Branched fluorine-containing poly (beta-amino ester) as well as preparation method and application thereof

Abstract

The invention discloses branched fluorine-containing poly (beta-amino ester) as well as a preparation method and application thereof, and belongs to the technical field of biomedical materials. The branched fluorine-containing poly (beta-amino ester) is prepared by polymerizing a diacrylate monomer, a triacrylate monomer, micromolecular organic amine and a fluorine-containing alkyl chain monomer through a Michael addition reaction and then blocking with an end-capping reagent. The preparation method is simple, and the process is controllable. The polymer has controllable molecular weight, branching degree and fluorine content, can be self-assembled with a gene drug to form a stable nano-composite, can mediate efficient gene transfection, has good in-vitro and in-vivo gene editing ability, and can be used for treating genetic metabolic diseases and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and in particular relates to a branched fluorine-containing poly (β-amino ester) and a preparation method thereof and application thereof in gene delivery. Background Art

[0002] Gene therapy refers to the delivery of exogenous nucleic acid fragments (DNA or RNA) into cells through certain means to achieve the purpose of gene supplementation, gene silencing or gene editing. However, due to the large molecular weight of exogenous nucleic acid fragments, their low delivery efficiency and other issues limit the application of gene therapy in clinical practice. In order to improve the therapeutic effect of gene therapy, efficient and safe delivery vectors are indispensable.

[0003] Currently, gene delivery vectors can be mainly divided into viral vectors and non-viral vectors.

[0004] Commonly used viral vectors can be divided into lentivirus, adenovirus and adeno-associated virus vectors. Although viral vectors are currently widely used in clinical trials of gene therapy, their in vivo applications are limited due to their potential immunogenicity, limited loading capacity and difficulty in production, and their application prospects are increasingly questioned.

[0005] In comparison, non-viral vectors have higher safety and biocompatibility and are more suitable for in vivo applications. Based on the type of material, non-viral vectors can be divided into lipid-based, inorganic material-based and polymer-based vectors.

[0006] Lipid-based carriers usually include four substances: ionizable lipids, neutral auxiliary lipids, cholesterol, and PEGylated lipids. Through the rational design of the formulation composition, lipid-based carriers can bind to nucleic acid fragments through electrostatic interactions and effectively deliver exogenous nucleic acids to achieve the purpose of treating diseases. However, the delivery efficiency of lipid-based carriers is heavily dependent on the type of ionizable lipids, and the relevant technical patents of commercial lipid nanotechnology are difficult to break through in a short period of time, and the delivery efficiency of large molecular nucleic acid fragments is low, which greatly limits the application of lipid-based carriers in clinical practice.

[0007] Carriers based on inorganic materials can be divided into gold nanocarriers, carbon nanocarriers, and metal organic framework carriers. The size and shape of nanoparticles can be quickly adjusted through the synthesis process. Among them, gold nanocarriers are the most widely used. In the application of gene therapy, gold nanocarriers are often modified with positively charged polypeptides to deliver negatively charged nucleic acid fragments. However, since inorganic materials are difficult to degrade in the body and related in vivo research data are limited, their safety and delivery efficiency remain to be investigated.

[0008] Polymer-based vectors are one of the most important vectors in the field of gene delivery due to their good safety, diverse chemical composition and easy production, and are also one of the vectors with the greatest potential for clinical transformation. Among them, poly(β-amino esters) (PBAEs) are a type of vector with excellent gene transfection efficiency. It was first synthesized by Professor Robert Langer and others at the Massachusetts Institute of Technology through the Michael addition reaction of diacrylates and amines. Due to its diverse monomer composition, easy-to-regulate structural properties and biodegradability, it has shown great application prospects in gene delivery in recent years.

[0009] To further enhance the application potential of poly(β-amino esters), researchers have tried a variety of functional modification strategies, such as fluorine modification, to improve the gene delivery ability of poly(β-amino esters).

[0010] Chinese patent CN 115626984 A discloses a fluorinated polymer and its synthesis method and application in gene delivery. The provided functional fluorinated poly (β-amino ester) polymer has an adjustable structure, and its main chain contains poly (β-amino ester) of multiple components, and the side chain is fluorinated and modified to form or not form an alkyl side chain structure, and the terminal structure contains primary amine, secondary amine or tertiary amine, which is a new type of functional polymer gene carrier. Chinese patent CN 115232306 A discloses a fluorinated linear poly (β-amino ester) and its preparation method and application. Poly (β-amino ester) is synthesized by Michael addition reaction using fluorinated diacrylate and amino alcohol as monomers, and then end-capped with amine molecules to obtain a fluorinated linear poly (β-amino ester).

[0011] In the literature, GONG et al. [Biocompatible fluorinated poly(β-amino ester)sfor safe and efficient gene therapy. International Journal of Pharmaceutics, 2018, 535(1): 180-93] reported a series of fluorinated polyurethanes designed and synthesized by Michael addition reaction, which were used as gene delivery vectors. The results of transfection efficiency determination showed that compared with commercially available transfection reagents polyethyleneimine (PEI, Mw=25K) and Lipo2000, the optimized fluorinated polyurethane showed higher GFP expression, and the transfection efficiency on HeLa cells and HEK-293T cell lines was 70% and 90% higher than that of non-fluorinated polyurethane, respectively. The article attributed its advantages to the presence of fluorine. In addition, DENG et al. [Fluorinated amphiphilic Poly (β-Amino ester) nanoparticle for highly efficient and specific delivery of nucleic acids to the Lung capillary endothelium. Bioactive Materials, 2023, 31: 1-17] used diacrylates and amines to gradually polymerize into a hydrophobic polyurethane backbone through a Michael addition reaction, and the end groups were modified with hydrophilic fluorinated polyethyleneimine and / or polyethylene glycol to form an amphiphilic structure, which was self-assembled to form nanoparticles loaded with nucleic acids, showing good nanoparticle colloidal stability and cationic density, promoting gene encapsulation, and achieving efficient and specific lung endothelial cell delivery.

[0012] However, although poly(β-amino esters) have demonstrated excellent green fluorescent protein (GFP) plasmid delivery efficiency in various cell lines, they perform poorly in the delivery of functional macromolecular nucleic acid fragments, and there is great room for improvement in delivery efficiency. Summary of the invention

[0013] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a fluorinated poly (β-amino ester) and its preparation method and application, which solves the problem of low delivery efficiency of poly (β-amino ester) in macromolecular nucleic acid fragments in the prior art. The functional fluorinated poly (β-amino ester) provided by the present invention is a polymer with a completely new structure, which can be used to deliver functional nucleic acid fragments.

[0014] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0015] The first aspect of the present invention provides a branched fluorinated poly(β-amino ester) having the following structure:

[0016]

[0017] Wherein, x, y, and z represent the degree of polymerization of each repeating unit in the molecule, respectively, x is an integer of 1-40, y is an integer of 1-20, and z is an integer of 1-20; R 1 Select one of the following structures (the wavy lines represent connecting bonds):

[0018]

[0019] R 2 At least two selected from the following groups, which must contain 1%-20% of fluorinated alkyl groups:

[0020]

[0021] R3 selects one of the following structures:

[0022]

[0023] Preferably, R 3 for

[0024] In an embodiment of the present invention, a branched fluorinated poly(β-amino ester) having the following structure was synthesized:

[0025]

[0026]

[0027]

[0028]

[0029]

[0030] The second aspect of the present invention provides a method for synthesizing the branched fluorinated poly(β-amino ester), comprising the following steps:

[0031] (1) Acrylate monomers (including diacrylate monomers and triacrylate monomers) and small molecule organic amines and fluorinated monomers (NH 2 -R 2 ) synthesizing a branched fluorinated poly(β-amino ester) backbone having a double bond at the end through a Michael addition reaction;

[0032]

[0033] NH 2-R 2 Represents a mixture of small molecule organic amine and fluorine-containing monomer, including fluorine-containing monomer with a molar content of 1%-20%, and the rest is fluorine-free small molecule organic amine;

[0034] (2) reacting the branched fluorinated poly(β-amino ester) backbone synthesized in step (1) with a capping agent monomer (NH 2 -R 3 ) through Michael addition reaction to obtain the branched fluorinated poly (β-amino ester).

[0035]

[0036] In the above reaction formula, the triacrylate monomer is trimethylolpropane triacrylate; preferably, the diacrylate monomer is selected from bisphenol A glycerol diacrylate, 1,4-butanediol diacrylate, 1,5-pentanediol diacrylate, 1,6-hexanediol diacrylate; the small molecule organic amine is selected from N-(3-aminopropyl)morpholine, N-(2-aminoethyl)morpholine, 3-amino-1-propanol, 4-amino-1-butanol, 5-amino-1-pentanol; the fluorine-containing monomer is selected from 1H, 1H-undecafluorohexylamine, 2,2,3,3,4,4,4-heptafluorobutylamine, 2,2,3,3,3-pentafluoropropylamine, 2,2,2-trifluoroethylamine; the end-capping agent monomer is selected from 2-hydroxyethylamine propylamine, 1-(3-aminopropyl)-4-methylpiperazine, 1-(3-aminopropyl)pyrrolidine, 1,3-diaminopentane, 2-methyl-1,5-diaminopentane, 1,11-diamino-3,6,9-trioxaundecane, diethylenetriamine, 1,4-bis(3-aminopropyl)piperazine. The structures of some compounds are shown below:

[0037]

[0038] Preferably, in step 1), the molar ratio of the diacrylate monomer to the triacrylate monomer is 7:1 to 1:1; the diacrylate monomer is reacted with the small molecule organic amine and the fluorinated monomer NH 2 -R 2 The feeding molar ratio is 0.4:1 to 1.2:1, wherein the feeding molar ratio of the small molecule organic amine to the fluorine-containing monomer is 19:1 to 7:3.

[0039] Furthermore, step 1) is carried out in a polar organic solvent (such as N,N-dimethylformamide, dimethyl sulfoxide, etc.) at a temperature of 80-110°C; step 2) is carried out in a polar organic solvent (such as N,N-dimethylformamide, dimethyl sulfoxide, etc.) at a temperature of 15-35°C.

[0040] The third aspect of the present invention provides the use of the branched fluorine-containing poly (β-amino ester) described in the first aspect in the preparation of gene therapy drugs.

[0041] Furthermore, the branched fluorinated poly(β-amino ester) is used as a carrier for delivering gene drugs.

[0042] Furthermore, the gene medicine is DNA or siRNA.

[0043] Furthermore, the branched fluorine-containing poly (β-amino ester) and the gene drug self-assemble to form a nanocomposite.

[0044] The beneficial effects of the present invention are:

[0045] 1. The present invention designs a new type of fluorinated hyperbranched poly (β-amino ester), which utilizes the unique fluorine effect of fluorine atoms and the fluorine-philic effect of fluorinated alkyl chains to enable the fluorinated hyperbranched poly (β-amino ester) to form a stable nanocomplex with gene drugs and mediate efficient gene transfection.

[0046] 2. The fluorinated hyperbranched poly(β-amino ester) of the present invention can be prepared by Michael addition reaction between acrylates, amine molecules and fluorinated alkyl chains. The preparation method is simple and the process is controllable. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the branched fluorinated poly(β-amino ester) prepared in Example 2.

[0048] Figure 2 This is the nuclear magnetic resonance fluorine spectrum of the branched fluorine-containing poly(β-amino ester) prepared in Example 2.

[0049] Figure 3 This is a diagram showing the transfection efficiency evaluation results of Example 8.

[0050] Figure 4 This is the particle size distribution diagram of the nanocomplex formed by assembling the branched fluorinated poly (β-amino ester) prepared in Example 2 and the pX458 plasmid.

[0051] Figure 5 This is a transmission electron microscopy image of a nanocomplex formed by assembling the branched fluorinated poly (β-amino ester) prepared in Example 2 and the pX458 plasmid.

[0052] Figure 6 When the branched fluorinated poly (β-amino ester) prepared in Example 2 and the pX458 plasmid were delivered, the expression of the plasmid after transfection was observed under a fluorescence microscope; Blank was a blank control; Lipo was a commercial transfection reagent Hieff Trans TMLiposomal Transfection Reagent (Yisheng Biotechnology (Shanghai) Co., Ltd.), the dosage is 3 μL Lipo / 1 μg plasmid; FHPAE (25) and FHPAE (50) represent the branched fluorinated poly (β-amino ester) prepared in Example 2, the dosage is 25 μg FHPAE / 1 μg plasmid and 50 μg FHPAE / 1 μg plasmid, respectively.

[0053] Figure 7 When the branched fluorinated poly (β-amino ester) prepared in Example 2 delivered the pX458 plasmid, the transfection efficiency of the nanocomplex was evaluated by flow cytometry; wherein Blank was a blank control; Lipo was a commercial transfection reagent Hieff Trans TM Liposomal Transfection Reagent (Yisheng Biotechnology (Shanghai) Co., Ltd.), the dosage is 3 μL Lipo / 1 μg plasmid; FHPAE (25) and FHPAE (50) represent the branched fluorinated poly (β-amino ester) prepared in Example 2, the dosage is 25 μg FHPAE / 1 μg plasmid and 50 μg FHPAE / 1 μg plasmid, respectively.

[0054] Figure 8 This is the Sanger sequencing map of the CD47 gene fragment after B16F10 cells were treated with PBS in Example 11.

[0055] Fig. 9 This is the Sanger sequencing map of the CD47 gene fragment after B16F10 cells were treated with the nanocomplex in Example 11.

[0056] Fig.10 The CD47 gene editing efficiency of the nanocomplex was analyzed using TIDE in Example 11.

[0057] Fig.11 Representative images and semi-quantitative results of immunofluorescence staining of mouse tumor sections; wherein FHPAE represents the branched fluorinated poly(β-amino ester) prepared in Example 2. DETAILED DESCRIPTION

[0058] In order to enable those skilled in the art to better understand the scheme of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the embodiments described herein are only used to explain the present invention and are not all embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work should belong to the scope of protection of the present invention.

[0059] Embodiment 1 to Embodiment 7

[0060] Preparation of branched fluorinated poly (β-amino ester), the general structure of which is shown below:

[0061]

[0062] The raw materials and their amounts are shown in the following table:

[0063]

[0064] The preparation steps are as follows:

[0065] (1) Dissolve bisphenol A glycerol diacrylate, trimethylolpropane triacrylate, N-(3-aminopropyl)morpholine and 1H,1H-undecylfluorohexylamine in N,N-dimethylformamide, stir and react at 95° C. for 24 hours, and cool to room temperature to obtain a brown-yellow liquid.

[0066] (2) The brown-yellow liquid obtained in step (1) and 2-hydroxyethylaminepropylamine are dissolved in N,N-dimethylformamide, and the mixture is stirred at room temperature for 48 hours, and then precipitated and washed with ice ether for three times, and the solvent is removed by vacuum drying to obtain a brown-yellow product, which is a fluorinated hyperbranched poly(β-amino ester).

[0067] The branched fluorinated poly(β-amino ester) prepared in each example was subjected to hydrogen nuclear magnetic resonance spectroscopy. 1 H NMR and fluorine NMR spectroscopy 19 F NMR characterization. The spectrum of the branched fluorinated poly(β-amino ester) prepared in Example 2 is as follows: Figure 1 , 2 shown.

[0068] In the hydrogen nuclear magnetic resonance spectrum, the two characteristic peaks with a chemical shift of 6.5-7.5 ppm are attributed to the two hydrogens on the benzene ring of bisphenol A glycerol diacrylate, the two characteristic peaks at 4.5-5.5 ppm are attributed to the hydroxyl groups of bisphenol A glycerol diacrylate and 2-hydroxyethylamine propylamine, and the characteristic peak with a chemical shift of 0.8 ppm is the methyl peak on trimethylolpropane triacrylate.

[0069] The characteristic peaks in the NMR fluorine spectrum come from the monomer 1H,1H-undecylfluorohexylamine. Taking the methyl carbon of 1H,1H-undecylfluorohexylamine as the α position, the characteristic peak with a chemical shift of -80ppm is attributed to the fluorine (-CF 3 ); The four characteristic peaks in the chemical shift range from -110 ppm to -130 ppm are attributed to the fluorine (-CF 2 -), from left to right they are ε, δ, γ, β.

[0070] C NMR 13The polymers prepared in each example were characterized by C NMR, and the characteristic peaks of each monomer were integrated. The degree of branching (DB) was calculated according to the following formula:

[0071]

[0072] Wherein, D, T, and L represent the characteristic peak areas of trimethylolpropane triacrylate (branching unit), 2-hydroxyethylamine propylamine (end group), and bisphenol A glycerol diacrylate (linear unit), respectively.

[0073] The calculation results are shown in the following table.

[0074] Example 1 Example 2 Example 3 Degree of branching 0.509 0.705 0.762

[0075] The branching degree of the branched fluorinated poly(β-amino ester) prepared in Examples 4 to 7 is the same as that in Example 2.

[0076] Example 8

[0077] Take 0.5 μg of pEGFP plasmid (Shanghai Jiying Biotechnology Co., Ltd.), add 12.5 μg of the polymer prepared in Examples 1-7, vortex thoroughly, and then let stand for 20 minutes to form a nanocomposite. Separately take a completely linear, completely branched, non-fluorinated branched poly (β-amino ester) to prepare a nanocomposite. The synthetic formula of the completely linear, completely branched, non-fluorinated branched poly (β-amino ester) is as follows:

[0078] Linear branching Not fluorinated Bisphenol A glycerol diacrylate 0.6mmol 0 0.7mmol Trimethylolpropane triacrylate 0 0.6mmol 0.2mmol N-(3-Aminopropyl)morpholine 0.4mmol 0.6mmol 0.83mmol 1H,1H-Undecafluorohexylamine 0.1mmol 0.15mmol 0 2-Hydroxyethylamine Propylamine 0.6mmol 1.2mmol 0.7mmol

[0079] Human melanoma cell line B16F10 was cultured at 10×10 4 The cells were seeded at a density of 0.5 mL / well in a 24-well plate and cultured for 24 hours. The nanocomplexes were then added to the 24-well plate and cultured for another 48 hours. The transfection efficiency was quantitatively evaluated by flow cytometry.

[0080] The results are as follows Figure 3 As shown in A, when the feed ratio of bisphenol A glycerol diacrylate and trimethylolpropane triacrylate is less than 7 / 3, the transfection efficiency of the branched structure polymer is significantly better than that of the linear structure; when the feed amount of trimethylolpropane triacrylate is further increased, the transfection efficiency of the polymer is significantly reduced, and the fully branched polymer shows an extremely low transfection efficiency, which has not been reported in previous studies.

[0081] In addition, if Figure 3As shown in Figure B, compared with the unfluorinated branched poly (β-amino ester), when the feed ratio of N-(3-aminopropyl)morpholine and 1H,1H-undecylfluorohexylamine is less than 4 / 1, the fluorine modification can significantly improve the transfection efficiency of the polymer, and the optimal transfection efficiency is shown when the feed ratio is 9 / 1-5 / 1, which is completely different from the ratio reported by Gong et al. (Gong JH, Wang Y, Xing L, Cui PF, Qiao JB, He YJ, Jiang HL. Biocompatible fluorinated poly (β-aminoester)s for safe and efficient gene therapy. Int J Pharm. 2018 Jan 15; 535(1-2): 180-193. doi: 10.1016 / j.ijpharm.2017.11.015. Epub 2017 Nov 10. PMID: 29129572.) (This document points out that the transfection efficiency is highest when the fluorinated monomer accounts for 20% of the side chain monomer feed amount).

[0082] Example 9

[0083] 25 μg of the polymer prepared in Example 2 was added with 1 μg of pX458 plasmid (Nano Today 42 (2022) 101359), vortexed thoroughly, and then allowed to stand for 20 minutes to form a nanocomposite. The hydrodynamic particle size was measured using a dynamic light scattering instrument (Malvern Zetasizer Nano-ZS laser particle size analyzer, Malvern Instruments, UK). The results are shown in Figure 4 As shown, the particle size of the nanocomposite prepared by the polymer prepared in Example 2 and the pX458 plasmid is 111.1 nm, and the polydispersity index PDI is 0.071.

[0084] The microscopic morphology of the nanocomposite formed by the polymer prepared in Example 2 and the pX458 plasmid was observed using a transmission electron microscope (JEM-1400PLUS transmission electron microscope, Leica, Germany). Figure 5 As shown, the composite is spherical and has a particle size of about 50 nm.

[0085] Example 10

[0086] Human cervical cancer cell line Hela was cultured at 10×10 4 The cells were inoculated in a 24-well plate at a density of 0.5 mL / well and cultured for 24 hours. The nanocomplex was prepared according to the method described in Example 9, and then the nanocomplex was added to the 24-well plate and cultured for 72 hours. The expression results of the plasmid were observed by fluorescence microscopy, and the transfection efficiency was quantitatively evaluated by flow cytometry.

[0087] The results of the test are as follows Figure 6 , Figure 7 As shown, the embodiments of the present invention mediate higher transfection efficiency, which is significantly better than commercial transfection reagents (Hieff Trans TM Liposomal Transfection Reagent (Lipo, Yisheng Biotechnology (Shanghai) Co., Ltd.).

[0088] Embodiment 11

[0089] Mouse melanoma cell line B16F10 was cultured at 10 × 10 4 The cells were inoculated at a density of 0.5 mL / well in a 24-well plate and cultured for 24 hours. According to the method described in Example 9, the pX458 plasmid [Nano Today, 2022, 42, 101359] targeting the CD47 gene was used to prepare the nanocomplex, and then the nanocomplex was added to the 24-well plate and cultured for 72 hours. The genome was extracted using a blood / cell / tissue genomic DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.), and a partial fragment of the CD47 gene was amplified by PCR. The PCR product was Sanger sequenced by Beijing Qingke Biotechnology Co., Ltd. The Sanger sequencing results were analyzed by the TIDE online analysis tool (https: / / tide.nki.nl / ).

[0090] The results are as follows Figure 8 , 9 As shown in Figure 10, the nanocomplex caused significant gene mutations in the CD47 genome, with a gene editing efficiency of 47.1%.

[0091] Example 12

[0092] Mouse melanoma cell line B16F10 was cultured at 1×10 6 Each mouse was subcutaneously inoculated with B16F10 tumor-bearing mice to establish a B16F10 tumor-bearing mouse model. When the tumor volume of the mouse grew to about 100 mm 3When the nanocomplex was prepared according to the method described in Example 9, the pX330 plasmid targeting the CD47 gene (Nano Today 42 (2022) 101359) was used, and the peritumoral administration was carried out, and PBS was used as the control group. Three days after administration, the mouse tumor was isolated and frozen sections were prepared. Immunofluorescence staining was performed using DAPI staining solution, Rabbit Anti-CD47antibody, and Goat Anti-Rabbit IgG H&L / Alexa Fluor 488 (Beijing Boosun Biotechnology Co., Ltd.), and the frozen sections were observed using a laser confocal microscope (STELLARIS 8 laser confocal microscope, Leica, Germany), and semi-quantitative statistics of the images were performed using ImageJ.

[0093] The results of the test are as follows Fig.11 As shown, after treatment with the nanocomplex, the CD47 fluorescence signal in the mouse tumor was significantly reduced, indicating that the branched fluorinated poly (β-amino ester) prepared in the embodiment of the present invention can efficiently deliver gene editing plasmids in vivo and has broad in vivo application prospects.

Claims

1. A branched fluorinated poly(β-amino ester) having a structure as shown in Formula I: in, x, y, and z represent the degree of polymerization of each repeating unit, respectively, x is an integer of 1-40, y is an integer of 1-20, and z is an integer of 1-20; R1 is selected from one of the following structures: R2 is selected from at least two of the following groups, which must contain 1%-20% of fluorinated alkyl groups: R3 represents a capping group selected from the following groups:

2. The branched fluorinated poly(β-amino ester) according to claim 1, characterized in that The branched fluorinated poly(β-amino ester) is selected from the following polymers:

3. The method for preparing the branched fluorinated poly(β-amino ester) according to claim 1 or 2, comprising the following steps: 1) synthesizing a branched fluorinated poly(β-amino ester) skeleton having a double bond at the end by reacting a triacrylate monomer represented by formula a and a diacrylate monomer represented by formula b with a small molecule organic amine and a fluorine-containing monomer through a Michael addition reaction; Wherein, NH2-R2 represents a mixture of small molecule organic amine and fluorine-containing monomer, including fluorine-containing monomer with a molar content of 1%-20%, and the rest is small molecule organic amine without fluorine; 2) subjecting the branched fluorinated poly(β-amino ester) backbone obtained in step 1) to a Michael addition reaction with the end-capping agent monomer NH2-R3 to obtain the branched fluorinated poly(β-amino ester); wherein R1, R2 and R3 are as defined in claim 1.

4. The preparation method according to claim 3, characterized in that: The diacrylate monomer represented by formula b is selected from bisphenol A glycerol diacrylate, 1,4-butanediol diacrylate, 1,5-pentanediol diacrylate, and 1,6-hexanediol diacrylate.

5. The preparation method according to claim 3, characterized in that: The small molecule organic amine is selected from N-(3-aminopropyl)morpholine, N-(2-aminoethyl)morpholine, 3-amino-1-propanol, 4-amino-1-butanol, and 5-amino-1-pentanol; the fluorine-containing monomer is selected from 1H,1H-undecafluorohexylamine, 2,2,3,3,4,4,4-heptafluorobutylamine, 2,2,3,3,3-pentafluoropropylamine, and 2,2,2-trifluoroethylamine.

6. The preparation method according to claim 3, characterized in that: The end-capping agent monomer is selected from 2-hydroxyethylamine propylamine, 1-(3-aminopropyl)-4-methylpiperazine, 1-(3-aminopropyl)pyrrolidine, 1,3-diaminopentane, 2-methyl-1,5-diaminopentane, 1,11-diamino-3,6,9-trioxaundecane, diethylenetriamine, and 1,4-bis(3-aminopropyl)piperazine.

7. The preparation method according to claim 3, characterized in that: In step 1), the molar ratio of the diacrylate monomer to the triacrylate monomer is 7:1 to 1:1; the molar ratio of the diacrylate monomer to the small molecule organic amine and the fluorine-containing monomer NH2-R2 is 0.4:1 to 1.2:1, wherein the molar ratio of the small molecule organic amine to the fluorine-containing monomer is 19:1 to 7:

3.

8. The preparation method according to claim 3, characterized in that: Step 1) carrying out Michael addition reaction in a polar organic solvent at a temperature of 80-110° C.; Step 2) carrying out Michael addition reaction in a polar organic solvent at a temperature of 15-35° C.

9. Use of the branched fluorinated poly (β-amino ester) according to claim 1 or 2 in the preparation of a gene therapy drug, wherein the branched fluorinated poly (β-amino ester) is used as a carrier for delivering gene drugs.

10. The use according to claim 9, characterized in that: The gene medicine is DNA or siRNA, and the branched fluorine-containing poly (β-amino ester) and the gene medicine are self-assembled to form a nanocomposite.

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