Preparation method of gene-loaded magnetic nano-micelle

Abstract

The invention discloses a preparation method of gene-loaded magnetic nano-micelles, and belongs to the field of biological material preparation. The gene-loaded magnetic nano-micelle is prepared by adopting a re-emulsification-solvent evaporation technology, taking a triblock polymer mPEG-PCL-PEI as a micelle material, and taking Fe3O4 / Dox / siRNA as a model drug. The particle size distribution range of the gene-loaded magnetic nano-micelle is 100-300nm, and the gene-loaded magnetic nano-micelle has good drug controlled release performance. Cell experiments show that the gene-loaded magnetic nano-micelle can effectively enter tumor cells and kill the tumor cells, and has a good application prospect and a high application value.

Description

Technical Field

[0001] The invention belongs to the field of biomaterial preparation, and in particular relates to a method for preparing gene-carrying magnetic nano micelles. Background Art

[0002] In the treatment of tumors, although drug therapy has made great progress, inherent acquired drug resistance still needs to be overcome, otherwise it is difficult to achieve successful treatment and will eventually lead to patient death. In addition, drug resistance usually occurs during chemotherapy or in chemotherapy for post-recurrence. Tumor cells produce antibodies to drugs of different structures and types, resulting in a high recurrence rate, making tumors more difficult to cure. Many factors are involved in the occurrence of inherent or acquired multidrug resistance, which hinders the delivery of drugs to targeted tumor tissues and reduces their killing effects on tumor cells, making tumor cells insensitive to drugs. However, these drug resistances are mainly closely related to the overexpression or non-expression of some genes in cells. Therefore, in order to obtain better tumor treatment, it is a very necessary strategy to develop gene therapy for different types of tumor cells. However, the most critical thing about gene therapy is to seek suitable therapeutic genes for specific targets, because the protein expression in tumor cells of the same type formed by different types or different individuals is not necessarily the same.

[0003] siRNA is a short double-stranded RNA molecule, usually composed of 21 to 23 base pairs, which can silence genes related to multidrug resistance mechanisms, and thus has attracted more and more attention. In addition, siRNA can be synthesized in vitro and directly transferred into target cells, or produced by genetically modified target cells. After entering the target tumor cells, siRNA can bind to ribozyme compounds to form RNA-mediated silencing complexes (RISCs), which then complementarily bind to the targeted mRNA, mediate the degradation of mRNA, and achieve the purpose of silencing specific genes to treat tumors. Amino-modified ferroferric oxide can electrostatically adsorb genes, while simplifying the separation process, which is conducive to protecting the gene structure from being destroyed.

[0004] Liver cancer is the most common malignant tumor in clinic. The main problem facing the current treatment of liver cancer is that the traditional chemotherapy method for liver cancer is often prone to recurrence, mainly because new tumors grow in the liver or tumor metastasis, which causes great harm to the patient's body and mind at the same time. In addition, there are characteristics such as unclear treatment targets, weak selectivity, and easy to produce drug resistance, which leads to low efficiency. While killing tumor cells, it will damage normal tissue cells. At present, targeted therapy has gradually developed into a cutting-edge technology for the treatment of liver cancer. Targeted therapy for liver cancer can be divided into drug therapy and gene therapy. Drug therapy can be delivered through a nano drug delivery system, that is, the therapeutic drug is loaded on a nano carrier by physical or chemical methods to form a carrier-drug complex. This method can significantly increase the drug concentration at the target site, improve drug utilization and treatment effect, and reduce adverse drug reactions. Gene therapy is to transfer functional genes to specific tissues or cells to exert their functions. It can be combined with the unique genetic characteristics of the patient's own cancer cells for diagnosis and formulate an efficient and accurate treatment plan. Cationic polymers such as polyethyleneimine are positively charged due to their special structure with several amine groups. The electrostatic interaction between transgenic and cationic polymers is essential for the formation of polyelectrolyte complexes. On the other hand, the disadvantages of cationic polymers, such as low bioavailability, high cytotoxicity and poor transfection efficiency, limit their scope of use. In addition, the non-degradable nature of cationic polymers further limits their application. Therefore, the development of biodegradable gene delivery polymers is the first choice. Polycaprolactone is a biodegradable and bioabsorbable polyester, which has been widely used in drug and gene delivery systems with polyethylene glycol. The combination of polyethyleneimine, polycaprolactone and polyethylene glycol reduces the toxicity of polyethyleneimine and enhances the gene delivery efficiency. In addition, magnetic nanoparticles can be used as gene carriers to maintain the carrier at the target site by an external magnetic field, thereby improving the effect of gene transfection. Since folic acid plays a key role in DNA synthesis, the siRNA cleavage rate is high in cancer cells, and folate receptors are overexpressed on the surface of cancer cells. Therefore, folic acid is a suitable ligand for micelle surface functionalization to deliver drugs to cancer cells. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing gene-carrying magnetic nano-micelles. The present invention adopts the double emulsification-solvent evaporation technology, uses the triblock polymer mPEG-PCL-PEI as the micelle material, and uses Fe3O4 / Dox / siRNA as the model drug to prepare gene-carrying magnetic nano-micelles. The particle size distribution range of the gene-carrying magnetic nano-micelles is 100 to 300nm, and it has good drug controlled release performance. Cell experiments show that the gene-carrying magnetic nano-micelles can effectively enter tumor cells and kill them, and have good application prospects and high application value.

[0006] To achieve the above object, the present invention adopts the following technical solution: The first aspect of the present invention provides a method for preparing gene-carrying magnetic nanomicelles, comprising the following steps: S1: Preparation of mPEG-PCL-PEI: S1-1: Mix polyethylene glycol monomethyl ether, ε-caprolactone and Sn(Oct)2, react in an oil bath at 130°C for 24 hours, cool naturally to room temperature, add dichloromethane to dissolve, then precipitate in ice anhydrous ether, filter, and dry the residue in vacuum at room temperature to obtain mPEG-PCL; S1-2: mPEG-PCL was added to dichloromethane, triethylamine and acryloyl chloride were added under N2 protection, and the mixture was reacted at room temperature for 6 h, then extracted with saturated NaCl solution for 3 times, the organic phase was collected, precipitated in ice anhydrous ether, filtered, and the residue was dried under vacuum at room temperature to obtain mPEG-PCL-COCHCH2; S1-3: mPEG-PCL-COCHCH2 and polyethyleneimine were dissolved in DMF, pyridine was added, and the mixture was reacted at 60°C for 12 h under N2 protection. After the reaction, the mixture was dialyzed with DMF at room temperature for 24 h, and then with deionized water at room temperature for 72 h, and freeze-dried to obtain mPEG-PCL-PEI. S2: Preparation of Fe3O4 / mPEG-PCL-PEI / Dox: S2-1: FeCl3·6H2O and FeCl2·4H2O were added to deionized water, and mechanically stirred and dissolved at room temperature for 30 min under N2 protection. A 10 mol / L NaOH solution was added, and mechanically stirred and reacted at room temperature for 1 h under N2 protection, and then the temperature was raised to 90°C and kept for 2 h. The mixture was naturally cooled to room temperature, and magnetic separation was performed using a magnet. The obtained solid product was washed until the pH value of the supernatant was neutral, and vacuum dried at 40°C to obtain Fe3O4 nanoparticles. S2-2: Add Dox·HCl to dichloromethane, add triethylamine, and stir at room temperature in the dark overnight to obtain Dox·HCl solution; S2-3: mPEG-PCL-PEI was added to Dox·HCl solution, and dichloromethane and acetone were added, and ultrasonicated at room temperature for 2 min to obtain mixed solution A; S2-4: Disperse Fe3O4 nanoparticles in deionized water, transfer to mixed solution A, and perform ultrasonic treatment at room temperature for 3 min to obtain mixed solution B; S2-5: Mixed solution B was added to 2 wt% PVA solution under ultrasonic conditions, and ultrasonicated at room temperature for 3 min to obtain mixed solution C; S2-6: Mixed solution C was dialyzed with deionized water at room temperature for 24 h, and then the precipitate was adsorbed by an external magnet, and the precipitate was washed to obtain Fe3O4 / mPEG-PCL-PEI / Dox; S3: Preparation of Fe3O4 / FA-mPEG-PCL-PEI / Dox: S3-1: Dissolve folic acid in DMSO, add N-hydroxysuccinimide and EDC hydrochloride, and stir at room temperature for 4 h to obtain FA solution; S3-2: Fe3O4 / mPEG-PCL-PEI / Dox was dispersed in deionized water, FA solution was added dropwise under mechanical stirring, the reaction was stirred at room temperature overnight, centrifuged, and the precipitate was collected to obtain Fe3O4 / FA-mPEG-PCL-PEI / Dox; S4: Preparation of Fe3O4 / FA-mPEG-PCL-PEI / Dox / siRNA: Fe3O4 / FA-mPEG-PCL-PEI / Dox was dispersed in deionized water, and a 20 μg / mL siRNA solution was added dropwise under mechanical stirring conditions. The mixture was stirred at room temperature for 6 h, and magnetic separation was performed using a magnet. The obtained solid product was washed to obtain Fe3O4 / FA-mPEG-PCL-PEI / Dox / siRNA, which is a gene-carrying magnetic nanomicelle. Further, in step S1-1, the amount of polyethylene glycol monomethyl ether is 2 to 10 g, the amount of ε-caprolactone is 1 to 8 mL, the amount of Sn(Oct)2 is 0.01 to 0.1 g, and the amount of dichloromethane is 10 to 25 mL; in step S1-2, the amount of mPEG-PCL is 2 to 10 g, the amount of dichloromethane is 10 to 100 mL, the amount of triethylamine is 1 to 10 mL, and the amount of acryloyl chloride is 0.1 to 1 mL; in step S1-3, the amount of mPEG-PCL-COCHCH2 is 2 to 10 g, the amount of polyethyleneimine is 1 to 5 g, the amount of DMF is 10 to 100 mL, and the amount of pyridine is 2 to 15 mL; Further, in step S2-1, the amount of FeCl3·6H2O is 5 to 20 g, the amount of FeCl2·4H2O is 1 to 8 g, the amount of deionized water is 50 to 200 mL, and the amount of NaOH solution is 20 to 100 mL; in step S2-2, the amount of Dox·HCl is 10 to 100 mg, the amount of dichloromethane is 10 to 150 mL, and the amount of triethylamine is 0.2 to 1.5 mL; in step S2-3, the amount of mPEG-PCL-PEI is 10 to 1 00mg, the amount of Dox·HCl solution is 0.2-2mL, the amount of dichloromethane solution is 1-8mL, and the amount of acetone is 1-2mL; in step S2-4, the amount of Fe3O4 nanoparticles is 10-100mg, the amount of deionized water is 1-5mL, and the amount of mixed solution A is 2-10mL; in step S2-5, the amount of mixed solution B is 6-15mL, and the amount of PVA solution is 10-100mL; in step S2-6, the pH value of 1×PBS buffer is 7.4; Further, in step S3-1, the amount of folic acid is 10-100 mg, the amount of DMSO is 5-50 mL, the amount of N-hydroxysuccinimide is 5-20 mg, and the amount of EDC hydrochloride is 20-150 mg; in step S3-2, the amount of Fe3O4 / mPEG-PCL-PEI / Dox is 10-20 mg, and the amount of deionized water is 1-10 mL; the amount of FA solution is 0.2-2 mL; further, in step S4, the amount of Fe3O4 / FA-mPEG-PCL-PEI / Dox is 10-20 mg, the amount of deionized water is 10-21 mL, and the amount of siRNA solution is 3-6.3 mL.

[0007] The second aspect of the present invention provides a gene-carrying magnetic nanomicelle, which is prepared by the above-mentioned preparation method.

[0008] The third aspect of the present invention provides the above-mentioned preparation method and application of the above-mentioned gene-carrying magnetic nano-micelles in the preparation of drugs for treating tumors.

[0009] The significant advantages of the present invention are: 1) The present invention uses the triblock polymer mPEG-PCL-PEI as the micelle material, taking advantage of its amphiphilicity, in vivo degradability and good biocompatibility, and uses polyethyleneimine to electrostatically adsorb genes, which has a higher nucleic acid loading capacity due to its more amino groups; 2) The present invention adopts the double emulsion-solvent evaporation method to load the hydrophobic Dox and the hydrophilic Fe3O4 / siRNA nanocomplex, and prepares a gene-carrying magnetic nanomicelle without using an emulsifier, which can achieve precise targeted treatment of liver cancer; 3) The present invention uses the triblock polymer mPEG-PCL-PEI as the micelle matrix to self-assemble and encapsulate gene drugs, and utilizes the versatility, biocompatibility and biodegradability of the amphiphilic copolymer micelle preparation technology to prepare gene-loaded magnetic nanomicelles, which are expected to be used for the treatment of liver cancer through in situ injection. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 : Particle size and PDI of Fe3O4 / FA-mPEG-PCL-PEI / Dox / siRNA of Example 1, mPEG-PCL-PEI of Comparative Example 1, mPEG-PCL-PEI of Comparative Example 2, Fe3O4 / mPEG-PCL-PEI / Dox of Comparative Example 3, and Fe3O4 / FA-mPEG-PCL-PEI / Dox of Comparative Example 4.

[0011] Figure 2 : Zeta potential of Fe3O4 / FA-mPEG-PCL-PEI / Dox / siRNA of Example 1, mPEG-PCL-PEI of Comparative Example 1, mPEG-PCL-PEI of Comparative Example 2, Fe3O4 / mPEG-PCL-PEI / Dox of Comparative Example 3, and Fe3O4 / FA-mPEG-PCL-PEI / Dox of Comparative Example 4.

[0012] Figure 3 : Doxorubicin release curves of Fe3O4 / FA-mPEG-PCL-PEI / Dox / siRNA of Example 1, mPEG-PCL-PEI / Dox / siRNA of Comparative Example 5, and Fe3O4 / mPEG-PCL-PEI / Dox / siRNA of Comparative Example 6.

[0013] Figure 4 : The MTT method was used to characterize the killing effects of Fe3O4 / FA-mPEG-PCL-PEI / Dox / siRNA in Example 1, mPEG-PCL-PEI in Comparative Example 1, Fe3O4 / mPEG-PCL-PEI / Dox in Comparative Example 3, Fe3O4 / mPEG-PCL-PEI / Dox / siRNA in Comparative Example 6, Fe3O4 / mPEG-PCL-PEI / siRNA in Comparative Example 7, free doxorubicin (free Dox), and free siRNA (free siRNA) on liver cancer cells.

[0014] Figure 5 : The dead-live staining method was used to characterize the killing effect of Fe3O4 / FA-mPEG-PCL-PEI / Dox / siRNA in Example 1 on liver cancer cells. DETAILED DESCRIPTION

[0015] To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. In addition, in order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. It should be understood by those skilled in the art that the present invention can be implemented without certain specific details. In certain embodiments, raw materials, components, methods, means, etc. known to those skilled in the art are not described in detail to highlight the main idea of ​​the present invention.

[0016] The polyethylene glycol monomethyl ether (mPEG) in the embodiment of the present invention is purchased from Shanghai McLean Biochemical Technology Co., Ltd., with a CAS number of 9004-74-4 and an average molecular weight of 2000. The ε-caprolactone (ε-CL) in the embodiment of the present invention is purchased from Shanghai McLean Biochemical Technology Co., Ltd., with a CAS number of 502-44-3. The polyethyleneimine (PEI) in the embodiment of the present invention is purchased from Shanghai McLean Biochemical Technology Co., Ltd., with a CAS number of 25987-06-8 and an average molecular weight of 800. The polyvinyl alcohol (PVA) in the embodiment of the present invention is purchased from Shanghai McLean Biochemical Technology Co., Ltd., with a CAS number of 9002-89-5 and an average molecular weight of 75000. The folic acid (FA) in the embodiment of the present invention is purchased from Shanghai McLean Biochemical Technology Co., Ltd., with a CAS number of 59-30-3. The siRNA in the present invention is a siRNA that targets and inhibits the expression of the bcl-2 gene, and its sense strand nucleotide sequence is: 5'-GUGGAUGACUGAGUACCUGTT-3', and its antisense strand nucleotide sequence is: 5'-CAGGUACUCAGUCAUCCACTT-3'. The pH value of the 1×PBS buffer in the present invention is 7.4.

[0017] Embodiment 1: A method for preparing gene-carrying magnetic nanomicelles is carried out according to the following steps: S1: Preparation of mPEG-PCL-PEI: S1-1: 2 g mPEG, 1 mL ε-CL and 0.01 g Sn(Oct)2 were mixed, reacted in an oil bath at 130 °C for 24 h, cooled naturally to room temperature, 10 mL dichloromethane was added to dissolve, and then precipitated in ice anhydrous ether for 30 min, filtered, and the residue was vacuum dried at room temperature for 24 h to obtain mPEG-PCL; S1-2: 2 g of mPEG-PCL was added to 10 mL of dichloromethane, and 1 mL of triethylamine and 0.1 mL of acryloyl chloride were added under N2 protection, and the mixture was reacted at room temperature for 6 h. The mixture was then extracted with saturated NaCl solution for 3 times, 10 ml each time. The organic phase was collected and precipitated in ice anhydrous ether for 30 min, and filtered. The residue was dried under vacuum at room temperature for 24 h to obtain mPEG-PCL-COCHCH2. S1-3: Dissolve 2 g mPEG-PCL-COCHCH2 and 1 g PEI in 10 mL N,N-dimethylformamide (DMF), add 2 mL pyridine, and react at 60 °C for 12 h under N2 protection. After the reaction, transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 2000 Da, dialyze with DMF at room temperature for 24 h, and then dialyze with deionized water at room temperature for 72 h. After dialyzation, freeze-dry the liquid in the dialysis bag to obtain mPEG-PCL-PEI.

[0018] S2: Preparation of Fe3O4 / mPEG-PCL-PEI / Dox: S2-1: 5g FeCl3·6H2O and 1g FeCl2·4H2O were added to 50mL deionized water, and mechanically stirred and dissolved at room temperature for 30min under N2 protection. 20mL 10mol / L NaOH solution was added, and mechanically stirred and reacted at room temperature for 1h under N2 protection, and then the temperature was raised to 90℃ and kept for 2h. The mixture was naturally cooled to room temperature and magnetically separated by a magnet. The solid product was alternately washed with deionized water and 2mol / L HNO3 solution until the pH of the supernatant was neutral, and vacuum dried at 40℃ for 24h to obtain Fe3O4 nanoparticles. S2-2: Add 10 mg of doxorubicin hydrochloride (Dox·HCl) to 10 mL of dichloromethane, add 0.2 mL of triethylamine, and stir overnight at room temperature in the dark to obtain a Dox·HCl solution; S2-3: 10 mg of mPEG-PCL-PEI was added to 0.2 mL of Dox·HCl solution, 1 mL of dichloromethane and 1 mL of acetone were added, and the mixture was sonicated at room temperature for 2 min using a cell disruptor at a power of 36 W to obtain a mixed solution A; S2-4: 10 mg of Fe3O4 nanoparticles were dispersed in 1 mL of deionized water, transferred to 2 mL of mixed solution A, and sonicated at room temperature for 3 min using a cell disruptor at a power of 54 W to obtain mixed solution B; S2-5: 3 mL of mixed solution B was added to 10 mL of 2 wt% PVA solution under ultrasonic conditions, and ultrasonicated at room temperature for 3 min at a power of 54 W using a cell disruptor to obtain mixed solution C; S2-6: The mixed solution C was transferred to a dialysis bag with a molecular weight cutoff of 2000Da, dialyzed with deionized water at room temperature for 24h, and then the precipitate was adsorbed using an external magnet. The precipitate was repeatedly washed with 1×PBS buffer to obtain Fe3O4 / mPEG-PCL-PEI / Dox.

[0019] S3: Preparation of Fe3O4 / FA-mPEG-PCL-PEI / Dox: S3-1: Dissolve 10 mg FA in 5 mL dimethyl sulfoxide (DMSO), add 5 mg N-hydroxysuccinimide (NHS) and 20 mg EDC hydrochloride (EDC·HCl), and stir at room temperature for 4 h to obtain a FA solution; S3-2: Disperse 10 mg of Fe3O4 / mPEG-PCL-PEI / Dox in 1 mL of deionized water, add 0.2 mL of FA solution dropwise under mechanical stirring, stir at room temperature for overnight reaction, then centrifuge at 8000 r / min for 5 min, collect the precipitate, and obtain Fe3O4 / FA-mPEG-PCL-PEI / Dox.

[0020] S4: Preparation of Fe3O4 / FA-mPEG-PCL-PEI / Dox / siRNA: 10 mg Fe3O4 / FA-mPEG-PCL-PEI / Dox was dispersed in 10 mL deionized water, and 3 mL of 20 μg / mL siRNA solution was added dropwise under mechanical stirring. The reaction was stirred at room temperature for 6 h, and magnetic separation was performed using a magnet. The solid product was repeatedly washed with deionized water to obtain Fe3O4 / FA-mPEG-PCL-PEI / Dox / siRNA, which is the gene-carrying magnetic nanomicelles.

[0021] Embodiment 2: A method for preparing gene-carrying magnetic nanomicelles is carried out according to the following steps: S1: Preparation of mPEG-PCL-PEI: S1-1: 10 g mPEG, 8 mL ε-CL and 0.1 g Sn(Oct)2 were mixed, reacted in an oil bath at 130 °C for 24 h, cooled naturally to room temperature, 25 mL dichloromethane was added to dissolve, and then precipitated in ice anhydrous ether for 30 min, filtered, and the residue was vacuum dried at room temperature for 24 h to obtain mPEG-PCL; S1-2: 10 g of mPEG-PCL was added to 100 mL of dichloromethane, and 10 mL of triethylamine and 1 mL of acryloyl chloride were added under N2 protection, and the mixture was reacted at room temperature for 6 h. The mixture was then extracted with saturated NaCl solution for 3 times, 100 ml each time. The organic phase was collected and precipitated in ice anhydrous ether for 30 min, and filtered. The residue was dried under vacuum at room temperature for 24 h to obtain mPEG-PCL-COCHCH2. S1-3: Dissolve 10 g mPEG-PCL-COCHCH2 and 5 g PEI in 100 mL DMF, add 15 mL pyridine, and react at 60 °C for 12 h under N2 protection. After the reaction, transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 2000 Da, dialyze with DMF at room temperature for 24 h, and then dialyze with deionized water at room temperature for 72 h. Freeze-dry the liquid in the dialysis bag after dialyzation to obtain mPEG-PCL-PEI.

[0022] S2: Preparation of Fe3O4 / mPEG-PCL-PEI / Dox: S2-1: 20 g FeCl3·6H2O and 8 g FeCl2·4H2O were added to 200 mL deionized water, and mechanically stirred and dissolved at room temperature for 30 min under N2 protection. 100 mL 10 mol / L NaOH solution was added, and mechanically stirred and reacted at room temperature for 1 h under N2 protection, and then the temperature was raised to 90°C and kept for 2 h. The mixture was naturally cooled to room temperature and magnetically separated by a magnet. The solid product was alternately washed with deionized water and 2 mol / L HNO3 solution until the pH of the supernatant was neutral, and vacuum dried at 40°C for 24 h to obtain Fe3O4 nanoparticles. S2-2: Add 100 mg of Dox·HCl to 150 mL of dichloromethane, add 1.5 mL of triethylamine, and stir overnight at room temperature in the dark to obtain a Dox·HCl solution; S2-3: 100 mg of mPEG-PCL-PEI was added to 2 mL of Dox·HCl solution, 8 mL of dichloromethane and 2 mL of acetone were added, and the mixture was sonicated at room temperature for 2 min using a cell disruptor at a power of 36 W to obtain a mixed solution A; S2-4: 100 mg of Fe3O4 nanoparticles were dispersed in 5 mL of deionized water, transferred to 10 mL of mixed solution A, and sonicated at room temperature for 3 min using a cell disruptor at a power of 54 W to obtain mixed solution B; S2-5: 15 mL of mixed solution B was added to 100 mL of 2 wt% PVA solution under ultrasonic conditions, and the mixture was ultrasonicated at room temperature for 3 min at a power of 54 W using a cell disruptor to obtain mixed solution C; S2-6: The mixed solution C was transferred to a dialysis bag with a molecular weight cutoff of 2000Da, dialyzed with deionized water at room temperature for 24h, and then the precipitate was adsorbed using an external magnet. The precipitate was repeatedly washed with 1×PBS buffer to obtain Fe3O4 / mPEG-PCL-PEI / Dox.

[0023] S3: Preparation of Fe3O4 / FA-mPEG-PCL-PEI / Dox: S3-1: Dissolve 100 mg FA in 50 mL DMSO, add 20 mg NHS and 150 mg EDC·HCl, and stir at room temperature for 4 h to obtain FA solution; S3-2: Disperse 20 mg of Fe3O4 / mPEG-PCL-PEI / Dox in 10 mL of deionized water, add 2 mL of FA solution dropwise under mechanical stirring, stir and react at room temperature overnight, then centrifuge at 8000 r / min for 5 min, collect the precipitate, and obtain Fe3O4 / FA-mPEG-PCL-PEI / Dox.

[0024] S4: Preparation of Fe3O4 / FA-mPEG-PCL-PEI / Dox / siRNA: 20 mg Fe3O4 / FA-mPEG-PCL-PEI / Dox was dispersed in 20 mL deionized water, and 6 mL of 20 μg / mL siRNA solution was added dropwise under mechanical stirring. The reaction was stirred at room temperature for 6 h, and magnetic separation was performed using a magnet. The solid product was repeatedly washed with deionized water to obtain Fe3O4 / FA-mPEG-PCL-PEI / Dox / siRNA, which is the gene-carrying magnetic nanomicelles.

[0025] Embodiment 3: A method for preparing gene-carrying magnetic nanomicelles is carried out according to the following steps: S1: Preparation of mPEG-PCL-PEI: S1-1: 5 g mPEG, 4 mL ε-CL and 0.04 g Sn(Oct)2 were mixed, reacted in an oil bath at 130 °C for 24 h, cooled naturally to room temperature, and 20 mL dichloromethane was added to dissolve, and then precipitated in ice anhydrous ether for 30 min, filtered, and the residue was vacuum dried at room temperature for 24 h to obtain mPEG-PCL; S1-2: 5 g of mPEG-PCL was added to 50 mL of dichloromethane, and 5 mL of triethylamine and 0.4 mL of acryloyl chloride were added under N2 protection, and the mixture was reacted at room temperature for 6 h. The mixture was then extracted with saturated NaCl solution for 3 times, 40 ml each time. The organic phase was collected and precipitated in ice anhydrous ether for 30 min, and filtered. The residue was dried under vacuum at room temperature for 24 h to obtain mPEG-PCL-COCHCH2. S1-3: Dissolve 5 g mPEG-PCL-COCHCH2 and 2 g PEI in 40 mL DMF, add 8 mL pyridine, and react at 60 °C for 12 h under N2 protection. After the reaction, transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 2000 Da, dialyze with DMF at room temperature for 24 h, and then dialyze with deionized water at room temperature for 72 h. Freeze-dry the liquid in the dialysis bag after dialyzation to obtain mPEG-PCL-PEI.

[0026] S2: Preparation of Fe3O4 / mPEG-PCL-PEI / Dox: S2-1: 10g FeCl3·6H2O and 4g FeCl2·4H2O were added to 100mL deionized water, and mechanically stirred and dissolved at room temperature for 30min under N2 protection. 50mL 10mol / L NaOH solution was added, and mechanically stirred and reacted at room temperature for 1h under N2 protection, and then the temperature was raised to 90℃ and kept for 2h. The mixture was naturally cooled to room temperature, and magnetic separation was performed using a magnet. The solid product was alternately washed with deionized water and 2mol / L HNO3 solution until the pH of the supernatant was neutral, and vacuum dried at 40℃ for 24h to obtain Fe3O4 nanoparticles. S2-2: Add 40 mg of Dox·HCl to 60 mL of dichloromethane, add 0.8 mL of triethylamine, and stir at room temperature in the dark overnight to obtain a Dox·HCl solution; S2-3: 40 mg of mPEG-PCL-PEI was added to 0.8 mL of Dox·HCl solution, 3 mL of dichloromethane and 1.4 mL of acetone were added, and the mixture was sonicated at room temperature for 2 min using a cell disruptor at a power of 36 W to obtain a mixed solution A; S2-4: 40 mg of Fe3O4 nanoparticles were dispersed in 2 mL of deionized water, transferred to 5 mL of mixed solution A, and sonicated at room temperature for 3 min using a cell disruptor at a power of 54 W to obtain mixed solution B; S2-5: 6 mL of mixed solution B was added to 40 mL of 2 wt% PVA solution under ultrasonic conditions, and ultrasonicated at room temperature for 3 min at a power of 54 W using a cell disruptor to obtain mixed solution C; S2-6: The mixed solution C was transferred to a dialysis bag with a molecular weight cutoff of 2000Da, dialyzed with deionized water at room temperature for 24h, and then the precipitate was adsorbed using an external magnet. The precipitate was repeatedly washed with 1×PBS buffer to obtain Fe3O4 / mPEG-PCL-PEI / Dox.

[0027] S3: Preparation of Fe3O4 / FA-mPEG-PCL-PEI / Dox: S3-1: Dissolve 40 mg FA in 20 mL DMSO, add 10 mg NHS and 60 mg EDC·HCl, and stir at room temperature for 4 h to obtain FA solution; S3-2: Disperse 14 mg of Fe3O4 / mPEG-PCL-PEI / Dox in 4 mL of deionized water, add 0.8 mL of FA solution dropwise under mechanical stirring, stir and react at room temperature overnight, then centrifuge at 8000 r / min for 5 min, collect the precipitate, and obtain Fe3O4 / FA-mPEG-PCL-PEI / Dox.

[0028] S4: Preparation of Fe3O4 / FA-mPEG-PCL-PEI / Dox / siRNA: 14 mg Fe3O4 / FA-mPEG-PCL-PEI / Dox was dispersed in 14 mL deionized water, and 4.2 mL 20 μg / mL siRNA solution was added dropwise under mechanical stirring conditions. The reaction was stirred at room temperature for 6 h, and magnetic separation was performed using a magnet. The solid product was repeatedly washed with deionized water to obtain Fe3O4 / FA-mPEG-PCL-PEI / Dox / siRNA, which is the gene-carrying magnetic nanomicelles.

[0029] Embodiment 4: A method for preparing gene-carrying magnetic nanomicelles is carried out according to the following steps: S1: Preparation of mPEG-PCL-PEI: S1-1: 7.5 g mPEG, 6 mL ε-CL and 0.06 g Sn(Oct)2 were mixed, reacted in an oil bath at 130 °C for 24 h, cooled naturally to room temperature, 22.5 mL dichloromethane was added to dissolve, and then precipitated in ice anhydrous ether for 30 min, filtered, and the residue was vacuum dried at room temperature for 24 h to obtain mPEG-PCL; S1-2: 7.5 g mPEG-PCL was added to 50 mL dichloromethane, and 7.5 mL triethylamine and 0.6 mL acryloyl chloride were added under N2 protection, and the mixture was reacted at room temperature for 6 h. Then, the mixture was extracted with saturated NaCl solution for 3 times, 40 ml each time, and the organic phase was collected. The mixture was precipitated in ice anhydrous ether for 30 min, filtered, and the residue was dried under vacuum at room temperature for 24 h to obtain mPEG-PCL-COCHCH2; S1-3: 7.5 g mPEG-PCL-COCHCH2 and 3 g PEI were dissolved in 60 mL DMF, 12 mL pyridine was added, and the mixture was reacted at 60 °C under N2 protection for 12 h. After the reaction, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 2000 Da, and dialyzed with DMF at room temperature for 24 h, and then dialyzed with deionized water at room temperature for 72 h. The liquid in the dialysis bag after dialysis was freeze-dried to obtain mPEG-PCL-PEI.

[0030] S2: Preparation of Fe3O4 / mPEG-PCL-PEI / Dox: S2-1: 15g FeCl3·6H2O and 6g FeCl2·4H2O were added to 100mL deionized water, and mechanically stirred and dissolved at room temperature for 30min under N2 protection. 75mL 10mol / L NaOH solution was added, and mechanically stirred and reacted at room temperature for 1h under N2 protection, and then the temperature was raised to 90℃ and kept for 2h. The mixture was naturally cooled to room temperature, and magnetic separation was performed using a magnet. The solid product was alternately washed with deionized water and 2mol / L HNO3 solution until the pH value of the supernatant was neutral, and vacuum dried at 40℃ for 24h to obtain Fe3O4 nanoparticles. S2-2: Add 60 mg of Dox·HCl to 90 mL of dichloromethane, add 1.2 mL of triethylamine, and stir at room temperature in the dark overnight to obtain a Dox·HCl solution; S2-3: 60 mg of mPEG-PCL-PEI was added to 1.2 mL of Dox·HCl solution, 4.5 mL of dichloromethane and 2.1 mL of acetone were added, and the mixture was sonicated at room temperature for 2 min using a cell disruptor at a power of 36 W to obtain a mixed solution A; S2-4: 60 mg of Fe3O4 nanoparticles were dispersed in 3 mL of deionized water, transferred to 7.5 mL of mixed solution A, and sonicated at room temperature for 3 min using a cell disruptor at a power of 54 W to obtain mixed solution B; S2-5: 10 mL of mixed solution B was added to 60 mL of 2 wt% PVA solution under ultrasonic conditions, and ultrasonicated at room temperature for 3 min at a power of 54 W using a cell disruptor to obtain mixed solution C; S2-6: The mixed solution C was transferred to a dialysis bag with a molecular weight cutoff of 2000Da, dialyzed with deionized water at room temperature for 24h, and then the precipitate was adsorbed using an external magnet. The precipitate was repeatedly washed with 1×PBS buffer to obtain Fe3O4 / mPEG-PCL-PEI / Dox.

[0031] S3: Preparation of Fe3O4 / FA-mPEG-PCL-PEI / Dox: S3-1: Dissolve 60 mg FA in 20 mL DMSO, add 15 mg NHS and 90 mg EDC·HCl, and stir at room temperature for 4 h to obtain FA solution; S3-2: Disperse 21 mg of Fe3O4 / mPEG-PCL-PEI / Dox in 6 mL of deionized water, add 1.2 mL of FA solution dropwise under mechanical stirring, stir at room temperature for overnight reaction, then centrifuge at 8000 r / min for 5 min, collect the precipitate, and obtain Fe3O4 / FA-mPEG-PCL-PEI / Dox.

[0032] S4: Preparation of Fe3O4 / FA-mPEG-PCL-PEI / Dox / siRNA: 21 mg Fe3O4 / FA-mPEG-PCL-PEI / Dox was dispersed in 21 mL deionized water, and 6.3 mL 20 μg / mL siRNA solution was added dropwise under mechanical stirring conditions. The reaction was stirred at room temperature for 6 h, and magnetic separation was performed using a magnet. The solid product was repeatedly washed with deionized water to obtain Fe3O4 / FA-mPEG-PCL-PEI / Dox / siRNA, which is the gene-carrying magnetic nanomicelles.

[0033] Comparative Example 1: A method for preparing mPEG-PCL-PEI, wherein the steps are the same as those of the method for preparing mPEG-PCL-PEI in Example 1.

[0034] Comparative Example 2: A method for preparing mPEG-PCL-PEI / Dox is carried out according to the following steps: S1: Preparation of mPEG-PCL-PEI: The preparation method is the same as that of mPEG-PCL-PEI in Example 1.

[0035] S2: Preparation of mPEG-PCL-PEI / Dox: S2-1: Add 10 mg of Dox·HCl to 10 mL of dichloromethane, add 0.2 mL of triethylamine, and stir at room temperature in the dark overnight to obtain a Dox·HCl solution; S2-2: 10 mg of mPEG-PCL-PEI was added to 0.2 mL of Dox·HCl solution, 1 mL of dichloromethane and 1 mL of acetone were added, and the mixture was sonicated at room temperature for 2 min using a cell disruptor at a power of 36 W to obtain a mixed solution A; S2-3: Add 1 mL of deionized water to 2 mL of mixed solution A, and use a cell disruptor to sonicate at room temperature for 3 min at a power of 54 W to obtain mixed solution B; S2-4: 3 mL of mixed solution B was added to 10 mL of 2 wt% PVA solution under ultrasonic conditions, and ultrasonicated at room temperature for 3 min at a power of 54 W using a cell disruptor to obtain mixed solution C; S2-5: The mixed solution C was transferred to a dialysis bag with a molecular weight cutoff of 2000Da, dialyzed with deionized water at room temperature for 24h, and then the precipitate was adsorbed using an external magnet. The precipitate was repeatedly washed with 1×PBS buffer to obtain mPEG-PCL-PEI / Dox.

[0036] Comparative Example 3: A method for preparing Fe3O4 / mPEG-PCL-PEI / Dox, wherein the steps are the same as those of the method for preparing Fe3O4 / mPEG-PCL-PEI / Dox in Example 1.

[0037] Comparative Example 4: A method for preparing Fe3O4 / FA-mPEG-PCL-PEI / Dox, wherein the steps are the same as those of the method for preparing Fe3O4 / FA-mPEG-PCL-PEI / Dox in Example 1.

[0038] Comparative Example 5: A method for preparing mPEG-PCL-PEI / Dox / siRNA is carried out according to the following steps: S1: Preparation of mPEG-PCL-PEI: The preparation method is the same as that of mPEG-PCL-PEI in Example 1.

[0039] S2: Preparation of mPEG-PCL-PEI / Dox: The preparation method is the same as that of mPEG-PCL-PEI / Dox in Comparative Example 2.

[0040] S3: Preparation of mPEG-PCL-PEI / Dox / siRNA: 10 mg of mPEG-PCL-PEI / Dox was dispersed in 10 mL of deionized water, and 3 mL of 20 μg / mL siRNA solution was added dropwise under mechanical stirring. The reaction was stirred at room temperature for 6 h, and centrifuged at 8000 r / min for 5 min. The precipitate was collected and washed repeatedly with deionized water to obtain mPEG-PCL-PEI / Dox / siRNA.

[0041] Comparative Example 6: A method for preparing Fe3O4 / mPEG-PCL-PEI / Dox / siRNA is carried out according to the following steps: S1: Preparation of mPEG-PCL-PEI: The preparation method is the same as that of mPEG-PCL-PEI in Example 1.

[0042] S2: Preparation of Fe3O4 / mPEG-PCL-PEI / Dox: The preparation method is the same as that of Fe3O4 / mPEG-PCL-PEI / Dox in Example 1.

[0043] S3: Preparation of Fe3O4 / mPEG-PCL-PEI / Dox / siRNA: 10 mg Fe3O4 / FA-mPEG-PCL-PEI / Dox was dispersed in 10 mL deionized water, and 3 mL of 20 μg / mL siRNA solution was added dropwise under mechanical stirring. The reaction was stirred at room temperature for 6 h, and magnetic separation was performed using a magnet. The solid product was repeatedly washed with deionized water to obtain Fe3O4 / mPEG-PCL-PEI / Dox / siRNA.

[0044] Comparative Example 7: A method for preparing Fe3O4 / mPEG-PCL-PEI / siRNA is carried out according to the following steps: S1: Preparation of mPEG-PCL-PEI: The preparation method is the same as that of mPEG-PCL-PEI in Example 1.

[0045] S2: Preparation of Fe3O4 / mPEG-PCL-PEI: S2-1: 5g FeCl3·6H2O and 1g FeCl2·4H2O were added to 50mL deionized water, and mechanically stirred and dissolved at room temperature for 30min under N2 protection. 20mL 10mol / L NaOH solution was added, and mechanically stirred and reacted at room temperature for 1h under N2 protection, and then the temperature was raised to 90℃ and kept for 2h. The mixture was naturally cooled to room temperature and magnetically separated by a magnet. The solid product was alternately washed with deionized water and 2mol / L HNO3 solution until the pH of the supernatant was neutral, and vacuum dried at 40℃ for 24h to obtain Fe3O4 nanoparticles. S2-2: Add 0.2 mL of triethylamine to 10 mL of dichloromethane, stir at room temperature in the dark overnight to obtain a dichloromethane solution; S2-3: 10 mg of mPEG-PCL-PEI was added to 0.2 mL of dichloromethane solution, 1 mL of dichloromethane and 1 mL of acetone were added, and the mixture was sonicated at room temperature for 2 min using a cell disruptor at a power of 36 W to obtain a mixed solution A; S2-4: 10 mg of Fe3O4 nanoparticles were dispersed in 1 mL of deionized water, transferred to 2 mL of mixed solution A, and sonicated at room temperature for 3 min using a cell disruptor at a power of 54 W to obtain mixed solution B; S2-5: 3 mL of mixed solution B was added to 10 mL of 2 wt% PVA solution under ultrasonic conditions, and ultrasonicated at room temperature for 3 min at a power of 54 W using a cell disruptor to obtain mixed solution C; S2-6: The mixed solution C was transferred to a dialysis bag with a molecular weight cutoff of 2000Da, dialyzed with deionized water at room temperature for 24h, and then the precipitate was adsorbed using an external magnet. The precipitate was repeatedly washed with 1×PBS buffer to obtain Fe3O4 / mPEG-PCL-PEI.

[0046] S3: Preparation of Fe3O4 / mPEG-PCL-PEI / siRNA 10 mg of Fe3O4 / mPEG-PCL-PEI was dispersed in 10 mL of deionized water, and 3 mL of 20 μg / mL siRNA solution was added dropwise under mechanical stirring. The reaction was stirred at room temperature for 6 h, and magnetic separation was performed using a magnet. The solid product was repeatedly washed with deionized water to obtain Fe3O4 / mPEG-PCL-PEI / siRNA.

[0047] Figure 1 The results show that the gene-carrying magnetic nanomicelles prepared by the present invention have a particle size of 100 to 300 nm and a uniform particle size distribution, which facilitates the effective entry of the gene-carrying magnetic nanomicelles into and killing of liver cancer cells.

[0048] Figure 2 The results show the change of the surface charge of the product during the preparation process of the present invention. The gene-carrying magnetic nanomicelles finally prepared by the present invention have a strong positive charge, which is beneficial to killing liver cancer cells.

[0049] The gene-carrying magnetic nanomicelles prepared by the present invention were dispersed in 1×PBS solution, kept at 37°C and oscillated in the dark, and samples were taken after 0.5h, 1h, 2h, 4h, 6h, 8h, 12h, 24h, 36h, 48h, 60h, 72h and 84h, respectively, and the amount of doxorubicin hydrochloride released was determined by fluorescence intensity method. The release curve of doxorubicin hydrochloride is shown in Figure 3 .from Figure 3 It can be seen that the release of the drug carrier is biphasic, with a burst release followed by a sustained release. The cumulative release percentages of mPEG-PCL-PEI / Dox / siRNA, Fe3O4 / mPEG-PCL-PEI / Dox / siRNA and Fe3O4 / FA-mPEG-PCL-PEI / Dox / siRNA at 6 h were 21.83%, 49.41% and 33.42%, respectively, and the cumulative release percentages at 24 h were 36.01%, 58.00% and 61.11%, respectively. The cumulative release percentages at 84 h were 41.90%, 60.88% and 62.56%, respectively, indicating that the gene-loaded magnetic nanomicelles prepared by the present invention have good drug controlled release performance.

[0050] The gene-carrying magnetic nanomicelles prepared by the present invention were mixed with Hep-G2 cells (Hep-G2 cell density was 10 6 / mL, the working concentration of gene-carrying magnetic nanomicelles was 60mg / mL) after 24h of co-culture, the survival rate of Hep-G2 cells was as follows Figure 4 As shown. Figure 4It can be seen that after Fe3O4 / FA-mPEG-PCL-PEI / Dox / siRNA was co-cultured with Hep-G2 cells for 24 hours, the survival rate of Hep-G2 cells was 32.31%, indicating that the killing rate of Fe3O4 / FA-mPEG-PCL-PEI / Dox / siRNA on Hep-G2 cells reached 67.69%.

[0051] At the same time, the gene-carrying magnetic nanomicelles prepared by the present invention were mixed with Hep-G2 cells (Hep-G2 cell density was 10 6 After 24 h of co-culture, the results of cell live and dead staining (dead cells are red) are as follows: Figure 5 As shown. Figure 5 It can be seen that the gene-carrying magnetic nanomicelles prepared by the present invention have a good killing effect on Hep-G2 cells.

[0052] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A method for preparing gene-carrying magnetic nanomicelles, which is characterized by comprising the following steps: S1: Preparation of mPEG-PCL-PEI: S1-1: Mix polyethylene glycol monomethyl ether, ε-caprolactone and Sn(Oct)2, react in an oil bath at 130°C for 24 hours, cool naturally to room temperature, add dichloromethane to dissolve, then precipitate in ice anhydrous ether, filter, and dry the residue in vacuum at room temperature to obtain mPEG-PCL; S1-2: mPEG-PCL was added to dichloromethane, triethylamine and acryloyl chloride were added under N2 protection, and the mixture was reacted at room temperature for 6 h, then extracted with saturated NaCl solution for 3 times, the organic phase was collected, precipitated in ice anhydrous ether, filtered, and the residue was dried under vacuum at room temperature to obtain mPEG-PCL-COCHCH2; S1-3: mPEG-PCL-COCHCH2 and polyethyleneimine were dissolved in DMF, pyridine was added, and the mixture was reacted at 60°C for 12 h under N2 protection. After the reaction, the mixture was dialyzed with DMF at room temperature for 24 h, and then with deionized water at room temperature for 72 h, and freeze-dried to obtain mPEG-PCL-PEI. S2: Preparation of Fe3O4 / mPEG-PCL-PEI / Dox: S2-1: FeCl3·6H2O and FeCl2·4H2O were added to deionized water, and mechanically stirred and dissolved at room temperature for 30 min under N2 protection. A 10 mol / L NaOH solution was added, and mechanically stirred and reacted at room temperature for 1 h under N2 protection, and then the temperature was raised to 90°C and kept for 2 h. The mixture was naturally cooled to room temperature, and magnetic separation was performed using a magnet. The obtained solid product was washed until the pH value of the supernatant was neutral, and vacuum dried at 40°C to obtain Fe3O4 nanoparticles. S2-2: Add Dox·HCl to dichloromethane, add triethylamine, and stir at room temperature in the dark overnight to obtain a Dox·HCl solution; S2-3: mPEG-PCL-PEI was added to Dox·HCl solution, and dichloromethane and acetone were added, and ultrasonicated at room temperature for 2 min to obtain mixed solution A; S2-4: Disperse Fe3O4 nanoparticles in deionized water, transfer to mixed solution A, and perform ultrasonic treatment at room temperature for 3 min to obtain mixed solution B; S2-5: adding the mixed solution B to the 2 wt % PVA solution under ultrasonic conditions, and ultrasonicating at room temperature for 3 min to obtain a mixed solution C; S2-6: The mixed solution C was dialyzed with deionized water at room temperature for 24 h, and then the precipitate was adsorbed by an external magnet, and the precipitate was washed to obtain Fe3O4 / mPEG-PCL-PEI / Dox; S3: Preparation of Fe3O4 / FA-mPEG-PCL-PEI / Dox: S3-1: Dissolve folic acid in DMSO, add N-hydroxysuccinimide and EDC hydrochloride, and stir at room temperature for 4 h to obtain FA solution; S3-2: Fe3O4 / mPEG-PCL-PEI / Dox was dispersed in deionized water, FA solution was added dropwise under mechanical stirring, the reaction was stirred at room temperature overnight, centrifuged, and the precipitate was collected to obtain Fe3O4 / FA-mPEG-PCL-PEI / Dox; S4: Preparation of Fe3O4 / FA-mPEG-PCL-PEI / Dox / siRNA: Fe3O4 / FA-mPEG-PCL-PEI / Dox was dispersed in deionized water, and 20 μg / mL siRNA solution was added dropwise under mechanical stirring conditions. The reaction was stirred at room temperature for 6 h, and magnetic separation was performed using a magnet. The solid product was washed to obtain Fe3O4 / FA-mPEG-PCL-PEI / Dox / siRNA, which is the gene-carrying magnetic nanomicelles.

2. The preparation method according to claim 1, characterized in that: In step S1-1, the amount of polyethylene glycol monomethyl ether is 2~10g, the amount of ε-caprolactone is 1~8mL, the amount of Sn(Oct)2 is 0.01~0.1g, and the amount of dichloromethane is 10~25mL; in step S1-2, the amount of mPEG-PCL is 2~10g, the amount of dichloromethane is 10~100mL, the amount of triethylamine is 1~10mL, and the amount of acryloyl chloride is 0.1~1mL; in step S1-3, the amount of mPEG-PCL-COCHCH2 is 2~10g, the amount of polyethyleneimine is 1~5g, the amount of DMF is 10~100mL, and the amount of pyridine is 2~15mL.

3. The preparation method according to claim 1, characterized in that: In step S2-1, the amount of FeCl3·6H2O is 5~20g, the amount of FeCl2·4H2O is 1~8g, the amount of deionized water is 50~200mL, and the amount of NaOH solution is 20~100mL; in step S2-2, the amount of Dox·HCl is 10~100mg, the amount of dichloromethane is 10~150mL, and the amount of triethylamine is 0.2~1.5mL; in step S2-3, the amount of mPEG-PCL-PEI is 10~100m g, the amount of Dox·HCl solution is 0.2~2mL, the amount of dichloromethane solution is 1~8mL, and the amount of acetone is 1~2mL; in step S2-4, the amount of Fe3O4 nanoparticles is 10~100mg, the amount of deionized water is 1~5mL, and the amount of mixed solution A is 2~10mL; in step S2-5, the amount of mixed solution B is 6~15mL, and the amount of PVA solution is 10~100mL; in step S2-6, the pH value of 1×PBS buffer is 7.

4.

4. The preparation method according to claim 1, characterized in that: In step S3-1, the amount of folic acid is 10-100 mg, the amount of DMSO is 5-50 mL, the amount of N-hydroxysuccinimide is 5-20 mg, and the amount of EDC hydrochloride is 20-150 mg; in step S3-2, the amount of Fe3O4 / mPEG-PCL-PEI / Dox is 10-20 mg, and the amount of deionized water is 1-10 mL; the amount of FA solution is 0.2-2 mL.

5. The preparation method according to claim 1, characterized in that: In step S4, the amount of Fe3O4 / FA-mPEG-PCL-PEI / Dox is 10-20 mg, the amount of deionized water is 10-21 mL, and the amount of siRNA solution is 3-6.3 mL.

6. A gene-carrying magnetic nanomicelle, characterized in that: The method is prepared according to any one of claims 1 to 5.

7. Use of the preparation method according to any one of claims 1 to 5 and the gene-carrying magnetic nanomicelles according to claim 6 in the preparation of drugs for treating tumors.

Images