles (GalNaC@MMs) were prepared using the same P123, F127, and LFT composition and the same method.

[0048] 3.3 In addition, we also used the same method to prepare coumarin-loaded nanomicelles for uptake studies.

[0049] like Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 , Fig. 9 , Fig.10 , Fig.11 and Fig.12 As shown, it is mainly used to experiment the characteristics of the nanomicelles prepared in this example.

[0050] 4. In vitro release characteristics: The in vitro release of LFT from LFT-MMs and GAlNAc@LFT-MMs / siCOP1 was studied by dialysis diffusion method in phosphate buffered saline (PBS, pH 7.4) containing 0.5% w / v Tween 80. Briefly, 3 mL of the suspension was added to a pre-swollen dialysis bag (MW = 3000 Da). After sealing, the bags were immersed in 40 mL of phosphate buffered saline (pH 7.4) respectively. The bags were shaken horizontally at 100 rpm at 37 °C. At predetermined time intervals (0.5, 1, 2, 4, 6, 8, 10, 12, 24, 48 h, and 96 h), the release medium outside the dialysis bag was replaced with an equal amount of fresh release medium to maintain the sinking state. The samples were filtered through a 0.22 mm syringe filter and injected into the HPLC system for analysis. In vitro release experiments The release experiments were repeated three times and the results are expressed as mean ± SD (n = 3).

[0051] 5. Determination of Cellular Activity of Nanomicelles In vitro cytotoxicity studies were performed using the cell counting kit-8 (CCK-8) method to determine cell viability. HepG2 cells, Huh7 cells, Hela cells, and MIHA cells in the logarithmic growth phase were plated at 5×10 3 The cells were seeded in a 96-well plate at a density of 100 μL and incubated overnight in 100 μL DMEM containing fetal bovine serum. The old culture medium was aspirated and 100 μL of different concentrations of drug-containing culture medium was added to each well. The experiments were divided into free LFT group (Free LFT), LFT mixed micelle group (LFT-MMs), N-acetylgalactosamine-modified LFT mixed micelle group (GalNaC@LFT-MMs), LFT / siRNA mixed micelle group (LFT / siRNA-MMs), and N-acetylgalactosamine-modified LFT / siRNA mixed micelle group (GalNaC@LFT / siRNA-MMs). Similarly, the micelles loaded with LFT were diluted to a series of LFT concentration gradients (3.125, 6.25, 12.5, 25, 50 and 100 μM) using culture medium. Six replicates were set for each concentration, and each group was set up with a positive control (culture medium and cells, no formula) and a negative control (culture medium only, no cells or formula) group. After culturing at 37°C and 5% carbon dioxide for 24 hours, 48 ​​hours, and 72 hours, 100 μL of DMEM CCK8 solution was added to each well and incubated for about 1 hour. The absorbance (OD) was measured at 490 nm on an ELISA reader, and the inhibition rate of drug on cell proliferation was calculated = [1-(OD experimental group-OD negative control group) / (OD positive control group-OD negative control group)] x 100%. The results are expressed as the mean and standard deviation of three repeated experiments.

[0052] 6. Cell Apoptosis Assay The cells were double-stained using the Annexin V-FITC / PI double staining kit and analyzed by flow cytometry. The specific operation is as follows: three human liver cancer cell suspensions, HepG2, Huh7 and LM3, were added at 1.6×10 5The cells were inoculated in a 6-well plate at a density of 1 ml per well and incubated for 24 h. The supernatant of each well was discarded, and the cells were rinsed with PBS and incubated for 24 h with the Free LFT, LFT-MMs, GalNaC@LFT-MMs, LFT / siRNA-MMs, and GalNaC@LFT / siRNA-MMs solutions containing 2 ug / ml LFT. The treatment group without any drug was used as the negative control group. After removing the culture medium, the cells were digested with trypsin and centrifuged, washed twice with PBS, dispersed in 250 μL of binding buffer, and then mixed with AnnexinV-FITC (5 μL) and PI (5 μL). After incubation for 10 min, the cells were detected by flow cytometry.

[0053] 7. Cell Cycle Assay Three types of human hepatoma cells, HepG2, Huh7 and LM3, were suspended in 1.6 × 10 5 The cells were inoculated in a 6-well plate at a density of 1 ml per well and incubated for 24 h. The supernatant of each well was discarded, and the cells were rinsed with PBS and incubated for 24 h with the above-mentioned Free LFT, LFT-MMs, GalNaC@LFT-MMs, LFT / siRNA-MMs, and GalNaC@LFT / siRNA-MMs solutions containing 2 ug / ml of LFT. The treatment group without any drug was used as the negative control group. The cells were then trypsinized, washed, and fixed in precooled ethanol (70%) at 4°C overnight. The fixed cells were washed twice with 500 μL PBS, resuspended with 500 μL PI / RNase A staining working solution, and detected by flow cytometry after 30 min at room temperature in the dark. For all samples, ModFitLT5.1 program was used for analysis.

[0054] 8. Cellular Uptake 8.1. Quantitative analysis by flow cytometry Human hepatoma cell lines HepG2, Huh-7 and Hela cells in logarithmic growth phase were seeded into 6-well plates at a density of 3×105 cells / ml and incubated in 1 mL of DMEM containing 10% fetal bovine serum. Green fluorescent coumarin 6 (Cou 6) was loaded into nanomicelles according to the preparation method under "2.5" to obtain free coumarin 6 group (Cou 6), coumarin 6 mixed micelle group (Cou6-MMs), N-acetylgalactosamine modified coumarin 6 mixed micelle group (GalNaC@Cou 6-MMs), coumarin 6 / siRNA mixed micelle group (Cou 6 / siRNA-MMs), and N-acetylgalactosamine modified coumarin 6 / siRNA mixed micelle group (GalNaC@Cou 6 / siRNA-MMs), and diluted with DMEM medium to micelles containing Cou6 at a concentration of 1 ug / ml and added to the corresponding wells. After 4 hours of culture, the drug-containing medium was discarded, the cells were washed 3 times with PBS, and the cells were digested with trypsin and prepared into 1×10 5 cells / ml cell suspension, and the fluorescence intensity was measured by flow cytometry.

[0055] 8.2. Competition Analysis Human hepatoma cells HepG-2, Huh7 (ASGPR positive) and cervical cancer cells (ASGPR negative) were cultured at 2.0×10 5 The cells / well were inoculated at a density of 100 μg / mL in a 6-well cell culture plate and cultured for 24 h. To further verify that the nanomicelles were targeted through ASGPR, the original cell culture medium was removed 2 h before administration, and the cell culture medium containing 1 μg / mL free GalNAc was added to saturate ASGPR. They were then incubated again for 4 h with the drug-containing culture medium of GalNaC@Cou 6 / siRNA-MMs containing 1 μg / mL Cou6. The GalNaC@Cou 6 / siRNA-MMs solution not incubated with free GalNAc was set as a positive control group and a negative control group without any treatment. After the incubation, the drug-containing culture medium was discarded, and the cells were washed 3 times with PBS. After trypsinization, 1×10 5 cells / ml cell suspension, and the receptor saturated and unsaturated drug uptake and its fluorescence intensity were detected by flow cytometry.

[0056] 8.3. Qualitative analysis by laser confocal microscopy Human hepatoma cell lines HepG2, Huh-7 and Hela cells in logarithmic growth phase were cultured at 3×10 5Cells were inoculated into 6-well plates at a density of 10 cells / ml and incubated in 1 mL of DMEM containing 10% fetal bovine serum. Cou6, Cou 6-MMs, GalNaC@Cou 6-MMs, Cou 6 / siRNA-MMs, and GalNaC@Cou6 / siRNA-MMs micelles diluted with the above DMEM medium to contain Cou 6 at a concentration of 1ug / ml were added to the corresponding wells. After 6 hours of culture, the cells were washed twice with PBS, fixed with paraformaldehyde for 30 minutes, stained with DAPI for 10 minutes, and observed and photographed with a laser confocal microscope.

[0057] 9. Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR) Three hepatoma cells, HepG2, Huh7 and LM3, in the logarithmic growth phase were plated at 1.6 × 10 5 The cells were placed in a 6-well plate at a density of 1.5 μg / ml and incubated overnight. The experimental group was replaced with a solution containing Free LFT, LFT-MMs, GalNaC@LFT-MMs, LFT / siRNA-MMs, and GalNaC@LFT / siRNA-MMs at a LFT concentration of 5 ug / ml. The blank control group was added with an equal volume of DMEM medium and cultured for 48 h. The cells after intervention were collected, and the total cell RNA was extracted using an RNA extraction kit. The RNA concentration and purity were determined using a nucleic acid protein analyzer. The RNA was reverse transcribed into cRNA using a reverse transcription kit, and then Real-time PCR amplification was performed. The amplification system used was 10 μL. 2 -ΔΔct The relative expression level of COP1 mRNA was calculated.

[0058] Primer sequence: COP1, sense, 5′-GCAGCAGGAAGCGACCTCTC-3′, antisense5′-AAGCAGATGGGGCATACGAAGTC-3′, GAPDH, sense,: 5′-CACCCACTCCTCCACCTTTGAC-3′, Antisense: 5′-GTCCACCACCCTGTTGCTGTAG-3′.

[0059] 10. Western Blot Analysis The cells treated with the same method as in Example 9 were added with RIPA cell lysis buffer and lysed on ice for 5 min. ◦The supernatant was collected by centrifugation at 400 °C to obtain total protein, and the protein concentration was determined using the bicinchoninic acid (BCA) protein quantification kit method. 20 μg of protein in each sample was separated by 10% SDS-PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking the membrane with 5% skim milk at room temperature for 2 hours, the membrane was incubated with mouse anti-COP1 antibody (1:1000) or rabbit anti-GAPDH antibody (1:1000) at 4 °C overnight. The secondary antibody (1:2000) was incubated at room temperature for 2 hours. Finally, the protein on the band was observed using an enhanced chemiluminescence (ECL) system, and the relative protein expression level was quantified and analyzed using Image J software.

[0060] From the above-mentioned detection experiments, it can be seen that polymer micelles can simultaneously target the chemotherapy drug LFT and siRNA to liver cancer cells, and effectively release them in tumor cells to achieve stronger tumor inhibition and lower systemic toxicity. It has multiple functions such as active targeting, chemotherapy and gene therapy, especially in stem cell tumor treatment, and has broad application prospects.

[0061] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0062] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing liver-targeted chemotherapy drug nano-micelles, characterized in that: The following steps are involved: S100. Preparation of Pluronic-formic acid succinimidyl ester (P123-NHS): S101. PluronicP123 was dried under vacuum and the moisture was removed; S102. The vacuum-dried PluronicP123 was dissolved in anhydrous toluene and anhydrous dichloromethane, triphosgene was added and magnetic stirring was performed until the reaction was completed, and then the mixture was rotary evaporated to evaporate the solvent, and then dissolved in a mixed solution of anhydrous toluene and anhydrous dichloromethane to prepare a mixed solution; S103. treating the mixed solution prepared in step S102 with N-hydroxysuccinimide, S104. After diluting anhydrous triethylamine with anhydrous dichloromethane, the mixture is added dropwise to the mixed solution treated with N-hydroxysuccinimide in step S103 to prepare a primary solution; S105. Stirring the primary solution prepared in step S104 until the reaction is complete, filtering the primary solution and evaporating it in a vacuum to dryness to form a residue; S106. The residue prepared in step S105 is dissolved in ethyl acetate, filtered, and the filtrate is subjected to rotary evaporation to remove the ethyl acetate, cooled and solidified to form P123-NHS, and then the P123-NHS is stored in a low temperature dry environment for standby use; S200. Preparation of Pluronic-polyethyleneimine copolymer (P123-PEI): S201. dissolving polyethyleneimine (PEI) in phosphate buffer; S202. Activating the P123-NHS prepared in step S106 and dissolving it in a phosphate buffer; S203. The P123-NHS mixed solution in step S202 is added dropwise to the polyethyleneimine (PEI) solution in step S201, and stirred to form a solution after saturation with nitrogen; S204. The stirred solution in step S203 is added to the activated dialysis bag to remove unreacted activated P123, and the ionized water is continuously replaced during the dialysis process. Finally, the solution is freeze-dried to prepare a P123-PEI polymer, which is then dried and stored at low temperature; S300. Preparation of divinyl sebacate: S301. Sebacic acid, vinyl acetate, mercuric acetate and copper acetate were added to the reaction flask and stirred in a constant temperature water bath to prepare a reaction solution; S302. Concentrated sulfuric acid is added dropwise to the reaction solution prepared in step S301, sodium acetate is added after the reaction is completed, and excess vinyl acetate is removed by vacuum distillation to prepare a secondary reaction solution; S303. The secondary reaction solution in step S302 is separated on a column using silica gel as the stationary phase and petroleum ether-ethyl acetate as the mobile phase to separate divinyl sebacate; S400. Enzymatic synthesis of Pluronic F127-SE: S401. Pluronic F127, divinyl sebacate prepared in step S303, lipase (CRL) and acetone are added to a reaction flask to prepare a mixed solution. S402. After the mixed solution in step S401 is reacted in an air bath shaker, the reaction solution is filtered to remove lipase (CRL), the filtrate is vacuum dried and sterile water for injection is added, and then dialyzed in a dialysis bag, and freeze-dried to prepare PluronicF127-SE powder; S500. Preparation of PluronicF127-C8-GalNAc nanomicelles: S501. Add GalNAc, Pluronic F127-SE prepared in step S401, Lipozyme TL IM lipase, and acetone to the reaction flask; S502. After the mixed solution in step S501 is reacted in an air bath shaker, the reaction solution is filtered to remove the enzyme to prepare an enzyme-free solution; S503. The enzyme-free solution in step S502 is vacuum dried, sterile water for injection is added, and the solution is placed in a dialysis bag for dialysis, and freeze-dried to prepare Pluronic F127-C8-GalNAc; S600. Preparation of GAlNAc@LFT-MMs / siCOP1 complex: S601. P123-PEI 2KDa, PluronicF127-C8-GalNAc and LFT were placed in a reaction vessel and methanol was added to dissolve to obtain an organic phase, which was rotary evaporated until the organic solvent was completely removed and then vacuum dried to obtain a drug film; S602. Add sterile water for injection to the drug film prepared in step S601, stir it magnetically and cool it to room temperature, then add siCOP1 to incubate to obtain a transparent micelle solution; S603. Filter the transparent micelle solution prepared in step S602 with a microporous filter membrane, and prepare a GalNAc@LFT-MMs / siCOP1 nanocomplex after freeze-drying.

2. The method for preparing liver-targeted chemotherapy drug nano-micelles according to claim 1, characterized in that: In step S501, the ratio of GalNAc, Pluronic F127-SE, Lipozyme TL IM lipase, and acetone is: 0.4mmol:0.8mmol:100mg:50ml.

3. The method for preparing liver-targeted chemotherapy drug nano-micelles according to claim 1, characterized in that: In step S401 , the mixing ratio of Pluronic F127, divinyl sebacate, lipase (CRL) and acetone is: 1.3 mmol: 11.6 mmol: 200 mg: 50 ml.

4. The method for preparing liver-targeted chemotherapy drug nano-micelles according to claim 1, characterized in that: In step S402, the molecular weight cut-off of the dialysis bag is 10000 Da.

5. The method for preparing liver-targeted chemotherapy drug nano-micelles according to claim 1, characterized in that: In step S303, the ratio of petroleum ether and ethyl acetate in the mobile phase is 9:

1.

6. The method for preparing liver-targeted chemotherapy drug nano-micelles according to claim 1, characterized in that: In step S603, the microporous filter membrane is a 0.20 μm to 0.24 μm microporous filter membrane.

7. The method for preparing liver-targeted chemotherapy drug nano-micelles according to claim 1, characterized in that: In step S602, after adding siCOP1, the incubation time is 20 min to 40 min.

8. The method for preparing liver-targeted chemotherapy drug nano-micelles according to claim 1, characterized in that: In step 402, the dialysis time is 45 hours to 51 hours.

9. A liver-targeted chemotherapy drug, characterized in that: The method comprises the liver-targeted chemotherapy drug nano-micelles as described in any one of claims 1 to 8, and further comprises N-acetylgalactosamine, siRNA and lenvatinib.

10. Use of the liver-targeted chemotherapy drug according to claim 9 in a product for synergistically treating liver cancer.

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