Cationic polymer gene vector with fluorinated end group as well as preparation method and application of cationic polymer gene vector

Abstract

The invention relates to the technical field of biopolymer materials, in particular to a preparation method and application of a cationic polymer gene vector with a fluorinated end group. The invention provides a cationic polymer with a fluorinated end group. The structure of the cationic polymer is shown in the specification. According to the method, a fluorinated group is introduced through a fluorinated initiator by a one-step method to initiate synthesis of various terminal group fluorinated linear polyethyleneimine F-PEI with different molecular weights, the gene transfection efficiency is improved by introduction of the fluorine group, and high-efficiency DNA transfection is realized. The gene vector can achieve an efficient transfection effect in the cell transfection process, the toxicity generated to cells in the transfection process is small, gene molecules can be effectively and safely conveyed into the cells, and the gene transfection vector has the advantages of being efficient, low in toxicity, low in price, easy to synthesize and the like. The method has the advantages of simple reaction, easily available raw materials, low preparation cost, easily controlled molecular weight, high yield and simple post-treatment, and is suitable for large-scale production. # imgabs0 #

Description

Technical Field

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

[0002] Gene therapy is the most popular technology in modern times, involving the delivery of genetic material within cells for the treatment of various diseases, such as cancer, viral infection, and genetic diseases. Although gene therapy is the latest and most promising strategy to combat various diseases, its application is limited due to the lack of safe and effective vector systems. Viruses are the most widely studied delivery vectors for gene transfection, but because viral vectors are very likely to cause severe immune and inflammatory reactions and are not safe enough, their use is limited, which has promoted the in-depth study of non-viral gene transfection vectors. 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 to achieve efficient gene carrying and efficient cell transfection. Among the various non-viral vectors developed, cationic polymers have great potential and advantages over similar vectors due to their low toxicity, cost-effectiveness, easy production, and versatility for different applications. Polyethyleneimine (PEI) is a commonly used cationic gene transfection system in the prior art, which can be divided into linear and branched types, and there are different molecular weight products.

[0003] PEI is one of the cationic polymer carriers with high transfection efficiency. Branched polyethyleneimine (PEI 25k) with a molecular weight of 25,000 is often used as a gene vector evaluation standard to compare newly designed and newly developed gene vectors with it. The positively charged functional groups on the surface of PEI can bind and concentrate nucleic acids with negative charges on the surface through ionic action, and form nano-sized complexes, thereby protecting nucleic acids from degradation by nucleases and facilitating their entry into cells. After entering the cell, the complex is mainly concentrated in the endosomes or lysosomes. Under the mechanism mediated by the "proton sponge" effect, the nucleic acid complex can escape from the endosomes or lysosomes and release nucleic acids. However, due to the chemical structure of PEI itself, it has a strong positive charge and is prone to cytotoxicity and cell death during gene transfection. Generally speaking, PEI below 2000kDa has no obvious cytotoxicity, but the transfection efficiency is also very low. The large molecular PEI has a higher transfection efficiency, but is accompanied by greater cytotoxicity. However, the small molecular linear polyethyleneimine has a very poor transfection effect, and the large molecular linear polyethyleneimine has a high toxicity. 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 group. The prepared preparatory solution, even if stored in a 4°C refrigerator, the side group is 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 the synthesis difficult.

[0004] Fluorinated compounds can give polymers excellent self-assembly properties, anti-pollution ability, chemical and biological inertness, low cytotoxicity, etc. It can greatly improve the transfection efficiency of polyethyleneimine, reduce the nitrogen-phosphorus ratio required for complete complexation with plasmid DNA during transfection of cells, and greatly improve the performance of polyethyleneimine in cell transfection. Side-fluorinated polyethyleneimine is post-modified on branched polyethyleneimine (BPEI). Due to the inaccurate molecular weight of BPEI, the molecular weight distribution (PDI) is very large, and the source of raw materials is unstable, which makes its industrial application difficult. 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 scheme has a large molecular weight and a large cytotoxicity. Chinese patent CN107556493A generates an amide bond by reacting the acyl chloride group of perfluorooctanoyl chloride with the primary amine of oligopolyethyleneimine (OEI) in a one-step reaction, and connects the perfluorooctane group to the branched amino group in the oligopolyethyleneimine structure, and also obtains a side-group fluorinated polyethyleneimine. In this technical solution, due to the hydrolysis of the amide bond, the toxicity of the polyethyleneimine product is significantly increased, and the transfection efficiency is sharply reduced. 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 a preparation method and application thereof. 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 above 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 in step (1) to fluoride ions in the fluorine-containing compound 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, the fluorine-containing compound and the 2-ethyl-2-oxazoline monomer are respectively 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 scheme, the step (1) of cooling the reaction solution to room temperature and then subjecting it to rotary evaporation is to dilute the reaction solution with a benign solvent such as dichloromethane, ethyl acetate or ethanol, and then subject it to 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, acetone, with methanol being taken as an example.

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

[0025] The cationic polymer prepared by the 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 point 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 time is preferably 15-20 min.

[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 incubation temperature for gene transfection is 37° C., and the 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 may 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 and a condensing agent), thereby reducing the complexity of the synthesis, having a simple reaction, readily available raw materials, low preparation cost, easy control of molecular weight, high yield, simple post-processing, and being suitable for large-scale production.

[0036] The end-group fluorinated cationic polymer nucleic acid carrier provided by the present invention introduces a fluorinated group on the end group of linear polyethyleneimine (LPEI), can compress nucleic acid substances to form complex particles, and has a low-toxic and high-performance transfection reagent. The gene carrier can achieve a high-efficiency transfection effect in the cell transfection process, the transfection process has less toxicity to cells, and can effectively and safely deliver gene molecules to cells. It is a gene transfection carrier with the advantages of high efficiency, low toxicity, low price, and simple synthesis.

[0037] The preparation process of the invention is simple and highly repeatable. The prepared terminal fluorinated cationic polymer gene vector can compress nucleic acid substances to form complex particles, reduce the cytotoxicity of the complex and have high transfection efficiency, which is of great significance for constructing high-efficiency and low-toxic cationic polymer nucleic acid vectors. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The invention discloses a synthetic route for the terminal fluorinated cationic polymer gene carrier.

[0039] Figure 2 The one-dimensional hydrogen nuclear magnetic resonance spectrum of the end-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-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-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 vector prepared in the examples 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 the terminal fluorinated cationic polymer gene vector prepared in the example of the present invention in the presence of serum; PEI without terminal fluorine was used as a negative control.

[0046] Fig. 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; the PEI without terminal fluorine is a negative control, and the cells used are B16F10 cells.

[0047] Fig.10 This is a graph showing the DNA transfection efficiency of the terminal fluorinated cationic polymer gene vector prepared in the examples of the present invention in the presence of serum.

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

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

[0050] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work 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 2-ethyl-2-oxazoline after dehydration. Dissolve the dehydrated 2-ethyl-2-oxazoline and the fluorine-containing compound (molar ratio of 1:0.1-1.3 fluoride ion) with a solvent and react in a closed container at a reaction temperature of 100°C for a reaction time of 24 to 96 hours. After the reaction is completed, dissolve it with 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) The terminal fluorinated poly-2-ethyl-2-oxazoline is reacted with 6 mol / L hydrochloric acid at 100° C. for 10 h. The methanol is cooled in an ice-water bath in advance, and the cooling time in the ice-water bath is not less than 15 min. After the reaction is completed, the product is precipitated in methanol, washed 3 times, and dried in a vacuum oven until the product is in the form of 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 marked as 17F10:

[0058]

[0059] The terminal fluorinated cationic polymer gene carrier is prepared by the following method, which specifically comprises 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 bottle 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 3 times, and dried in a vacuum oven until the product was in the form of 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 residual solvent was removed by rotary evaporation to obtain terminal fluorinated poly-2-ethyl-2-oxazoline.

[0061] (2) The terminal fluorinated poly-2-ethyl-2-oxazoline was prepared with 6 mol / L hydrochloric acid to prepare a 50 g / L solution; the reaction was carried out at 100° C. for 10 h. After the reaction was completed, the product was precipitated in methanol, washed 3 times, and dried in a vacuum oven until the product was in the form of a white powder, thereby obtaining a terminal fluorinated cationic polymer with a yield of 92%.

[0062] Example 2

[0063]

[0064] The difference from Example 1 is that in the preparation process of 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 a structure of formula V was prepared by the same steps as in Example 1, the segment length of 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 in the preparation process of 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 a structure of formula VI was prepared by the same steps as in Example 1, the segment length of 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, in the preparation process of 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 a structure of formula VII was prepared by the same steps as in Example 1, the segment length of 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 fluorine-containing compound is 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) are dissolved in 10 mL of anhydrous chlorobenzene, and a terminal fluorinated cationic polymer gene vector having a structure of formula VIII is prepared by the same steps as in Example 1, labeled as 9F10, with a yield of 96%.

[0074] Example 6

[0075]

[0076]

[0077] The difference from Example 5 is that, in the preparation process of 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 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 in the preparation process of 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 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 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 comprises 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), reacted at 80°C for 24 h, and then the intermediate product was dissolved in dichloromethane. After 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 a structure shown in formula XII, labeled as 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 comprises 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, reacted at 80°C for 24 h, and the intermediate product was dissolved in dichloromethane. After rotary evaporation to remove the solvent, 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 a structure shown in formula XIII, labeled as PEI20, with a yield of 96%.

[0092] Embodiment 11

[0093]

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

[0095] Dissolve 10 mL of anhydrous 2-ethyl-2-oxazoline and 0.79 g of methyl p-toluenesulfonate (molar ratio 0.11 mol:0.0021 mol) in 10 mL of anhydrous acetonitrile, react at 80°C for 24 h, dissolve the intermediate product in dichloromethane, remove the solvent by rotary evaporation, add 6 mol / L hydrochloric acid and hydrolyze at 100°C for 8 h, precipitate the final product in ice-cold methanol, filter, and dry to obtain a cationic polymer having a 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 comprises 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, reacted at 80° C. for 24 h, and then the intermediate product was dissolved in dichloromethane. After 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 a structure shown in formula XV, labeled as PEI100, with a yield of 93%.

[0100] Example 13

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

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

[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-fluorinated cationic polymer gene carrier. Figure 3 It can be seen that the particle size of the terminal 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 terminal fluorinated cationic polymer gene carriers prepared in Examples 1-12 were subjected to potential measurement using a particle size analyzer. Figure 4This is the potential characterization result of the end-fluorinated cationic polymer gene carrier. 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, meeting the conditions for cell transfection.

[0105] Embodiment 14

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

[0107] Prepare 40ng / μL GFP plasmid aqueous solution and different concentrations of cationic polymer aqueous solution. Take 5μL of GFP plasmid aqueous solution and 5μL of cationic polymer aqueous solution, mix and vortex, let stand at room temperature for 15 minutes, add 2μL 6×DNA Loading Buffer, mix well and add to the wells of the prepared 1% agarose gel to test the complexing ability of cationic polymer to plasmid. Figure 5 The agarose gel electrophoresis results of the terminal fluorinated cationic polymer gene carrier prepared in Example 1-12. 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] Embodiment 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, digested with trypsin, and diluted with 10% fetal bovine serum culture medium at 0.5×10 4 The density of cells / well was plated 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 and cells were co-cultured for 24h or 48h, and then 10μL CCK-8 solution (mass fraction 0.5%) was added to each well and cultured for 4h at 37°C. The absorption of each well of the culture plate was then detected by an ELISA reader, and the detection wavelength was selected to be 450nm. The cell survival rate 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 material added. Each group of material contains 3 replicate wells.

[0113] The end-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, 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 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, mix and vortex, let stand at room temperature for 15 min, add 0.8 μg pDNA per well to a 24-well plate, and incubate in a constant temperature incubator for 24 h for transfection.

[0118] Take out the cells after transfection for 24 hours from the incubator, remove the culture medium in the well plate, add 500uL PBS to each well, and observe the green fluorescent protein signal under a fluorescence microscope. Transfected positive cells can produce green fluorescence, while untransfected negative cells do not produce green fluorescence. Aspirate the solution in the well plate, add 100μL of trypsin to each well, put it in the incubator for digestion for 2min, add 200μL of complete culture medium containing serum, collect the cells completely into a 2mL disposable sterile plastic centrifuge tube, discard the supernatant after centrifugation, add 500μL PBS to resuspend, and use flow cytometry to detect the percentage of transfected positive cells and determine the transfection efficiency.

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

[0120] Embodiment 17

[0121] 0.5 g of LPEI with a molecular weight of 30 k Da 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 it was 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. Fig.11 , 12, it can be seen that although the transfection efficiency is improved after fluorination, it is still lower than that of the end-fluorinated LPEI prepared by the present invention.

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

Claims

1. A cationic polymer, characterized in that It has the structure shown in formula (I): ， Formula (I) Among them, 0≤n≤7, 0≤m≤3, 10≤p≤500.

2. The cationic polymer according to claim 1, characterized in that 10≤p≤100。 3. The method for preparing a cationic polymer according to claim 1 or 2, characterized in that: The following steps are involved: (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 it 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, 0≤n≤7, 0≤m≤3.

4. The method for preparing a cationic polymer according to claim 3, characterized in that: 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.

5. The method for preparing a cationic polymer according to claim 3, characterized in that: In the precursor solution, the concentration of 2-ethyl-2-oxazoline is 5-12 mol / L.

6. The method for preparing a cationic polymer according to claim 3, characterized in that: The solvent is any one of chlorobenzene, acetonitrile, dichlorobenzene and DMF.

7. The method for preparing a cationic polymer according to claim 3, characterized in that: The reaction time of step (1) is 24 to 96 h.

8. The method for preparing a cationic polymer according to claim 3, characterized in that: The reaction time of step (2) is 8-24 hours.

9. Use of the cationic polymer according to claim 1 or 2 or the cationic polymer prepared by the method according to any one of claims 2 to 8 as a gene delivery vector.

10. The use according to claim 9, characterized in that: The mass ratio of the cationic polymer to the gene is 1-10:1.

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