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] This invention relates to the field of pharmaceutical application technology, and in particular to a method for preparing liver-targeted chemotherapy drug nanomicelles, the chemotherapy drug, and its application. Background Technology

[0002] Hepatocellular carcinoma is one of the most common malignant tumors worldwide, with a poor prognosis and high recurrence rate. In recent years, the multi-kinase inhibitor lenvatinib (LFT) has attracted much attention for its superior efficacy over sorafenib in improving patient prognosis and mitigating side effects. However, due to its low oral bioavailability, higher doses are required to produce anti-tumor effects, leading to side effects such as diarrhea and vomiting.

[0003] Small interfering RNA (siRNA) is used to silence cancer-related genes, thereby activating apoptosis pathways in cancer cells or inhibiting anti-apoptotic pathways. COP1 is a highly conserved E3-ubiquitin ligase. For the tumor suppressor protein p53, it is a key negative regulatory protease that affects 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, free siRNA faces challenges due to its hydrophilicity, negative charge, and sensitivity to RNases, making it difficult for siRNA to cross negatively charged cell membranes and circulate stably in vivo. Therefore, researchers have focused on constructing co-delivery vectors that can efficiently encapsulate anticancer drugs and siRNA, ensuring precise release of these therapeutic molecules at the tumor site. Polyethyleneimine (PEI), as a non-viral vector, is widely used for nucleic acid delivery due to its unique proton sponge effect and superior transfection efficiency.

[0004] To improve the targeting of nanoparticles in tumor therapy, researchers have used chemical methods to modify polymer micelle carriers with ligands that can bind to tumor-specific receptors, achieving active targeted delivery to tumors. Specifically, in the field of liver therapy, desialylate glycoprotein receptors, widely expressed on the hepatocyte membrane, have become ideal targets for liver-specific drug delivery, reducing systemic toxicity and improving efficacy. Furthermore, N-acetylgalactosamine, a ligand with high affinity for ASGPR on the hepatocyte surface, can effectively and specifically deliver chemotherapeutic drugs to ASGPR-expressing hepatocytes. This precise targeted delivery method ensures a higher concentration of therapeutic drugs at the tumor site, thereby improving therapeutic efficacy while significantly reducing the risk of toxicity to healthy cells. However, currently, the preparation efficiency of nanocarriers for liver-targeted chemotherapeutic drugs is low, and their targeting is insufficient, hindering large-scale application.

[0005] Therefore, those skilled in the art are dedicated to developing a method for preparing liver-targeted chemotherapy drug nanomicelles, chemotherapy drugs, and their applications, which will facilitate the rapid preparation of nanomicelles with multiple functions including active targeting, chemotherapy, and gene therapy. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for preparing liver-targeted chemotherapy drug nanomicelles, chemotherapy drugs and their applications, which facilitates the rapid preparation of nanomicelles with multiple functions of active targeting, chemotherapy and gene therapy.

[0007] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0008] A method for preparing liver-targeted chemotherapy drug nanomicelles, characterized by comprising the following steps:

[0009] S100. Preparation of Pranic-formate succinimide ester (P123-NHS):

[0010] S101. Pluronic P123 vacuum drying and moisture removal;

[0011] S102. Dissolve vacuum-dried Pluronic P123 in anhydrous toluene and anhydrous dichloromethane, add triphosgene and stir magnetically until the reaction is complete, then evaporate the solvent by rotary evaporation, and dissolve it in a mixed solution of anhydrous toluene and anhydrous dichloromethane to prepare a mixed solution.

[0012] S103. Treat the mixture prepared in step S102 with N-hydroxysuccinimide.

[0013] S104. Anhydrous triethylamine is diluted with anhydrous dichloromethane and then added dropwise to the mixture treated with N-hydroxysuccinimide in step S103 to prepare a primary solution.

[0014] S105. Stir the primary solution prepared in step S104 until the reaction is complete, then filter the primary solution and evaporate it under vacuum until dry to form a residue.

[0015] S106. Dissolve the residue prepared in step S105 in ethyl acetate, filter, and evaporate the ethyl acetate by rotary evaporation of the filtrate. After cooling and solidification, P123-NHS is formed. P123-NHS is then stored in a low-temperature and dry environment for later use.

[0016] S200. Preparation of Pluronic-polyethyleneimine copolymer (P123-PEI):

[0017] S201. Dissolve polyethyleneimine (PEI) in phosphate buffer;

[0018] S202. Activate the P123-NHS prepared in step S106 and dissolve it in phosphate buffer;

[0019] S203. The P123-NHS mixed solution from step S202 is added dropwise to the polyethyleneimine (PEI) solution from step S201, and the solution is formed by magnetic stirring after nitrogen saturation.

[0020] S204. Add the solution stirred in step S203 to the activated dialysis bag to remove unreacted activated P123. During the dialysis process, the deionized water is continuously replaced. Finally, the solution is freeze-dried to prepare P123-PEI polymer and stored at low temperature.

[0021] S300. Preparation of diethylene sebacic acid:

[0022] S301. Add sebacic acid, vinyl acetate, mercuric acetate and copper acetate to a reaction flask and stir in a constant temperature water bath to prepare a reaction solution;

[0023] S302. Add concentrated sulfuric acid dropwise to the reaction solution prepared in step S301. After the reaction is complete, add sodium acetate and remove excess vinyl acetate by vacuum distillation to prepare a secondary reaction solution.

[0024] S303. Separate the reaction secondary solution from step S302 onto a column using silica gel as the stationary phase and petroleum ether-ethyl acetate as the mobile phase to obtain divinyl sebacate.

[0025] S400. Enzymatic synthesis of Pluronic F127-SE:

[0026] S401. Add Pluronic F127, diethylene sebacic acid ester prepared in step S303, lipase (CRL), and acetone to a reaction flask to prepare a mixed solution.

[0027] S402. After the mixed solution in step S401 is reacted by air bath, the reaction solution is filtered to remove lipase (CRL), the filtrate is vacuum dried and then sterile water for injection is added, then it is placed in a dialysis bag for dialysis, and then freeze-dried to prepare Pluronic F127-SE powder.

[0028] S500. Preparation of Pluronic F127-C8-Gal NAc nanomicelles:

[0029] S501. Add Gal NAc, Pluronic F127-SE prepared in step S401, Lipozyme TL IM lipase, and acetone to the reaction flask;

[0030] S502. After the mixed solution in step S501 is reacted by air bath shaking, the enzyme is filtered out from the reaction solution to prepare an enzyme-free solution;

[0031] S503. After vacuum drying the enzyme-free solution from step S502, add sterile water for injection and place it into a dialysis bag for dialysis, and freeze-dry to prepare Pluronic F127-C8-Gal NAc;

[0032] S600. Preparation of Gal NAc@LFT-MMs / si COP1 complex:

[0033] S601. Place P123-PEI 2KDa, Pluronic F127-C8-Gal NAc and LFT in a reaction vessel and add methanol to dissolve them to obtain an organic phase. Rotary evaporate until the organic solvent is completely removed and then vacuum dry to obtain a drug film.

[0034] S602. After adding the drug film prepared in step S601 to sterile water for injection, stir magnetically and cool to room temperature, add Si COP1 and incubate to obtain a transparent micelle solution;

[0035] S603. The transparent micelle solution prepared in step S602 is filtered with a microporous filter membrane and then freeze-dried to prepare a GAlNAc@LFT-MMs / si COP1 nanocomposite.

[0036] The beneficial effects of the above scheme are: the polymer micelles prepared by the above steps can simultaneously target the chemotherapeutic drugs LFT and siRNA to liver cancer cells, effectively releasing them into the tumor cells to achieve stronger tumor inhibition and lower systemic toxicity.

[0037] By leveraging the advantages of nanomicelles and combining them with the combined effects of siRNA and chemotherapy drugs, the optimal conditions for the combined use of the two drugs can be fully explored.

[0038] Meanwhile, the preparation method of this invention is simple, which is of great significance for developing new anti-liver cancer gene drugs and improving the treatment effect of liver cancer, and has broad application prospects.

[0039] Based on the above technical solution, the present invention can be further improved as follows.

[0040] Furthermore, in step S501, the ratio of Gal NAc, Pluronic F127-SE, Lipozyme TL IM lipase, and acetone is 0.4 mmol: 0.8 mmol: 100 mg: 50 ml.

[0041] The beneficial effect of adopting the above-mentioned further scheme is that the appropriate component ratio is conducive to the subsequent preparation of PluronicF127-C8-Gal NAc.

[0042] Furthermore, in step S401, the mixing ratio of Pluronic F127, diethylene sebacate, lipase (CRL), and acetone is 1.3 mmol: 11.6 mmol: 200 mg: 50 ml.

[0043] The beneficial effect of adopting the above-mentioned further scheme is that the appropriate component ratio is conducive to the subsequent preparation of Pluronic F127-SE.

[0044] Furthermore, in step S402, the molecular weight cutoff of the dialysis bag is 10000 Da.

[0045] The beneficial effect of adopting the above-mentioned further scheme is that the molecular weight cutoff of the dialysis bag is 10000 Da, which is conducive to removing impurities and water from the solution.

[0046] Furthermore, in step S303, the ratio of the mobile phase petroleum ether to ethyl acetate is 9:1.

[0047] The beneficial effect of adopting the above-mentioned further scheme is that it facilitates the preparation of pure divinyl sebacate.

[0048] Furthermore, in step S603, the microporous filter membrane is a microporous filter membrane with a diameter of 0.20 μm to 0.24 μm.

[0049] The beneficial effect of adopting the above-mentioned further scheme is that it further purifies the reaction solution.

[0050] Furthermore, in step S602, after adding Si COP1, the incubation time is 20 min to 40 min.

[0051] The beneficial effect of adopting the above-mentioned further scheme is to improve the synthesis efficiency of transparent micelle solutions.

[0052] Furthermore, in step 402, the dialysis time is 45 hours to 51 hours.

[0053] The beneficial effect of adopting the above-mentioned further solution is to remove impurities and water from the solution.

[0054] A liver-targeted chemotherapy drug comprising liver-targeted chemotherapy drug nanomicelles as described above, and further comprising N-acetylgalactosamine, siRNA, and lenvatinib.

[0055] Furthermore, the use of liver-targeted chemotherapy drugs in products for the synergistic treatment of liver cancer. Attached Figure Description

[0056] Figure 1 This is a flowchart illustrating the steps of an embodiment of the present invention;

[0057] Figure 2 This is a synthetic route diagram of Pluronic P123-PEI according to Embodiment 1 of the present invention;

[0058] Figure 3 This is an example of Fourier transform infrared spectroscopy in an embodiment of the present invention. Figure 1 ;

[0059] Figure 4 This is an example of Fourier transform infrared spectroscopy in an embodiment of the present invention. Figure 2 ;

[0060] Figure 5 This is a diagram showing the particle size and Zeta potential distribution of nanomicelles in Embodiment 1 of the present invention;

[0061] Figure 6 This is a diagram showing the in vitro release results of nanomicelles in one embodiment of the present invention;

[0062] Figure 7 The relative survival rates of human hepatocellular carcinoma HepG2 and Huh7 cells, human cervical carcinoma HeLa cells, and normal hepatocytes MIHA after culturing for 24, 48, and 72 hours under different concentrations of nano-prepared agents are shown in the figure below, representing embodiments of the present invention.

[0063] Figure 8 This is a diagram showing the apoptosis results of HepG2, Huh7, and LM3 liver cancer cells after incubation with the drug formulation in an embodiment of the present invention.

[0064] Figure 9 Flow cytometry results of human liver cancer cells HepG2 and Huh7 cells, and human cervical cancer cells HeLa cells, as described in this embodiment of the invention.

[0065] Figure 10 This is a cell cycle diagram of HepG2, Huh7 and LM3 human liver cancer cells after incubation with the drug preparation in an embodiment of the present invention;

[0066] Figure 11 The image shows 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 the drug preparation, according to an embodiment of the present invention.

[0067] Figure 12 The image shows the Western blot (WB) results of HepG2, Huh7, and LM3 liver cancer cells in an embodiment of the present invention after incubation with the drug preparation. Detailed Implementation

[0068] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0069] In the description of this invention, it should be understood that the terms "center," "length," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "inner," "outer," "circumferential," and "circumferential" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing this invention and simplifying the description, and are not intended to indicate or imply that the system or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0070] In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0071] In this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components, unless otherwise explicitly limited. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0072] like Figure 1 As shown, a method for preparing liver-targeted chemotherapy drug nanomicelles includes the following steps:

[0073] S100. Preparation of succinimide praseodymide formate (P123-NHS):

[0074] S101. Pluronic P123 vacuum drying and moisture removal;

[0075] S102. Dissolve vacuum-dried Pluronic P123 in anhydrous toluene and anhydrous dichloromethane, add triphosgene and stir magnetically until the reaction is complete, then evaporate the solvent by rotary evaporation, and dissolve it in a mixed solution of anhydrous toluene and anhydrous dichloromethane to prepare a mixed solution.

[0076] S103. Treat the mixture prepared in step S102 with N-hydroxysuccinimide.

[0077] S104. Anhydrous triethylamine is diluted with anhydrous dichloromethane and then added dropwise to the mixture treated with N-hydroxysuccinimide in step S103 to prepare a primary solution.

[0078] S105. Stir the primary solution prepared in step S104 until the reaction is complete, then filter the primary solution and evaporate it under vacuum until dry to form a residue.

[0079] S106. Dissolve the residue prepared in step S105 in ethyl acetate, filter, and evaporate the ethyl acetate by rotary evaporation of the filtrate. After cooling and solidification, P123-NHS is formed. P123-NHS is then stored in a low-temperature and dry environment for later use.

[0080] S200. Preparation of Pluronic-polyethyleneimine copolymer (P123-PEI):

[0081] S201. Dissolve polyethyleneimine (PEI) in phosphate buffer;

[0082] S202. Activate the P123-NHS prepared in step S106 and dissolve it in phosphate buffer;

[0083] S203. The P123-NHS mixed solution from step S202 is added dropwise to the polyethyleneimine (PEI) solution from step S201, and stirred to form a solution after nitrogen saturation;

[0084] S204. Add the solution stirred in step S203 to the activated dialysis bag to remove unreacted activated P123. During the activation process, the deionized water is continuously replaced. Finally, the solution is freeze-dried to prepare P123-PEI polymer and stored at low temperature.

[0085] S300. Preparation of diethylene sebacic acid:

[0086] S301. Add sebacic acid, vinyl acetate, mercuric acetate and copper acetate to a reaction flask and stir in a constant temperature water bath to prepare a reaction solution;

[0087] S302. Add concentrated sulfuric acid dropwise to the reaction solution prepared in step S301. After the reaction is complete, add sodium acetate and remove excess vinyl acetate by vacuum distillation to prepare a secondary reaction solution.

[0088] S303. Separate the reaction secondary solution from step S302 onto a column using 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. Diethylene sebacate is obtained by separation.

[0089] S400. Enzymatic synthesis of Pluronic F127-SE:

[0090] S401. Add Pluronic F127, diethylene sebacate prepared in step S303, lipase (CRL), and acetone to a reaction flask to prepare a mixed solution. Specifically, the mixing ratio of Pluronic F127, diethylene sebacate, lipase (CRL), and acetone is 1.3 mmol: 11.6 mmol: 200 mg: 50 ml.

[0091] S402. After the mixed solution in step S401 is reacted by air bath, the reaction solution is filtered to remove lipase (CRL). The filtrate is vacuum dried and then sterile water for injection is added. After dialysis in a dialysis bag, the solution is freeze-dried to prepare Pluronic F127-SE powder. The molecular weight cutoff of the dialysis bag is 10000 Da, and the dialysis time is 45 h to 51 h.

[0092] S500. Preparation of Pluronic F127-C8-Gal NAc nanomicelles:

[0093] S501. Add Gal NAc, Pluronic F127-SE prepared in step S401, Lipozyme TL IM lipase, and acetone to the reaction flask. Specifically, the ratio of Gal NAc, Pluronic F127-SE, Lipozyme TL IM lipase, and acetone is 0.4 mmol: 0.8 mmol: 100 mg: 50 ml.

[0094] S502. After the mixed solution in step S501 is reacted by air bath shaking, the enzyme is filtered out from the reaction solution to prepare an enzyme-free solution;

[0095] S503. After vacuum drying the enzyme-free solution from step S502, add sterile water for injection and place it into a dialysis bag for dialysis, and freeze-dry to prepare Pluronic F127-C8-Gal NAc;

[0096] S600. Preparation of GAl NAc@LFT-MMs / si COP1 complex:

[0097] S601. Place P123-PEI 2KDa, PluronicF127-C8-Gal NAc and LFT in a reaction vessel and add methanol to dissolve them to obtain an organic phase. Rotary evaporate until the organic solvent is completely removed and then vacuum dry to obtain a drug film.

[0098] S602. Add the drug film prepared in step S601 to sterile water for injection, stir magnetically and cool to room temperature, add Si COP1 and incubate to obtain a transparent micelle solution for 20 min to 40 min.

[0099] S603. Filter the transparent micelle solution prepared in step S602 using a microporous filter membrane, wherein the microporous filter membrane is a 0.20 μm to 0.24 μm microporous filter membrane, and freeze-dry it to prepare a GAl NAc@LFT-MMs / si COP1 nanocomposite.

[0100] A liver-targeted chemotherapy drug comprising liver-targeted chemotherapy drug nanomicelles as described above, and further comprising N-acetylgalactosamine, siRNA, and lenvatinib.

[0101] The use of liver-targeted chemotherapy drugs in products for the synergistic treatment of liver cancer.

[0102] Example 1

[0103] 1. Synthesis of P123-PEI / siRNA nanopolymers

[0104] 1.1 Synthesis of Pranic succinimide formate (P123-NHS):

[0105] Pluronic P123 was vacuum dried at 50 °C to remove moisture. 6.96 g (1.2 mmol) of the dehydrated P123 was weighed and dissolved in a 3:1 mixture of anhydrous toluene and anhydrous dichloromethane (40 mL). Triphosgene (0.356 g, 1.2 mmol) was added, and the mixture was magnetically stirred overnight at room temperature. The solvent was removed by rotary evaporation, and the residue was redissolved in a 2:1 mixture of anhydrous toluene and anhydrous dichloromethane (30 mL). The residue was 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 mixture. The reaction was continued with stirring for approximately 4 hours. After the reaction is complete, the reaction solution is filtered, evaporated to dryness under vacuum, and the resulting residue is dissolved in 100 ml of ethyl acetate at 50 °C. The filtrate is then evaporated by rotary evaporation to remove the ethyl acetate. The reactants are then cooled and solidified and stored under dry conditions at -20 °C.

[0106] 1.2. Synthesis of Pluronic-polyethyleneimine copolymer (P123-PEI):

[0107] PEI (0.2 g, 0.1 mmol) was dissolved in 10 mL of phosphate-buffered saline (PBS, pH 7.4). Activated P123-NHS (0.58 g, 0.1 mmol) was dissolved in an appropriate amount of phosphate-buffered saline (PBS, pH 7.4) and slowly added dropwise to the continuously stirred PEI solution. After nitrogen saturation, the solution was stirred at room temperature for 24 h. The reaction solution was then added to an activated dialysis bag and dialyzed for 72 h to remove unreacted activated P123. Deionized water was replaced every 24 h. The resulting Pluronic P123-PEI polymer was then freeze-dried and stored at -20 °C. The results were characterized using infrared spectroscopy and nuclear magnetic resonance spectroscopy.

[0108] 2. Synthesis and characterization of Pluronic F127-Gal NaC:

[0109] 2.1. Preparation of divinyl sebacate:

[0110] 72.8 g (0.36 mol) sebacic acid, 150 mL vinyl acetate (1.62 mol), 2.1 g mercuric acetate, and a trace amount of copper acetate were added to a 250 mL three-necked flask. The flask containing the above substances was heated and stirred in a constant temperature water bath at 50 °C. After a few minutes, 0.25 mL of concentrated sulfuric acid was added dropwise, and the reaction was allowed to proceed for 8 hours. After the reaction was complete, about 2 g of sodium acetate was added and the mixture was shaken thoroughly to neutralize the sulfuric acid. Excess vinyl acetate was removed by vacuum distillation using a water pump, and the remaining blue solution was separated by column chromatography. Using silica gel as the stationary phase and petroleum ether-ethyl acetate (9:1) as the mobile phase, the pure product was obtained.

[0111] 2.2. Enzymatic synthesis of Pluronic F127-SE:

[0112] Accurately weigh 1.3 mmol of Pluronic F127, 11.6 mmol of diethylene sebate, 200 mg of CRL (lipase), and 50 mL of acetone, and add them to a stoppered conical flask. Place the flask in an air bath shaker at 35°C and 250 rpm for 11.5 h. After the reaction is complete, filter the reaction solution to remove the enzyme. After vacuum drying the filtrate, add an appropriate amount of sterile water for injection, place it in a pre-treated dialysis bag (molecular weight cutoff of 10000 Da), dialyze for 48 h, and freeze-dry to obtain Pluronic F127-SE powder.

[0113] 2.3. Enzymatic synthesis of Pluronic F127-C8-Gal NAc:

[0114] 0.4 mmol of GalNAc, 0.8 mmol of Pluronic F127-SE, 100 mg of Lipozyme TL IM lipase, and 50 mL of acetone were added to a stoppered conical flask and placed in an air bath shaker at 45°C and 250 rpm for 24 h. After the reaction, the reaction solution was filtered to remove the enzyme. The filtrate was then evaporated under vacuum, and an appropriate amount of sterile water for injection was added. The solution was then placed in a pre-treated dialysis bag (molecular weight cutoff of 10000 Da), dialyzed for 48 h, and freeze-dried to obtain Pluronic F127-C8-GalNAc. The flask was characterized by infrared spectroscopy and nuclear magnetic resonance spectroscopy.

[0115] like Figure 2 As shown, Figure 2 Synthetic route diagram for Pluronic P123-PEI:

[0116] Where A on the left is Pluronic P123;

[0117] Left B. is three-dimensional light;

[0118] Left C represents NHS;

[0119] Left D is P123-NHS;

[0120] Left E is a Pluronic P123-PEI copolymer;

[0121] Right A is sebacic acid;

[0122] B on the right is vinyl acetate;

[0123] C on the right is divinyl sebacate;

[0124] The rightmost D is the Puluonic F127;

[0125] The right-hand E is the Puluonic F127-SE;

[0126] F on the right is N-acetylgalactosamine;

[0127] The right G is Pluronic F127-C8-Gal NAc.

[0128] 3. Preparation of Gal NAc@LFT-MMs / si COP1 complex

[0129] 3.1. Weigh the prescribed amounts of P123-PEI 2 kDa and Pluronic F127-C8-Gal NAc, LFT into a round-bottom flask, add 10 ml of methanol, and sonicate until dissolved to obtain the organic phase. Evaporate at 50°C for 30 min until the organic solvent is completely removed, obtaining a dry, transparent film. Vacuum dry overnight in a vacuum drying oven. Heat in a 50°C water bath and add an appropriate amount of sterile water for injection at the same temperature, stirring magnetically for 1 h to hydrate. After cooling to room temperature, add an appropriate amount of si COP1 and incubate for 30 min to obtain a transparent micelle solution. Filter through a 0.22 μm microporous membrane and freeze-dry overnight to obtain the Gal NAc@LFT-MMs / si COP1 nanocomposite; wherein the nucleotide RNA sequence of si COP1 (from 5' to 3') is: CCUGUACUAUAAAGGACUUTT.

[0130] 3.2. Using the same P123, F127, LFT composition and the same method, LFT mixed micelles (LFT-MMs), N-acetylgalactosamine modified LFT mixed micelles (Gal NaC@LFT-MMs), LFT / siRNA mixed micelles (LFT / siRNA-MMs) and blank nanomicelles (Gal NaC@MMs) were prepared.

[0131] 3.3 In addition, we also used the same method to prepare coumarin-loaded nanomicelles for uptake studies.

[0132] like Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 , Figure 9 , Figure 10 , Figure 11 and Figure 12 As shown, it is mainly used to experiment with the properties of the nanomicelles prepared in this embodiment.

[0133] 4. In vitro release characteristics:

[0134] The in vitro release of LFT from LFT-MMs and GA1 NAc@LFT-MMs / siCOP1 was investigated using dialysis diffusion in phosphate-buffered saline (PBS, pH 7.4) containing 0.5% w / v Tween 80. Briefly, 3 mL of suspension was added to a pre-expanded dialysis bag (MW = 3000 Da). After sealing, the bag was immersed in 40 mL of phosphate-buffered saline (pH 7.4). The bag was then horizontally shaken 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 volume of fresh release medium to maintain a settling state. Samples were filtered through a 0.22 mm syringe filter and injected into an HPLC system for analysis. The in vitro release experiment was repeated three times, and results are expressed as mean ± SD (n = 3).

[0135] 5. Assay of cell viability of nanomicelles

[0136] In vitro cytotoxicity studies were performed using the Cell Counting Kit-8 (CCK-8) assay to determine cell viability. HepG2 cells, Huh7 cells, HeLa cells, and MIHA cells in logarithmic growth phase were cultured at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of 100 μL in 96-well plates and incubated overnight in 100 μL of DMEM containing fetal bovine serum. The old medium was removed, and 100 μL of different concentrations of drug-containing medium was added to each well. The experiment included free LFT, LFT-mixed micelles (LFT-MMs), N-acetylgalactosamine-modified LFT-mixed micelles (Gal NaC@LFT-MMs), LFT / siRNA-mixed micelles (LFT / siRNA-MMs), and N-acetylgalactosamine-modified LFT / siRNA-mixed micelles (Gal NaC@LFT / siRNA-MMs). Similarly, the LFT-loaded micelles were diluted to a series of LFT concentration gradients (3.125, 6.25, 12.5, 25, 50, and 100 μM) using medium. Each concentration was used in 6 replicates, and each group included a positive control (medium and cells, no formulation) and a negative control (medium only, no cells or formulation). After culturing at 37℃ and 5% CO2 for 24, 48, and 72 hours respectively, 100 μL of DMEM CCK8 solution was added to each well, and incubation was carried out for approximately 1 hour. The absorbance (OD) was measured at 490 nm using a microplate reader. The inhibition rate of cell proliferation by the drug was calculated as [1 - (OD experimental group - OD negative control group) / (OD positive control group - OD negative control group)] x 100%. Results are expressed as the mean and standard deviation of three replicate experiments.

[0137] 6. Apoptosis assay

[0138] Cells were double-stained using the Annexin V-FITC / PI double staining kit and analyzed by flow cytometry.

[0139] The effects of each drug administration group on cell apoptosis were investigated. The specific procedure was as follows: Human liver cancer cell suspensions of three types (HepG2, Huh7, and LM3) were injected with 1.6 × 10⁻⁶ cells / mL. 5 Cells were seeded at a density of 1 ml per well in 6-well plates and incubated for 24 h. The supernatant from each well was discarded, and the cells were washed with PBS. Then, solutions containing 2 μg / ml of Free LFT, LFT-MMs, Gal NaC@LFT-MMs, LFT / siRNA-MMs, and Gal NaC@LFT / siRNA-MMs were added and incubated for 24 h. A negative control group (without any drugs) was used. After removing the culture medium, the cells were trypsinized, centrifuged, washed twice with PBS, and dispersed in 250 μL of binding buffer. This was then mixed with Annexin V-FITC (5 μL) and PI (5 μL). After incubation for 10 min, the cells were analyzed by flow cytometry.

[0140] 7. Cell cycle assay

[0141] Suspensions of three types of human liver cancer cells, HepG2, Huh7, and LM3, were prepared at 1.6 × 10⁻⁶ cells / mL. 5 Cells were seeded at a density of 1 ml per well in 6-well plates and incubated for 24 h. The supernatant in each well was discarded, and the cells were washed with PBS. Then, solutions containing 2 μg / ml of Free LFT, LFT-MMs, Gal NaC@LFT-MMs, LFT / siRNA-MMs, or Gal NaC@LFT / siRNA-MMs were added and incubated for 24 h. A negative control group was used, without any added drugs. Cells were then trypsinized, washed, and fixed overnight in pre-chilled 70% ethanol at 4°C. The fixed cells were washed twice with 500 μL of PBS, resuspended in 500 μL of PI / RNase A staining working solution, and analyzed by flow cytometry after 30 min in the dark at room temperature. All samples were analyzed using the ModFit LT5.1 program.

[0142] 8. Cellular uptake

[0143] 8.1. Quantitative analysis by flow cytometry

[0144] Human hepatocellular carcinoma 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-banded fluorescent coumarin 6 (Cou6) was loaded into nanomicelles according to the preparation method described in section "2.5" to obtain free coumarin 6 (Cou 6), coumarin 6 mixed micelles (Cou 6-MMs), N-acetylgalactosamine-modified coumarin 6 mixed micelles (Gal NaC@Cou 6-MMs), coumarin 6 / siRNA mixed micelles (Cou6 / siRNA-MMs), and N-acetylgalactosamine-modified coumarin 6 / siRNA mixed micelles (Gal NaC@Cou 6 / siRNA-MMs). These micelles were diluted with DMEM to a concentration of 1 μg / ml of Cou6 and added to the corresponding wells. After culturing for 4 hours, the drug-containing culture medium was discarded, and the cells were washed three times with PBS. The cells were then digested with trypsin to prepare 1×10⁶ cells / day. 5 Cells / ml of cell suspension were used to measure fluorescence intensity using flow cytometry.

[0145] 8.2. Competition Analysis

[0146] Human liver cancer cells HepG-2, Huh7 (ASGPR positive) and cervical cancer cells (ASGPR negative) were cultured at a concentration of 2.0 × 10⁻⁶. 5 Cells were seeded at a density of 1 / well in 6-well cell culture plates and cultured for 24 h. To further verify that the nanomicelles target the cells via ASGPR-mediated targeting, the original cell culture medium was aspirated 2 h before drug administration, and cell culture medium containing 1 μg / mL free GalNAC was added for incubation to saturate ASGPR. Then, they were incubated again for 4 h with drug-containing culture medium containing 1 μg / mL Cou6 GalNaC@Cou6 / siRNA-MMs. A GalNaC@Cou6 / siRNA-MMs solution not incubated with free GalNAC was set up as a positive control and an untreated negative control. After incubation, the drug-containing culture medium was discarded, and the cells were washed three times with PBS, digested with trypsin, and then prepared into 1×10⁶ cells / wells. 5 Cells / ml cell suspension was used to detect drug uptake by receptor saturation and unsaturation and its fluorescence intensity using flow cytometry.

[0147] 8.3. Qualitative Analysis by Laser Confocal Microscopy

[0148] Human liver cancer cell lines HepG2, Huh-7, and HeLa cells in logarithmic growth phase were used at a rate of 3 × 10⁻⁶. 5The cells / ml were seeded into 6-well plates and incubated in 1 mL of DMEM containing 10% fetal bovine serum. The above-mentioned cells were diluted with DMEM to prepare micelles containing 1 μg / ml of Cou6, including Cou6, Cou6-MMs, Gal NaC@Cou6-MMs, Cou6 / siRNA-MMs, and Gal NaC@Cou6 / siRNA-MMs, and added to the corresponding wells. After 6 h of incubation, the cells were washed twice with PBS, fixed with paraformaldehyde for 30 min, stained with DAPI for 10 min, and observed and photographed using a laser confocal microscope.

[0149] 9. Quantitative reverse transcription polymerase chain reaction (RT-qPCR)

[0150] Three types of liver cancer cells in logarithmic growth phase, HepG2, Huh7, and LM3, were cultured at 1.6 × 10⁻⁶ cells per well. 5 Cells were placed in 6-well plates at a density of 5 μg / ml and incubated overnight. The experimental groups were replaced with solutions containing Free LFT, LFT-MMs, Gal NaC@LFT-MMs, LFT / siRNA-MMs, and Gal NaC@LFT / siRNA-MMs at a concentration of 5 μg / ml. The blank control group was added with an equal volume of DMEM medium, and the cells were cultured for another 48 h. Cells were collected after intervention, and total RNA was extracted using an RNA extraction kit. RNA concentration and purity were measured using a nucleic acid protein analyzer. RNA was reverse transcribed into cRNA using a reverse transcription kit, followed by Real-time PCR amplification in 10 μL amplification volumes. -ΔΔct Calculate the relative expression level of COP1 mRNA.

[0151] Primer sequence: COP1, sense, 5′-GCAGCAGGAAGCGACCTCTC-3′, antisense5′-AAGCAGATGGGGCATACGAAGTC-3′, GAPDH, sense,: 5′-CACCCACTCCTCCACCTTTGAC-3′, Antisense: 5′-GTCCACCACCCTGTTGCTGTAG-3′.

[0152] 10. Western blot analysis

[0153] Cells treated with the same method as in Example 9 were added to RIPA cell lysis buffer and lysed on ice for 5 min. The supernatant was collected by centrifugation at 4°C to obtain total protein, and protein concentration was determined using a quinolinic acid (BCA) protein quantification kit. 20 μg of protein from each sample was separated by 10% SDS-PAGE gel electrophoresis 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 overnight at 4°C with mouse anti-COP1 antibody (1:1000) or rabbit anti-GAPDH antibody (1:1000). Secondary antibody (1:2000) was then incubated at room temperature for 2 hours. Finally, the proteins on the bands were observed using an enhanced chemiluminescence (ECL) system, and the relative protein expression levels were quantified and analyzed using ImageJ software.

[0154] The above tests show that polymer micelles can simultaneously target the chemotherapy drugs LFT and siRNA to liver cancer cells, effectively releasing them within the tumor cells to achieve stronger tumor suppression and lower systemic toxicity. They have multiple effects, including active targeting, chemotherapy, and gene therapy, and have broad application prospects, especially in stem cell tumor therapy.

[0155] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0156] 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 principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a liver-targeting chemotherapeutic drug nanomicelle, characterized in that, Comprising the following steps: S100. Preparation of Pluronic-formic acid succinimidyl ester (P123-NHS): S101. Pluronic P123 is vacuum dried and moisture is removed; S102. The vacuum-dried Pluronic P123 is dissolved in anhydrous toluene and anhydrous dichloromethane, and after adding triphosgene, magnetic stirring is performed until the reaction is complete, then the mixed solution is prepared by dissolving in a mixed solution of anhydrous toluene and anhydrous dichloromethane after rotary evaporation of the mixed solution to volatilize the solvent; S103. The mixed solution prepared in step S102 is treated with N-hydroxysuccinimide, S104. After diluting anhydrous triethylamine with anhydrous dichloromethane, a primary solution is prepared by adding it dropwise to the mixed solution treated with N-hydroxysuccinimide in step S103; S105. The primary solution prepared in step S104 is stirred until the reaction is complete, and then the primary solution is filtered and evaporated to dryness in a vacuum to form a residue; S106. The residue prepared in step S105 is dissolved in ethyl acetate, filtered, and after rotary evaporation of the filtrate to volatilize the ethyl acetate, P123-NHS is formed after cooling and solidification, and the P123-NHS is stored in a low-temperature dry environment for future use; S200. Preparation of Pluronic-polyethyleneimine copolymer (P123-PEI): S201. Polyethyleneimine (PEI) is dissolved in a phosphate buffer solution; S202. P123-NHS prepared in step S106 is activated and dissolved in a phosphate buffer solution; S203. The P123-NHS mixed solution in step S202 is added dropwise to the polyethyleneimine (PEI) solution in step S201, and after saturation with nitrogen, stirring is performed to form a solution; S204. The solution after stirring in step S203 is added to an activated dialysis bag to remove unreacted activated P123, and ionized water is continuously replaced during dialysis, and finally the solution is freeze-dried to prepare P123-PEI polymer, which is stored in a low-temperature dry environment; S300. Preparation of divinyl sebacate: S301. Sebacic acid, vinyl acetate, mercury acetate, and copper acetate are added to a reaction bottle, and a reaction solution is prepared by constant temperature water bath stirring; S302. Concentrated sulfuric acid is added dropwise to the reaction solution prepared in step S301, and after the reaction is complete, sodium acetate is added, and excess vinyl acetate is removed by distillation under reduced pressure to prepare a reaction secondary solution; S303. The reaction secondary solution in step S302 is separated by column separation, with silica gel as the stationary phase and petroleum ether-acetic ether as the mobile phase, and divinyl sebacate is separated; 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 bottle to prepare a mixed solution, S402. After the air bath shaker reaction of the mixed solution in step S401, the reaction solution was filtered to remove lipase (CRL), and the filtrate was vacuum rotary evaporated, then sterilized water was added, and the solution was dialyzed in a dialysis bag, and then freeze-dried to prepare Pluronic F127-SE powder; S500. Preparation of Pluronic F127-C8-Gal NAc nanomicelles: S501. Gal NAc, Pluronic F127-SE prepared in step S401, Lipozyme TL IM lipase, and acetone were added to a reaction bottle; S502. After the air bath shaker reaction of the mixed solution in step S501, the reaction solution was filtered to remove lipase (CRL), and the filtrate was vacuum rotary evaporated, then sterilized water was added, and the solution was dialyzed in a dialysis bag, and then freeze-dried to prepare Pluronic F127-C8-Gal NAc; S600. Preparation of GAl NAc@LFT-MMs / siCOP1 complex: S601. P123-PEI 2KDa, Pluronic F127-C8-Gal NAc, and LFT were placed in a reaction container and dissolved in methanol to obtain an organic phase, and then rotary evaporated until the organic solvent was completely removed, and then vacuum dried to obtain a drug film; S602. The drug film prepared in step S601 was added to sterilized water, magnetically stirred and cooled to room temperature, and then siCOP1 was added and incubated to obtain a transparent micelle solution; S603. The transparent micelle solution prepared in step S602 was filtered with a microporous filter membrane, and then freeze-dried to prepare a nanocomposite of Gal NAc@LFT-MMs / siCOP1. In step S501, the ratio of Gal NAc, Pluronic F127-SE, Lipozyme TL IM lipase, and acetone was:

2. The method for preparing liver-targeted chemotherapy drug nanomicelles according to claim 1, characterized in that: 0.4mmol:0.8mmol:100mg:50ml. In step S401, the mixing ratio of Pluronic F127, divinyl sebacate, lipase (CRL), and acetone was: 1.3mmol:11.6mmol:200mg:50ml.

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

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

1.

5. The method for preparing liver-targeted chemotherapy drug nanomicelles according to claim 1, characterized in that: In step S603, the microporous filter membrane was 0.20μm to 0.24μm microporous filter membrane.

6. The method for preparing liver-targeted chemotherapy drug nanomicelles according to claim 1, characterized in that: In step S602, after the addition of si COP1, the incubation time was 20min to 40min.

7. The method for preparing liver-targeted chemotherapy drug nanomicelles according to claim 1, characterized in that: In step 402, the dialysis time was 45h to 51h.

8. The method for preparing liver-targeted chemotherapy drug nanomicelles according to claim 1, characterized in that: The liver-targeting chemotherapy drug nanomicelles as claimed in any one of claims 1 to 8, further comprising N-acetylgalactosamine, si RNA, and lenvatinib.

9. A liver-targeted chemotherapeutic drug, characterized by:

10. Use of the liver-targeting chemotherapy drug of claim 9 in the preparation of a product for synergistic treatment of liver cancer. ​

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