A cationic polymer gene carrier with end group fluorination and a preparation method and application thereof

Abstract

The application relates to the technical field of biological macromolecular materials, in particular to a preparation method and application of a cationic polymer gene carrier with a fluorinated end group. The cationic polymer gene carrier with a fluorinated end group has the following structure. The fluorinated initiator is used to introduce fluorinated groups in one step to initiate the synthesis of linear polyethylene imine F-PEI with various molecular weights, the introduction of the fluorinated groups improves the gene transfection efficiency, and high-efficiency DNA transfection is realized. The gene carrier can achieve high-efficiency transfection effect in the cell transfection process, the transfection process has small toxicity to cells, gene molecules can be effectively and safely delivered into cells, and the gene carrier has the advantages of high efficiency, low toxicity, low price, simple synthesis and the like. The application has the advantages of simple reaction, easily-obtained raw materials, low preparation cost, easy control of molecular weight, high yield, simple post-treatment and suitability for large-scale production.

Description

Technical Field

[0001] The present invention relates to the technical field of biopolymer materials, in particular to a terminal fluorinated cationic polymer gene carrier and a preparation method and application thereof. Background Art

[0002] Gene therapy is one of the most sought-after technologies today, involving the intracellular delivery of genetic material for the treatment of various diseases, such as cancer, viral infections, and hereditary disorders. Although gene therapy is the newest and most promising strategy for combating these diverse diseases, its application is limited by the lack of safe and effective delivery systems. Viruses are the most widely studied delivery vehicles for gene transfection, but their use is limited by their high risk of causing severe immune and inflammatory responses and safety concerns. This has prompted in-depth research into non-viral gene delivery vehicles. Cationic polymers in non-viral gene delivery systems can electrostatically adsorb negatively charged nucleic acid macromolecules, such as plasmid DNA or antisense oligonucleotides, to form microparticles, micelles, or liposomes, enabling efficient gene delivery and cell transfection. Among the various non-viral delivery vehicles under development, cationic polymers hold significant potential and advantages over their counterparts due to their low toxicity, cost-effectiveness, ease of production, and versatility in diverse applications. Polyethyleneimine (PEI) is a commonly used cationic gene delivery system. PEI can be classified into linear and branched types, and is available in various molecular weights.

[0003] PEI is a cationic polymer carrier with high transfection efficiency. Branched polyethyleneimine (PEI 25k), with a molecular weight of 25,000, is often used as a benchmark for evaluating gene vectors and comparing newly designed and developed gene vectors. The positively charged functional groups on the PEI surface bind and concentrate negatively charged nucleic acids through ionic interactions, forming nanoscale complexes that protect them from nuclease degradation and facilitate their entry into cells. Once inside cells, the complexes are primarily concentrated in endosomes or lysosomes. Through a "proton sponge" effect, the nucleic acid complexes can escape from these endosomes or lysosomes, releasing the nucleic acid. However, due to its inherent chemical structure, PEI is highly positively charged, making it susceptible to cytotoxicity and cell death during gene transfection. Generally, PEI below 2000 kDa exhibits no significant cytotoxicity, but also exhibits low transfection efficiency. Larger PEI molecules offer higher transfection efficiency but are associated with greater cytotoxicity. However, small linear polyethyleneimine molecules exhibit poor transfection efficiency, while larger linear polyethyleneimine molecules exhibit high cytotoxicity. Among them, branched polyethyleneimine (PEI 25k) with a molecular weight of 25,000 is known as the "gold standard" for cationic polymer vector transfection efficiency. However, PEI 25k has high cytotoxicity, which severely limits its clinical application. Therefore, the development and preparation of cationic polymer nucleic acid vectors with high transfection efficiency and low cytotoxicity has become an urgent need. Commercial linear LPEI transfection reagents have better transfection performance. The degree of polymerization of commercial PEI 22k and PEI MAX are both around 500, and the degree of polymerization of PEI pro is even higher. PEI 22k is a partial hydrolysis product of the side groups. Even if the prepared solution is stored in a 4°C refrigerator, the side groups are unstable and cannot be stored for a long time. PEI MAX and PEI pro are very successful commercial products. The side groups are completely hydrolyzed and the degree of polymerization is high, which makes their synthesis difficult.

[0004] Fluorochemicals can give polymers excellent self-assembly properties, anti-pollution ability, chemical and biological inertness, low cytotoxicity, etc. They can greatly improve the transfection efficiency of polyethyleneimine, reduce the nitrogen-phosphorus ratio required for complete complexation with plasmid DNA during cell transfection, and greatly improve the performance of polyethyleneimine in cell transfection. Side-fluorinated polyethyleneimine is post-modified on branched polyethyleneimine (BPEI). Due to the imprecise molecular weight of BPEI, the molecular weight distribution (PDI) is very large, and the source of raw materials is unstable, resulting in difficulties in its industrial application. For example, Chinese patent CN106188537A uses heptafluorobutyric anhydride to replace the primary amine hydrogen in branched polyethyleneimine, and Chinese patent CN110467540A uses perfluoroalkyl acyl halide or perfluoroalkyl sulfonyl halide to replace the primary amine hydrogen of polyethyleneimine. The side-fluorinated polyethyleneimine obtained by the above technical solution has a large molecular weight and is more cytotoxic. Chinese patent CN107556493A uses a one-step reaction between the acyl chloride group of perfluorooctanoyl chloride and the primary amine of oligopolyethyleneimine (OEI) to form an amide bond. This perfluorooctane group is then attached to the branched amino group within the OEI structure, similarly yielding a pendant fluorinated polyethyleneimine. However, hydrolysis of the amide bond in this solution significantly increases the toxicity of the polyethyleneimine product and dramatically reduces transfection efficiency. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a terminal fluorinated cationic polymer gene carrier and its preparation method and application. The cationic polymer provided by the present invention can be used as a gene carrier to achieve efficient and low-toxic DNA transfection.

[0006] The present invention first provides a cationic polymer, the structure of which is shown in the following formula (I):

[0007]

[0008] Wherein, 0≤n≤7, 0≤m≤3, 10≤p≤500. Preferably, 10≤p≤100.

[0009] The present invention further provides a method for preparing the cationic polymer, comprising the following steps:

[0010] (1) dissolving a fluorine-containing compound having a structure represented by formula (II) and a 2-ethyl-2-oxazoline monomer in an anhydrous solvent to obtain a precursor solution; reacting the precursor solution in a sealed container at 60-120° C., cooling the reaction solution to room temperature, and then rotary evaporating to obtain a terminal fluorinated poly-2-ethyl-2-oxazoline intermediate;

[0011] (2) reacting the terminal fluorinated poly-2-ethyl-2-oxazoline intermediate with hydrochloric acid at 100° C.; settling the reaction solution, washing, drying, and pulverizing to obtain the cationic polymer;

[0012]

[0013] Wherein, X=I, Br or p-toluenesulfonate, 0≤n≤7, 0≤m≤3.

[0014] In the above technical solution, the 2-ethyl-2-oxazoline monomer is dried over anhydrous magnesium sulfate or calcium hydride for more than 4 hours to remove water.

[0015] As one of the preferred technical solutions of the present invention, the molar ratio of 2-ethyl-2-oxazoline to fluoride ions in the fluorine-containing compound in step (1) is 1:0.1-1.3.

[0016] As one of the preferred technical solutions of the present invention, the anhydrous solvent is any one of chlorobenzene, dichlorobenzene, and acetonitrile.

[0017] As one of the preferred technical solutions of the present invention, a fluorine-containing compound and a 2-ethyl-2-oxazoline monomer are separately dissolved in an anhydrous solvent and then mixed to obtain a precursor solution, wherein the concentration of the 2-ethyl-2-oxazoline is 5-12 mol / L, preferably 10-12 mol / L.

[0018] As one of the preferred technical solutions of the present invention, the reaction temperature in step (1) is 100° C., and the reaction time is 24-96 h, preferably 24 h.

[0019] In the above technical solution, the step (1) of cooling the reaction solution to room temperature and then performing rotary evaporation is to dilute the reaction solution with a benign solvent such as dichloromethane, ethyl acetate or ethanol, and then perform rotary evaporation in a water bath at a temperature of 30°C to 70°C.

[0020] As one of the preferred technical solutions of the present invention, the concentration of the hydrochloric acid in step (2) is 2-6 mol / L, preferably 6 mol / L.

[0021] As one of the preferred technical solutions of the present invention, the concentration of the terminal fluorinated poly-2-ethyl-2-oxazoline intermediate in the hydrochloric acid solution in step (2) is 50 g / L.

[0022] As one of the preferred technical solutions of the present invention, the reaction time in step (2) is 8-24 hours, preferably 10 hours.

[0023] As one of the preferred technical solutions of the present invention, the precipitation in step (2) is as follows: adding the reaction solution to a low-boiling-point water-soluble solvent pre-cooled in an ice-water bath as a precipitant, such as methanol, ethanol, acetonitrile, or acetone, with methanol being taken as an example.

[0024] The terminal-fluorinated cationic polymer prepared by the present invention has a molecular weight of 10,000-30,000.

[0025] The cationic polymer prepared by the present invention has a very narrow molecular weight distribution (PDI) and is particularly suitable for the highly repeatable production of medical raw materials.

[0026] The present invention also provides the use of the cationic polymer as a gene delivery vector.

[0027] Specifically, the application is to use the cationic polymer as a gene delivery vector and incubate it with nucleic acid to obtain a nucleic acid complex, wherein the nucleic acid is DNA, RNA or plasmid.

[0028] More specifically, the mass ratio of the cationic polymer to the nucleic acid is preferably (1-10):1; specifically, it can be 10 / 1, 5 / 1, 2.5 / 1 or 1 / 1; or any value between the above two.

[0029] The cationic polymer of the present invention is prepared in water with a concentration of 0.5-1 mg / mL.

[0030] The nucleic acid diluent of the present invention is DMEM, and the concentration of the nucleic acid is 0.05-0.1 mg / mL.

[0031] The incubation temperature of the present invention is 20-30° C., and the incubation time is preferably 15-20 minutes.

[0032] The present invention also provides a gene transfection method, wherein the nucleic acid complex described in the above technical solution is mixed with cells and then incubated, wherein the cells are animal cells, wherein the gene transfection incubation temperature is 37° C. and the incubation time is preferably 24 to 48 hours.

[0033] The present invention does not limit the culture medium, and any conventional culture medium well known to those skilled in the art can be used.

[0034] The terminal-fluorinated cationic polymer gene carrier provided by the present invention uses a perfluoro or polyfluoroalkyl compound as an initiator to be grafted onto the end of a 2-ethyl-2-oxazoline polymer to obtain a terminal-fluorinated poly-2-ethyl-2-oxazoline intermediate, which is then hydrolyzed to obtain a terminal-fluorinated cationic polymer having the structural characteristics of linear polyethyleneimine.

[0035] The present invention introduces a fluorinated group in a one-step method without introducing other reagents (such as an activator or a condensing agent), thereby reducing the complexity of the synthesis, simplifying the reaction, making the raw materials readily available, reducing the preparation cost, making the molecular weight easily controllable, and increasing the yield. The post-processing is simple and the preparation is suitable for large-scale production.

[0036] The present invention provides a fluorinated end-group cationic polymer nucleic acid carrier. Fluorinated groups are introduced into the end groups of linear polyethyleneimine (LPEI), compressing nucleic acid substances to form complex particles. This makes it a high-performance transfection reagent with low toxicity and high efficiency. This gene carrier can achieve efficient transfection during cell transfection, with minimal toxicity to cells. It can effectively and safely deliver gene molecules into cells, making it a gene transfection vector with the advantages of high efficiency, low toxicity, low cost, and simple synthesis.

[0037] The preparation process of the present invention is simple and highly reproducible. The prepared cationic polymer gene carrier with fluorinated terminal groups can compress nucleic acid substances to form complex particles, reduce the cytotoxicity of the complex, and has high transfection efficiency, which is of great significance for the construction of high-efficiency and low-toxic cationic polymer nucleic acid carriers. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a synthetic route for the terminal fluorinated cationic polymer gene carrier of the present invention.

[0039] Figure 2 This is a one-dimensional H NMR spectrum of the end-group fluorinated cationic polymer gene carrier prepared in the embodiment of the present invention ( 1 1H NMR).

[0040] Figure 3 This is a graph showing the particle size characterization results of the end-group fluorinated cationic polymer gene carrier prepared in an example of the present invention.

[0041] Figure 4 This is a graph showing the zeta potential characterization results of the end-group fluorinated cationic polymer gene carrier prepared in an example of the present invention.

[0042] Figure 5 This is a graph showing the agarose gel electrophoresis results of the terminal fluorinated cationic polymer gene carrier prepared in an example of the present invention.

[0043] Figure 6 This is a 24h cytotoxicity graph of the terminal fluorinated cationic polymer gene carrier prepared in the present invention.

[0044] Figure 7 This is a 48h cytotoxicity graph of the terminal fluorinated cationic polymer gene carrier prepared in the examples of the present invention.

[0045] Figure 8This is a fluorescence image of DNA transfection of a cationic polymer gene vector with fluorinated terminal groups prepared in an example of the present invention in the presence of serum; PEI without fluorinated terminal groups was used as a negative control.

[0046] Figure 9 The figure is a flow cytometric graph of DNA transfection of the terminal fluorinated cationic polymer gene vector prepared in the example of the present invention in the presence of serum; PEI without terminal fluorine is a negative control, and the cells used are B16F10 cells.

[0047] Figure 10 This is a graph showing the DNA transfection efficiency of the terminal fluorinated cationic polymer gene carrier prepared in an example of the present invention in the presence of serum.

[0048] Figure 11 Flow cytometry plot of 30 kDa LPEI transfected with post-fluorinated 30 kDa-5% 9F DNA.

[0049] Figure 12 Figure 2 shows the DNA transfection efficiency of 30 kDa LPEI and post-fluorinated 30 kDa-5% 9F. DETAILED DESCRIPTION

[0050] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0051] The synthetic routes in the following specific embodiments of the present invention are as follows:

[0052]

[0053] The synthesis method comprises the following steps:

[0054] (1) Dry 2-ethyl-2-oxazoline with anhydrous magnesium sulfate or calcium hydride for more than 4 hours to obtain dehydrated 2-ethyl-2-oxazoline. Dissolve the dehydrated 2-ethyl-2-oxazoline and a fluorine-containing compound (molar ratio of 1:0.1-1.3 fluoride ion) in a solvent and react in a sealed container at a reaction temperature of 100°C for 24 to 96 hours. After the reaction is completed, dissolve the mixture in dichloromethane and transfer it to a reaction bottle. Use a rotary evaporator at 30°C to 70°C to remove the residual solvent to obtain terminal fluorinated poly-2-ethyl-2-oxazoline.

[0055] (2) Reacting terminal-fluorinated poly-2-ethyl-2-oxazoline with 6 mol / L hydrochloric acid at 100°C for 10 hours. Cooling the methanol in an ice-water bath for at least 15 minutes. After the reaction, the product is precipitated in methanol, washed three times, and dried in a vacuum oven until the product is a white powder, thereby obtaining a terminal-fluorinated cationic polymer.

[0056] Example 1

[0057] The present invention provides a terminal fluorinated cationic polymer gene carrier having a structure shown in Formula IV and labeled as 17F10:

[0058]

[0059] A terminal fluorinated cationic polymer gene carrier is prepared by the following method, which specifically includes the following steps:

[0060] (1) 10 mL of anhydrous 2-ethyl-2-oxazoline and 3.97 g of 1-iodo-1H,1H,2H,2H-perfluorodecane (molar ratio 0.11 mol:0.0069 mol) were dissolved in 10 mL of anhydrous chlorobenzene and reacted at 100°C for 24 h. After the reaction, the mixture was dissolved in dichloromethane and transferred to a reaction flask and the residual solvent was removed by rotary evaporation to obtain terminal fluorinated poly-2-ethyl-2-oxazoline. The intermediate product was then reacted with 6 mol / L hydrochloric acid at 100°C for 10 h. After the reaction, the product was precipitated in methanol, washed three times, and dried in a vacuum oven until the product was a white powder. The product was reacted in a sealed container at 100°C for 24 h. After the reaction, the mixture was dissolved in dichloromethane and the mixture was transferred to a reaction flask and the residual solvent was removed by rotary evaporation to obtain terminal fluorinated poly-2-ethyl-2-oxazoline.

[0061] (2) A 50 g / L solution of terminally fluorinated poly (2-ethyl-2-oxazoline) was prepared with 6 mol / L hydrochloric acid and reacted at 100°C for 10 h. After the reaction, the product was precipitated in methanol, washed three times, and dried in a vacuum oven until it became a white powder. The yield of the terminally fluorinated cationic polymer was 92%.

[0062] Example 2

[0063]

[0064] The difference from Example 1 is that during the preparation of the terminal fluorinated poly 2-ethyl-2-oxazoline, 10 mL of anhydrous 2-ethyl-2-oxazoline and 1.98 g of 1-iodo-1H,1H,2H,2H-perfluorodecane (molar ratio 0.11 mol:0.0034 mol) were dissolved in 10 mL of anhydrous chlorobenzene, and a terminal fluorinated cationic polymer gene carrier having the structure of Formula V was prepared by the same steps as in Example 1. The segment length of the polyethyleneimine was changed from 10 to 20, i.e., p = 20, marked as 17F20, and the yield was 95%.

[0065] Example 3

[0066]

[0067] The difference from Example 1 is that during the preparation of the terminal fluorinated poly 2-ethyl-2-oxazoline, 10 mL of anhydrous 2-ethyl-2-oxazoline and 0.79 g of 1-iodo-1H,1H,2H,2H-perfluorodecane (molar ratio 0.11 mol:0.0013 mol) were dissolved in 10 mL of anhydrous chlorobenzene, and a terminal fluorinated cationic polymer gene carrier having the structure of Formula VI was prepared by the same steps as in Example 1. The segment length of the polyethyleneimine was changed from 10 to 50, i.e., p = 50, marked as 17F50, and the yield was 92%.

[0068] Example 4

[0069]

[0070] The difference from Example 1 is that during the preparation of the terminal fluorinated poly 2-ethyl-2-oxazoline, 10 mL of anhydrous 2-ethyl-2-oxazoline and 0.40 g of 1-iodo-1H,1H,2H,2H-perfluorodecane (molar ratio 0.11 mol:0.00069 mol) were dissolved in 10 mL of anhydrous chlorobenzene, and a terminal fluorinated cationic polymer gene carrier having the structure of Formula VII was prepared by the same steps as in Example 1. The segment length of the polyethyleneimine was changed from 10 to 100, i.e., p = 100, marked as 17F100, and the yield was 93%.

[0071] Example 5

[0072]

[0073] The difference from Example 1 is that during the preparation process, the fluorinated compound was changed to 1,1,2,2-tetrahydroperfluorohexyl iodide. 10 mL of anhydrous 2-ethyl-2-oxazoline and 6.10 g of 1,1,2,2-tetrahydroperfluorohexyl iodide (molar ratio 0.11 mol:0.016 mol) were dissolved in 10 mL of anhydrous chlorobenzene. Following the same steps as in Example 1, a terminally fluorinated cationic polymer gene vector having the structure of Formula VIII, labeled 9F10, was prepared with a yield of 96%.

[0074] Example 6

[0075]

[0076]

[0077] The difference from Example 5 is that during the preparation of the terminal fluorinated poly 2-ethyl-2-oxazoline, 10 mL of anhydrous 2-ethyl-2-oxazoline and 3.00 g of 1,1,2,2-tetrahydroperfluorohexyl iodide (molar ratio 0.11 mol:0.0080 mol) were dissolved in 10 mL of anhydrous chlorobenzene, and the terminal fluorinated cationic polymer gene carrier having the structure of Formula IX was prepared by the same steps as in Example 1. The segment length of the polyethyleneimine was changed from 10 to 20, i.e., p = 20, marked as 9F20, and the yield was 93%.

[0078] Example 7

[0079]

[0080] The difference from Example 5 is that during the preparation of the terminal fluorinated poly 2-ethyl-2-oxazoline, 10 mL of anhydrous 2-ethyl-2-oxazoline and 1.21 g of 1,1,2,2-tetrahydroperfluorohexyl iodide (molar ratio 0.11 mol:0.0032 mol) were dissolved in 10 mL of anhydrous chlorobenzene, and the terminal fluorinated cationic polymer gene carrier having the structure of Formula X was prepared by the same steps as in Example 1. The segment length of the polyethyleneimine was changed from 10 to 50, i.e., p=50, marked as 9F50, and the yield was 93%.

[0081] Example 8

[0082]

[0083] The difference from Example 5 is that during the preparation of the terminal fluorinated poly 2-ethyl-2-oxazoline, 10 mL of anhydrous 2-ethyl-2-oxazoline and 0.61 g of 1,1,2,2-tetrahydroperfluorohexyl iodide (molar ratio 0.11 mol:0.0016 mol) were dissolved in 10 mL of anhydrous chlorobenzene, and the terminal fluorinated cationic polymer gene carrier having the structure of formula XI was prepared by the same steps as in Example 1. The segment length of the polyethyleneimine was changed from 10 to 100, i.e., p = 100, marked as 9F100, and the yield was 95%.

[0084] Example 9

[0085]

[0086] The cationic polymer gene carrier LPEI is prepared by the following method, which specifically includes the following steps:

[0087] 10 mL of anhydrous 2-ethyl-2-oxazoline and 3.9 g of methyl p-toluenesulfonate were dissolved in 10 mL of anhydrous acetonitrile (molar ratio 0.11 mol:0.010 mol). The reaction was carried out at 80°C for 24 h. The intermediate product was dissolved in dichloromethane, and the solvent was removed by rotary evaporation. 6 mol / L hydrochloric acid was added and hydrolyzed at 100°C for 8 h. The final product was precipitated in ice-cold methanol, filtered, and dried to obtain a cationic polymer having the structure shown in Formula XII, labeled PEI10, with a yield of 94%.

[0088] Example 10

[0089]

[0090] The cationic polymer gene carrier LPEI is prepared by the following method, which specifically includes the following steps:

[0091] 10 mL of anhydrous 2-ethyl-2-oxazoline and 1.96 g of methyl p-toluenesulfonate (molar ratio 0.11 mol:0.0053 mol) were dissolved in 10 mL of anhydrous acetonitrile and reacted at 80°C for 24 h. The intermediate product was dissolved in dichloromethane, and the solvent was removed by rotary evaporation. 6 mol / L hydrochloric acid was added and hydrolyzed at 100°C for 8 h. The final product was precipitated in ice-cold methanol, filtered, and dried to obtain a cationic polymer having the structure of Formula XIII, labeled PEI20, with a yield of 96%.

[0092] Example 11

[0093]

[0094] The cationic polymer gene carrier LPEI was prepared by the following method, which specifically includes the following steps:

[0095] 10 mL of anhydrous 2-ethyl-2-oxazoline and 0.79 g of methyl p-toluenesulfonate (molar ratio 0.11 mol:0.0021 mol) were dissolved in 10 mL of anhydrous acetonitrile and reacted at 80°C for 24 h. The intermediate product was dissolved in dichloromethane, and the solvent was removed by rotary evaporation. 6 mol / L hydrochloric acid was added and hydrolyzed at 100°C for 8 h. The final product was precipitated in ice-cold methanol, filtered, and dried to obtain a cationic polymer having the structure shown in Formula XIV, labeled PEI50, with a final yield of 94%.

[0096] Example 12

[0097]

[0098] The cationic polymer gene carrier LPEI is prepared by the following method, which specifically includes the following steps:

[0099] 10 mL of anhydrous 2-ethyl-2-oxazoline and 0.39 g of methyl p-toluenesulfonate (molar ratio 0.11 mol:0.0010 mol) were dissolved in 10 mL of anhydrous acetonitrile and reacted at 80°C for 24 h. The intermediate product was dissolved in dichloromethane, and the solvent was removed by rotary evaporation. 6 mol / L hydrochloric acid was added and hydrolyzed at 100°C for 8 h. The final product was precipitated in ice-cold methanol, filtered, and dried to obtain a cationic polymer having the structure of Formula XV, labeled PEI100, with a yield of 93%.

[0100] Example 13

[0101] The particle size and potential characterization of the complexed cationic polymer with terminal fluorination and plasmid are illustrated using the green fluorescent protein (GFP) plasmid as an example.

[0102] Prepare a 0.05 mg / mL aqueous solution of green fluorescent protein (GFP) plasmid and a 1 mg / mL aqueous solution of cationic polymer. Take 1.3 mL of the GFP plasmid solution and 2.89 mL of the cationic polymer solution, vortex and let stand at room temperature for 15 minutes. The particle size and potential of the vector / DNA complex are then characterized.

[0103] The particle size of the terminal fluorinated cationic polymer gene carriers prepared in Examples 1-12 was measured using a particle size analyzer. Figure 3 The figure shows the particle size characterization results of the end-group fluorinated cationic polymer gene carrier. Figure 3 It can be seen that the particle size of the end-fluorinated cationic polymer gene carriers is between 200-300 nm, which can be internalized by cells and meets the conditions for cell transfection.

[0104] The potential of the terminal fluorinated cationic polymer gene carriers prepared in Examples 1-12 was measured using a particle size analyzer. Figure 4The figure shows the potential characterization results of the cationic polymer gene carrier with fluorinated end groups. Figure 4 It can be seen that the potential of the end-fluorinated cationic polymer gene carriers is positive, which can bind to the cell surface with a negative potential and enter the cell interior, meeting the conditions for cell transfection.

[0105] Example 14

[0106] Agarose gel electrophoresis of a fluorinated cationic polymer complexed with a plasmid, using a green fluorescent protein (GFP) plasmid as an example.

[0107] Prepare a 40ng / μL aqueous solution of GFP plasmid and various concentrations of cationic polymer solutions. Vortex and mix 5μL of each GFP plasmid solution and 5μL of the cationic polymer solution. After standing at room temperature for 15 minutes, add 2μL of 6× DNA Loading Buffer, mix thoroughly, and then apply to the wells of a prepared 1% agarose gel to test the cationic polymer's ability to bind to the plasmid. Figure 5 The agarose gel electrophoresis results of the terminal fluorinated cationic polymer gene carrier prepared in Example 1-12 are shown. Figure 5 It can be seen that when the nitrogen-phosphorus ratio N / P>4, the end-fluorinated cationic polymer can completely complex the plasmid, which is specifically manifested as the complex remaining in the gel pores, while the non-fluorinated polyethyleneimine cannot completely complex with the plasmid when N / P=15.

[0108] Example 15

[0109] Cytotoxicity of end-fluorinated cationic polymers.

[0110] Taking B16F10 cells as an example, the cells were cultured with 10% fetal bovine serum culture medium and placed in a constant temperature incubator at 37°C and 5% CO2. 24 hours before transfection, cells in logarithmic growth phase were obtained, trypsinized, and diluted with 10% fetal bovine serum culture medium. The cells were then diluted to 0.5×10 4 Cells were plated at a density of 100 cells / well in a 96-well cell culture plate and cultured in a constant temperature incubator at 37°C and a CO2 volume fraction of 5% until the cell confluence reached 80% to 90%. Different concentrations of cationic polymers were co-cultured with cells for 24 or 48 hours, and then 10 μL of CCK-8 solution (mass fraction 0.5%) was added to each well. The cells were cultured at 37°C for another 4 hours. The absorbance of each well of the culture plate was then measured using a microplate reader at a wavelength of 450 nm. The cell viability was calculated according to formula (1):

[0111] Cell survival rate (%) = (A sample / A control )×100(1)

[0112] A sample is the absorption of the transfected cell sample well, A control It is the absorption of sample wells in the group without adding materials. Each group of materials contains 3 replicate wells.

[0113] The end-group fluorinated cationic polymer was prepared into solutions of 640 mg / mL, 320 mg / mL, 160 mg / mL, 80 mg / mL, 40 mg / mL, 20 mg / mL, 10 mg / mL, and 5 mg / mL using culture medium and added to a 96-well plate. The cytotoxicity was detected after 24 h and 48 h, respectively. Figure 6 , Figure 7 The cytotoxicity of 24h and 48h respectively was determined by Figure 6 , Figure 7 It can be seen that the terminal fluorinated cationic polymer has almost no toxicity at a concentration of less than 40 mg / mL.

[0114] Example 16

[0115] Transfection efficiency of terminally fluorinated cationic polymers using green fluorescent protein (GFP) plasmids.

[0116] B16F10 cells in the logarithmic phase were selected and cultured in a constant temperature incubator until the cell confluence reached 80-90%.

[0117] Prepare a 0.05 mg / mL green fluorescent protein plasmid (GFP) aqueous solution, take 1.3 μL and add 2.89 uL of 1 mg / mL terminal fluorinated cationic polymer at a mass ratio of N / P = 15 m(plasmid): m(FPEI) = 1:3.61. After mixing and vortexing, let it stand at room temperature for 15 minutes, add 0.8 μg pDNA per well to a 24-well plate and incubate in a constant temperature incubator for 24 hours for transfection.

[0118] Remove the cells from the incubator 24 hours after transfection, aspirate the culture medium from the plate, add 500 μL of PBS to each well, and observe the expressed green fluorescent protein signal under a fluorescence microscope. Transfected positive cells will produce green fluorescence, while untransfected negative cells will not produce green fluorescence. Aspirate the solution from the plate, add 100 μL of trypsin to each well, and place the plate in the incubator for digestion for 2 minutes. Add 200 μL of complete culture medium containing serum, and completely collect the cells into a 2mL disposable sterile plastic centrifuge tube. After centrifugation, discard the supernatant and resuspend in 500 μL of PBS. Determine the percentage of transfected positive cells using flow cytometry to determine transfection efficiency.

[0119] Figure 8 、 Figure 9 、 Figure 10The results of the GFP (green fluorescent protein) DNA transfection efficiency of the terminal fluorinated cationic polymer gene carrier prepared in Examples 1-12 in the presence of serum are shown in the figure; PEI without terminal fluorinated groups is used as a negative control, and PEI 10k and PEI 25k are used as positive controls. Figure 8 、 Figure 9 、 Figure 10 It can be seen that the transfection efficiency of cationic polymers with fluorinated terminal groups is greatly improved.

[0120] Example 17

[0121] 0.5 g of LPEI with a molecular weight of 30 kDa and 0.159 g of 1,1,2,2-tetrahydroperfluorohexyl iodide (molar ratio 0.000017 mol: 0.00043 mol) were dissolved in 10 mL of ethanol and reacted at 70 °C for 24 h. After the reaction was completed, the ethanol was spin-dried and dissolved in 5 mL of deionized water. 0.5 mL of concentrated hydrochloric acid was added to acidify the product and precipitated in ice-cold acetone. Filtered and dried to obtain the product 30 kDa-5% 9F.

[0122] 30kDa LPEI and 30kDa-5% 9F were used for gene transfection. Figure 11 , 12, it can be seen that although the transfection efficiency is improved after fluorination, it is still lower than the end-fluorinated LPEI prepared by the present invention.

[0123] The present invention is not limited to the above-described embodiments, which does not necessarily mean that the present invention must rely on the above-described embodiments to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacements for the raw materials used in the present invention, additions of auxiliary components, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

Claims

1. A cationic polymer used as a gene delivery vector, characterized in that: The preparation method of the cationic polymer comprises the following steps: (1) dissolving a fluorine-containing compound having a structure represented by formula (II) and a 2-ethyl-2-oxazoline monomer in an anhydrous solvent to obtain a precursor solution; reacting the precursor solution in a sealed container at 60-120° C., cooling the reaction solution to room temperature, and then subjecting the reaction solution to rotary evaporation to obtain a terminal fluorinated poly-2-ethyl-2-oxazoline intermediate; (2) reacting the terminal fluorinated poly-2-ethyl-2-oxazoline intermediate with hydrochloric acid at 100° C.; settling the reaction solution, washing, drying, and pulverizing to obtain the cationic polymer; ， Formula (II) Wherein, X=I, Br or p-toluenesulfonate group, 0≤n≤7, 0≤m≤3.

2. The use according to claim 1, characterized in that The molar ratio of 2-ethyl-2-oxazoline in step (1) to fluoride ions in the fluorine-containing compound is 1:0.1-1.

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3. The use according to claim 1, characterized in that In the precursor solution, the concentration of 2-ethyl-2-oxazoline is 5-12 mol / L.

4. The use according to claim 1, characterized in that The solvent is any one of chlorobenzene, acetonitrile, dichlorobenzene and DMF.

5. The use according to claim 1, characterized in that The reaction time of step (1) is 24 to 96 h.

6. The use according to claim 1, characterized in that The reaction time of step (2) is 8-24 hours.

7. The use according to claim 1, characterized in that The mass ratio of the cationic polymer to the gene is 1-10:1.

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