Preparation method and application of gold / fluorinated PEI composite gene vector

Abstract

The invention provides a preparation method and application of a gold / fluorinated PEI composite gene vector. The gold / fluorinated PEI composite gene vector is gold nanoparticle modified sulfhydrylated fluorine-containing polyethyleneimine (F-LPEI-SH-Au for short), a fluorine group is modified at one end of linear polyethyleneimine, a sulfydryl group is modified at the other end of the linear polyethyleneimine, and gold nanoparticles are connected with the polyethyleneimine through the sulfydryl group; the gold nanoparticles are prepared by reducing chloroauric acid with citric acid. Gold nanoparticles are introduced into sulfhydrylated end group fluorinated linear polyethyleneimine, so that a gene delivery carrier with excellent transfection performance and small cytotoxicity is synthesized. The method is simple and convenient in synthesis process, has high repeatability, and is beneficial to construction of a high-efficiency and low-toxicity cationic polymer gene delivery carrier.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical polymer materials, and in particular relates to a preparation method and application of a gold / fluorinated PEI composite gene carrier. Background Art

[0002] Gene therapy is the transfer of nucleic acids to target cells and is considered an advanced treatment for other difficult or incurable diseases, such as cancer. Among them, cationic polymers have gradually become the most promising non-viral vectors for nucleic acid delivery because of their stability, simple synthesis, easy chemical modification and high transfection efficiency. Polyethyleneimine (PEI) is one of the most widely studied cationic polymers for transfection. PEI on the market is divided into linear polyethyleneimine (LPEI) and branched polyethyleneimine (BPEI) according to its structure. Branched polyethyleneimine 25k (BPEI 25k) has become the "gold standard" for transfection due to its efficient transfection ability. However, BPEI 25k has high cytotoxicity, which severely limits its clinical application. After discovering that BPEI is an effective transfection agent, it was found that LPEI obtained by hydrolysis of poly (2-ethyl-2-oxazoline) is a safer and more efficient transfection reagent.

[0003] Low molecular weight LPEI has almost no cytotoxicity, but has a low transfection efficiency; high molecular weight LPEI has a high transfection efficiency, but also has high cytotoxicity. In order to reduce the cytotoxicity of high molecular weight LPEI, people have tried to modify the structure and surface function of PEI.

[0004] Gold nanoparticles have the outstanding advantages of biological inertness, biocompatibility, low immunogenicity and simple synthesis. In addition, the controllable surface chemical properties make gold nanoparticles highly versatile because they are easy to functionalize, bind targeting ligands and construct complex conjugated nanostructures. Cationic capping (such as LPEI) is conducive to electrostatic interaction with DNA, thereby ensuring effective gene delivery into cells. Gold nanoparticles chemically functionalized with LPEI show better DNA binding ability, thereby achieving more efficient in vitro transfection. In addition, the special optical properties exhibited by gold nanoparticles can also be used to improve the efficiency of gene delivery under near-ultraviolet irradiation. Therefore, it is very appropriate to choose gold nanoparticles combined with LPEI for modification.

[0005] Most of the existing technologies are based on BPEI modified gold nanoparticles, involving direct complexation of BPEI with chloroauric acid or coating of BPEI with chloroauric acid. The molecular weight and molecular weight distribution (PDI) of BPEI are uncontrollable, BPEI-SH is a side chain modification, which is random and uncontrollable, and the process repeatability and batch stability are very low. Therefore, it is an urgent need to develop a cationic polymer gene carrier with high transfection efficiency and low cytotoxicity. Summary of the invention

[0006] The main purpose of the present invention is to provide a preparation method and application of a gold / fluorinated polyethyleneimine composite gene vector, so as to provide a cationic polymer gene vector with low cytotoxicity and high transfection efficiency, which can safely and efficiently perform cell transfection.

[0007] In the first aspect, the present invention provides a gold / fluorinated polyethyleneimine complex, i.e., a thiol-modified fluorinated polyethyleneimine modified with gold nanoparticles, referred to as F-LPEI-SH-Au, wherein a fluorine group is modified at one end of the linear polyethyleneimine, a thiol group is modified at the other end of the linear polyethyleneimine, and the gold nanoparticles are connected to the polyethyleneimine via the thiol group; the thiol-modified fluorinated polyethyleneimine has a structure as shown in Formula I; the gold nanoparticles are prepared by reducing chloroauric acid with citric acid.

[0008]

[0009] Among them, 0≤n≤7, 0≤m≤3, 10≤p≤500.

[0010] Preferably, n=3 or 7, m=1, 50≤p≤100.

[0011] The synthesis route of the thiol-containing fluorinated polyethyleneimine (F-LPEI-SH) as shown in Formula I is as follows:

[0012]

[0013] The preparation method of the compound of formula I comprises the following steps:

[0014] (1) Dissolve 2-ethyl-2-oxazoline and a perfluoroalkyl compound in anhydrous chlorobenzene, and react at 100° C. in a sealed container for 24 hours. After the reaction is completed, add dichloromethane to dissolve, and evaporate the mixture to obtain compound A.

[0015] (2) Potassium ethyl xanthate and acetonitrile were added in a molar ratio of potassium ethyl xanthate to 2-ethyl-2-oxazoline of 5 to 1:1, and the mixture was reacted at room temperature for 24 hours. After the reaction was completed, the acetonitrile was removed by rotary evaporation, and dichloromethane was added to dissolve the mixture. The mixture was washed with deionized water, and the organic phase was extracted. The mixture was dried over anhydrous magnesium sulfate, filtered, and rotary evaporated to obtain an intermediate compound B.

[0016] (3) Tetrahydrofuran is added at a mass ratio of 0.05 to 0.025:1 to compound B, and n-butylamine is added after dissolution, and the molar ratio of compound B to n-butylamine is 1 to 0.5:1; the reaction is carried out at room temperature for 12 hours. After the reaction is completed, rotary evaporation is performed to obtain a thiolated terminal fluorinated polyoxazoline compound C.

[0017] (4) Add 2-6 mol / L hydrochloric acid to make the concentration of compound C in the hydrochloric acid solution about 50 mg / mL, react at 100° C. for 10 h, and after the reaction is completed, precipitate in methanol precooled in an ice water bath for more than 15 min, wash, and dry in a vacuum oven to obtain a compound of formula I.

[0018] Furthermore, the raw materials or solvents used in step (1) are all dehydrated and do not contain water.

[0019] Furthermore, the perfluoroalkyl compound in step (1) is 1-iodo-1H,1H,2H,2H-perfluorodecane or 1,1,2,2-tetrahydroperfluorohexyl iodide.

[0020] Furthermore, the molar ratio of 2-ethyl-2-oxazoline to the fluoride ion in the perfluoroalkyl compound in step (1) is 1:0.1-0.3. When the molar ratio of 2-ethyl-2-oxazoline to the fluoride ion in the perfluoroalkyl compound is 1:0.1-0.15, the chain length p of the polyethyleneimine group in the synthesized compound of formula I is about 100; when the molar ratio of 2-ethyl-2-oxazoline to the fluoride ion in the perfluoroalkyl compound is 1:0.2-0.3, the chain length p of the polyethyleneimine group in the synthesized compound of formula I is about 50.

[0021] Furthermore, in step (2), acetonitrile is added to dissolve potassium ethyl xanthate, wherein the molar concentration of potassium ethyl xanthate in acetonitrile is 20-30 mol / L.

[0022] The method for preparing the gold nanoparticles of the present invention comprises the following steps:

[0023] After the trisodium citrate solution is boiled, a chloroauric acid solution is added, the reaction is carried out for 10 minutes, and the reaction is terminated in an ice water bath to obtain a gold nanoparticle solution, wherein the molar ratio of the chloroauric acid to the trisodium citrate is 1:4, and the concentration of the chloroauric acid in the reaction system is 0.25 mol / L; and the particle size of the gold nanoparticles is about 5 nm.

[0024] The 5 nm gold nanoparticle solution prepared by the above method is a gold seed solution, and the particle size of the gold nanoparticles is regulated by adjusting the ratio of trisodium citrate, chloroauric acid, gold seed solution, and hydroquinone in the reaction system. In an exemplary embodiment of the present invention, a 50 nm gold nanoparticle solution and a 100 nm gold nanoparticle solution were prepared by the following preparation methods.

[0025] Preparation of 50 nm gold nanoparticle solution: Add 0.887 mL of 15 mmoL / L trisodium citrate solution, 0.887 mL of 25 mmoL / L chloroauric acid solution, 11.29 mL of 5 nm gold nanoparticle solution and 0.887 mL of 25 mmoL / L hydroquinone solution to 86.05 mL of ultrapure water, stir at room temperature overnight to obtain a 50 nm gold nanoparticle solution.

[0026] Preparation of 100 nm gold nanoparticle solution: Add 0.986 mL of 15 mmoL / L trisodium citrate solution, 0.986 mL of 25 mmoL / L chloroauric acid solution, 1.14 mL of 5 nm gold nanoparticle solution and 0.967 mL of 25 mmoL / L hydroquinone solution to 95.63 mL of ultrapure water and stir at room temperature overnight to obtain a 100 nm gold nanoparticle solution.

[0027] In a second aspect, the present invention provides a method for preparing F-LPEI-SH-Au, comprising the following specific steps: mixing an F-LPEI-SH aqueous solution with a gold nanoparticle solution for reaction, and obtaining a F-LPEI-SH-Au cationic polymer gene carrier after the reaction is completed.

[0028] Preferably, the concentration of the F-LPEI-SH is 0.1 mg / mL.

[0029] Preferably, the concentration of the gold nanoparticle solution is 0.05 mg / mL.

[0030] Preferably, the mass ratio of the F-LPEI-SH to the gold nanoparticles is 1 to 5:1.

[0031] Preferably, the molecular weight of the F-LPEI-SH is 500-10000.

[0032] Preferably, the particle size of the gold nanoparticles is 5-100 nm.

[0033] The particle size of the F-LPEI-SH-Au cationic polymer prepared by the above method is 80-450 nm, and the potential is 0-40 mV.

[0034] In a third aspect, the present invention provides the application of F-LPEI-SH or F-LPEI-SH-Au in gene delivery. F-LPEI-SH-Au can also be applied to the field of cell imaging. The present invention blends F-LPEI-SH with gold nanoparticles having photothermal effect and biological inertness to prepare F-LPEI-SH-Au cationic polymers. The introduced gold nanoparticles can be applied to the field of cell imaging because of their own photothermal effect, and can significantly improve the transfection effect of cells, and can be applied to the field of gene delivery. In addition, due to the biological inertness of gold nanoparticles, they have low cytotoxicity, thereby preparing safe and efficient F-LPEI-SH-Au cationic polymer gene carriers.

[0035] In some exemplary specific embodiments of the present invention, the effects of F-LPEI-SH and F-LPEI-SH-Au on gene transfection efficiency were investigated. The results showed that both F-LPEI-SH and F-LPEI-SH-Au provided by the present invention had excellent gene transfection effects.

[0036] The present invention provides a cationic polymer gene carrier formed by the co-blending reaction of F-LPEI-SH and gold nanoparticles. The co-blending gene carrier has the advantages of both F-LPEI-SH and gold nanoparticles, and utilizes the advantage of high transfection efficiency of FPEI as a cationic polymer gene delivery carrier; and utilizes the photothermal effect of gold nanoparticles to promote cell transfection. The biological inertness of the gold nanoparticles makes it biocompatible and weakly cytotoxic, and the cationic polymer gene carrier is safe and efficient.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The invention uses a perfluoroalkyl compound as a fluorination initiator, introduces S into potassium ethyl xanthate, and synthesizes F-LPEI-SH for the first time, wherein the fluorinated group and the thiol group are respectively modified at both ends of LPEI.

[0039] It is based on low molecular weight F-LPEI-SH, with a clear chemical structure, uniform and controllable molecular weight, and fluorinated groups and thiol groups modified at both ends of LPEI, which has a high degree of controllability, high process repeatability and batch stability.

[0040] The F-LPEI-SH prepared by the present invention can be used directly after being modified with gold nanoparticles without centrifugal operation, which greatly reduces the cost and process difficulty, can be prepared on a large scale, and is convenient for quantitative control.

[0041] The gold nanoparticles in the present invention are prepared by reducing chloroauric acid with sodium citrate, have no cytotoxicity, do not require centrifugation, can be directly compounded with PEI-SH, and the compound does not require post-treatment and can be directly used in cells. The present invention uses trisodium citrate-modified gold nanoparticles, has high conversion rate, is simple and convenient to prepare, has low capital cost, low equipment cost, can be prepared in large quantities, and is suitable for commercial production.

[0042] The terminal fluorinated linear polyethyleneimine (F-LPEI) prepared in the present invention can give the polymer excellent self-assembly performance, anti-pollution ability and low cytotoxicity, etc.; gold nanoparticles have biocompatibility and weak cytotoxicity, and the combination of the two can prepare a cationic polymer gene vector with high transfection efficiency and low cytotoxicity. In addition, the raw materials of the gold / fluorinated PEI composite gene vector are easily available, the synthesis method is very simple, and the repeatability is high. It is a cationic polymer gene vector suitable for commercialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 TEM image of the gold nanoparticles used in Example 2 of the present invention (scale: 50 nm).

[0044] Figure 2 The product photos of gold nanoparticles and Au-FPEI-SH prepared in the present invention.

[0045] Figure 3 This is a particle size diagram of the Au-FPEI-SH cationic polymer gene carrier prepared in the present invention.

[0046] Figure 4 This is the potential diagram of the Au-FPEI-SH cationic polymer gene carrier prepared in the present invention.

[0047] Figure 5 The figure is an agarose gel electrophoresis diagram of the Au-FPEI-SH cationic polymer gene vector prepared by the present invention and the plasmid DNA after complexing into a complex.

[0048] Figure 6 This is a fluorescence diagram of GFP (green fluorescent protein) DNA transfection in the presence of serum using the Au-FPEI-SH cationic polymer gene vector prepared in the present invention (scale: 50 μm).

[0049] Figure 7 The figure is a flow chart of transfection of GFP (green fluorescent protein) DNA of the Au-FPEI-SH cationic polymer gene vector prepared by the present invention in the presence of serum.

[0050] Figure 8 This is a graph showing the transfection efficiency of GFP (green fluorescent protein) DNA of the Au-FPEI-SH cationic polymer gene vector prepared in the present invention in the presence of serum.

[0051] Fig. 9 The transfection efficiency of the FPEI prepared in Comparative Example 1 after direct reaction with aqueous chloroauric acid solution.

[0052] Fig.10 This is a graph showing the transfection efficiency of the CTAB-assisted chloroauric acid-modified Au-FPEI-SH cationic polymer gene vector prepared in Comparative Example 2. DETAILED DESCRIPTION

[0053] 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.

[0054] The present invention blends FPEI-SH with gold nanoparticles having photothermal effect and biological inertness, thereby preparing Au-FPEI-SH cationic polymer gene vectors. The introduced gold nanoparticles can significantly improve the transfection effect of cells due to their own photothermal effect. In addition, due to the biological inertness of the gold nanoparticles, they have low cytotoxicity, thereby preparing safe and efficient Au-FPEI-SH cationic polymer gene vectors.

[0055] The F-LPEI-SH-Au or Au-F-LPEI-SH or Au-FPEI-SH described in the following specific embodiments of the present invention all represent thiol-terminated fluorinated linear polyethyleneimine modified with gold nanoparticles, that is, the gold / fluorinated polyethyleneimine complex described in the present invention, which can also be called a gold / fluorinated polyethyleneimine composite gene carrier.

[0056] Example 1

[0057] The cationic polymer with fluorinated end groups (F-LPEI-SH) has a structural formula as shown in Formula I and a synthetic route as follows:

[0058]

[0059] Wherein, n=3 or 7, m=1, p=50 or 100, and X is iodine.

[0060] The fluorine-containing compound is 1-iodo-1H,1H,2H,2H-perfluorodecane or 1,1,2,2-tetrahydroperfluorohexyl iodide.

[0061] The preparation method of F-LPEI-SH comprises the following steps:

[0062] (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 10 mL of dehydrated 2-ethyl-2-oxazoline (0.11 mol) and different molar amounts of fluorine-containing compounds with 10 mL of anhydrous chlorobenzene in a sealed container and react at 100° C. for 24 hours. After the reaction is completed, dissolve it in dichloromethane and transfer it to a reaction bottle. Use a rotary evaporator at 30° C. to remove the residual solvent to obtain terminal fluorinated poly-2-ethyl-2-oxazoline (compound A).

[0063] (2) Compound A was reacted with potassium ethyl xanthate, the molar ratio of potassium ethyl xanthate to 2-ethyl-2-oxazoline was 5:1, and 20 mL of acetonitrile was added, and the reaction was carried out at room temperature for 24 hours. After the reaction was completed, the acetonitrile was evaporated, dissolved in dichloromethane, and transferred to a separatory funnel, and an equal volume of saturated sodium chloride solution was added. The organic phase was extracted with dichloromethane 5 times, dried over anhydrous magnesium sulfate, filtered, and evaporated to obtain the intermediate compound B.

[0064] (3) Compound B was dissolved in tetrahydrofuran and then n-butylamine was added, the mass ratio of compound B to tetrahydrofuran was 0.025:1; the molar ratio of compound B to n-butylamine was 0.5:1; the reaction was carried out at room temperature for 12 hours. After the reaction was completed, rotary evaporation was performed to obtain a thiolated terminal fluorinated polyoxazoline compound C.

[0065] (4) Add 6 mol / L hydrochloric acid to make the concentration of compound C in the hydrochloric acid solution about 50 mg / mL, react at 100° C. for 10 h, and after the reaction is completed, precipitate in methanol precooled in an ice water bath for more than 15 min, wash, and dry in a vacuum oven to obtain a product in the form of a white powder, i.e., the compound of formula I.

[0066] The polyethyleneimine chain length of compound A is controlled by adjusting the molar ratio of 2-ethyl-2-oxazoline to the fluorine-containing compound. When the molar ratio of 2-ethyl-2-oxazoline to the fluorine ion in the fluorine-containing compound is 1:0.2-0.3, p=50 in the synthesized compound A; when the molar ratio is 1:0.1-0.15, p=100 in the synthesized compound A.

[0067] The compound of formula I is synthesized according to the above preparation method, wherein

[0068] n = 3, m = 1, p = 50, labeled as 9F50-SH, yield 95%;

[0069] n = 3, m = 1, p = 100, labeled as 9F100-SH, yield 93%;

[0070] n = 7, m = 1, p = 50, labeled as 17F50-SH, yield 96%;

[0071] n=7, m=1, p=100, labeled as 17F100-SH, yield 93%.

[0072] Example 2

[0073] The method for preparing gold nanoparticles comprises the following steps:

[0074] (1) Preparation of 5 nm gold nanoparticle solution:

[0075] Add 1 mL of 0.1 mol / L trisodium citrate solution to 98.9 mL of ultrapure water, boil, then add 100 μL of 0.25 mol / L chloroauric acid solution, react for 10 min, and terminate in an ice water bath to obtain a 5 nm gold nanoparticle solution.

[0076] (2) Preparation of 50 nm gold nanoparticle solution:

[0077] Add 0.887 mL of 15 mmoL / L trisodium citrate solution, 0.887 mL of 25 mmoL / L chloroauric acid solution, 11.29 mL of 5 nm gold nanoparticle solution and 0.887 mL of 25 mmoL / L hydroquinone solution to 86.05 mL of ultrapure water and stir at room temperature overnight to obtain a 50 nm gold nanoparticle solution.

[0078] (3) Preparation of 100 nm gold nanoparticle solution:

[0079] Add 0.986 mL of 15 mmoL / L trisodium citrate solution, 0.986 mL of 25 mmoL / L chloroauric acid solution, 1.14 mL of 5 nm gold nanoparticle solution, and 0.967 mL of 25 mmoL / L hydroquinone solution to 95.63 mL of ultrapure water and stir at room temperature overnight to obtain a 100 nm gold nanoparticle solution.

[0080] TEM images of gold nanoparticles of different particle sizes prepared in this example are shown in FIG. Figure 1 As shown, sample photos see Figure 2 .

[0081] Example 3

[0082] The gold nanoparticle solution used in this example is a fresh solution prepared according to the method of Example 2, and water is added to adjust its concentration to 0.05 mg / mL.

[0083] To 500 μL of 0.1 mg / mL F-LPEI-SH aqueous solution, add 500 μL of 0.05 mg / mL 5 nm gold nanoparticle aqueous solution (Au 5) or 50 nm gold nanoparticle aqueous solution (Au 50) or 100 nm gold nanoparticle aqueous solution (Au100), and stir at room temperature overnight to obtain 1:1 Au-FPEI-SH.

[0084] F-LPEI-SH is 9F50-SH, that is, Au 5-9F50-SH, Au 50-9F50-SH, and Au100-9F50-SH are obtained in a 1:1 ratio.

[0085] F-LPEI-SH is 9F100-SH, and Au 5-9F100-SH, Au 50-9F100-SH, and Au100-9F100-SH were prepared in a 1:1 ratio.

[0086] F-LPEI-SH is 17F50-SH, that is, Au 5-17F50-SH, Au 50-17F50-SH, and Au 100-17F50-SH are obtained in a 1:1 ratio.

[0087] F-LPEI-SH is 17F100-SH, that is, Au 5-17F100-SH, Au 50-17F100-SH, and Au100-17F100-SH are obtained in a 1:1 ratio.

[0088] The product photo of Au-FPEI-SH prepared in this example is shown in Figure 2 .

[0089] Example 4

[0090] Add 500 μL of 0.05 mg / mL 5 nm gold nanoparticle aqueous solution (Au 5) or 50 nm gold nanoparticle aqueous solution (Au 50) or 100 nm gold nanoparticle aqueous solution (Au100) to 250 μL of 0.1 mg / mL F-LPEI-SH aqueous solution and stir at room temperature overnight to obtain 1:0.5 Au-FPEI-SH.

[0091] F-LPEI-SH is 9F50-SH, that is, Au 5-9F50-SH, Au 50-9F50-SH, and Au 100-9F50-SH are obtained in a ratio of 1:0.5.

[0092] F-LPEI-SH is 9F100-SH, and Au 5-9F100-SH, Au50-9F100-SH, and Au100-9F100-SH with a ratio of 1:0.5 were prepared.

[0093] F-LPEI-SH is 17F50-SH, that is, Au 5-17F50-SH, Au 50-17F50-SH, and Au 100-17F50-SH are obtained in a ratio of 1:0.5.

[0094] F-LPEI-SH is 17F100-SH, that is, Au 5-17F100-SH, Au 50-17F100-SH, and Au100-17F100-SH are obtained in a ratio of 1:0.5.

[0095] Example 5

[0096] Characterization of the particle size and potential of Au-FPEI-SH cationic polymer gene carrier.

[0097] 1 mL of Au-FPEI-SH cationic polymer gene carrier was used to characterize the particle size and potential using a particle size meter.

[0098] The particle size of the Au-FPEI-SH cationic polymer gene carrier prepared in the above example was measured using a particle size analyzer. Figure 3 The figure shows the particle size characterization results of Au-FPEI-SH cationic polymer gene carrier. Figure 3 It can be seen that the particle size of the Au-FPEI-SH cationic polymer gene carrier is between 80-450 nm, which can be internalized by cells and meets the conditions for cell transfection.

[0099] The Au-FPEI-SH cationic polymer gene carrier prepared in the above example was subjected to potential measurement using a particle size analyzer. Figure 4 The figure shows the potential characterization results of Au-FPEI-SH cationic polymer gene carrier. Figure 4 It can be seen that the potential of gold nanoparticles is negative, and the potential of Au-FPEI-SH cationic polymer gene carriers is positive, which can be internalized by cells and meets the conditions for cell transfection.

[0100] Example 6

[0101] Agarose gel electrophoresis of the complex of Au-FPEI-SH cationic polymer gene vector and plasmid, taking green fluorescent protein plasmid (GFP) as an example.

[0102] Prepare 20ng / μL GFP plasmid aqueous solution and different concentrations of Au-FPEI-SH cationic polymer aqueous solution. Take 5μL of GFP plasmid aqueous solution and 5μL of Au-FPEI-SH 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 Au-FPEI-SH cationic polymer to plasmid.

[0103] Figure 5 This is the agarose gel electrophoresis result of the 1:1 Au-FPEI-SH cationic polymer gene carrier prepared in Example 3. Figure 5 It can be seen that the Au-FPEI-SH cationic polymer can completely complex the plasmid, which is specifically manifested in that the complex remains in the gel pores.

[0104] Example 7

[0105] Transfection efficiency of Au-FPEI-SH cationic polymer using green fluorescent protein plasmid (GFP).

[0106] Taking B16F10 cells as an example, logarithmic phase B16F10 cells were selected, digested with trypsin for 2 minutes, neutralized with cell culture medium, centrifuged, washed twice with PBS, suspended in fresh cell culture medium, and counted with a cell counting plate under an inverted fluorescence microscope. After calculation, 2×10 4 The cells were plated at a density of 100 μg / mL in a 24-well plate and cultured in a constant temperature incubator at 37°C and 5% CO2 until the cell confluence reached 80-90%.

[0107] Prepare a 0.05 mg / mL green fluorescent protein (GFP) aqueous solution, take 1.3 μL and add Au-FPEI-SH cationic polymer at N / P=15, mix and vortex, let stand at room temperature for 15 minutes, add 0.8 μg pDNA per well to a 24-well plate, incubate in a constant temperature incubator for 24 hours for transfection.

[0108] 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. The transfected cells can produce green fluorescence, while the untransfected cells do not produce green fluorescence.

[0109] Aspirate the solution in the well plate, add 100 μL of trypsin to each well, place in the incubator for digestion for 2 minutes, add 200 μL of complete culture medium containing serum, collect the cells completely into a 2 mL disposable sterile plastic centrifuge tube, discard the supernatant after centrifugation, add 500 μL of PBS to resuspend, and use flow cytometer to detect the percentage of transfected positive cells to determine the transfection efficiency.

[0110] Figure 6 , Figure 7 , Figure 8 This is a graph showing the transfection efficiency of GFP (green fluorescent protein) DNA in B16F10 cells in the presence of serum using the Au-FPEI-SH cationic polymer gene vector prepared in the above example; FPEI-SH is used as a negative control, and BPEI 10k and BPEI 25k are used as positive controls. Figure 6 , Figure 7 , Figure 8It can be seen that the transfection efficiency of Au-FPEI-SH cationic polymer is greatly improved.

[0111] Comparative Example 1

[0112] Referring to the commonly used method of polyethyleneimine to modify gold nanoparticles, the terminal fluorinated polyethyleneimine was directly complexed with chloroauric acid.

[0113] Specific steps: Compound A is prepared according to the preparation method of 9F50-SH in Example 1, and compound A is directly hydrolyzed with hydrochloric acid without thiolation to obtain fluorinated polyethyleneimine, which is marked as 9F50. In 4.75mL of 50mg / L HAuCl4·3H2O aqueous solution, 0.25mL of 0.5mg / mL, 1mg / mL, 1.5mg / mL, 2mg / mL, 2.5mg / mL of 9F50 is added, and the reaction is carried out at 37℃ for 24h or 100℃ for 20min. The FPEI prepared in this example is reacted with chloroauric acid at different ratios at 37℃ for 24h or 100℃ for 20min. The transfection efficiency is as follows: Fig. 9 As shown, it can be seen that the transfection effect of FPEI modified by chloroauric acid treatment alone is low.

[0114] Comparative Example 2

[0115] Preparation of gold nanoparticles: 15mL 1mM HAuCl4·3H2O aqueous solution, add 2mL 10mM CTAB solution and stir at room temperature for 15min, the solution turns orange-yellow, then add 2mL 100mM NaBH4 solution, the solution turns orange-red, keep stirring at room temperature, observe that it does not change color within 15min, and the preparation of CTAB-modified gold nanoparticles is completed, marked as Au15. Referring to the method of Example 3, 1:1 Au 15-17F50-SH was prepared.

[0116] from Fig.10 It can be seen that Au 5 is a gold nanoparticle modified by sodium citrate, and Au 15 is a gold nanoparticle modified by CTAB. After the gold nanoparticles modified by sodium citrate are complexed with F-LPEI-SH, the transfection efficiency is significantly improved, while the transfection efficiency of F-LPEI-SH modified by CTAB gold nanoparticles is extremely low.

[0117] 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 gold / fluorinated polyethyleneimine composite, characterized in that The gold / fluorinated polyethyleneimine complex is a thiol-terminated fluorinated polyethyleneimine modified with gold nanoparticles, referred to as F-LPEI-SH-Au; the gold nanoparticles are connected to the polyethyleneimine via thiol groups; the gold nanoparticles are prepared by reducing chloroauric acid with citric acid; the thiol-terminated fluorinated polyethyleneimine has a structure as shown in Formula I: Among them, 0≤n≤7, 0≤m≤3, 10≤p≤500.

2. The gold / fluorinated polyethyleneimine composite according to claim 1, characterized in that The compound of formula I has n=3 or 7, m=1, and 50≤p≤100.

3. The gold / fluorinated polyethyleneimine composite according to claim 1, characterized in that: The preparation method of the compound of formula I comprises the following steps: (1) dissolving 2-ethyl-2-oxazoline and a perfluoroalkyl compound in anhydrous chlorobenzene, reacting at 100° C. in a sealed container for 24 hours, adding dichloromethane to dissolve after the reaction, and performing rotary evaporation to obtain compound A; (2) adding potassium ethyl xanthate and acetonitrile in a molar ratio of potassium ethyl xanthate to 2-ethyl-2-oxazoline of 5 to 1:1, reacting at room temperature for 24 hours, and after the reaction, removing the acetonitrile by rotary evaporation, adding dichloromethane to dissolve, washing with deionized water, extracting the organic phase, drying over anhydrous magnesium sulfate, filtering, and rotary evaporation to obtain an intermediate compound B; (3) Tetrahydrofuran is added at a mass ratio of 0.05 to 0.025:1, and after dissolving, n-butylamine is added, and the molar ratio of compound B to n-butylamine is 1 to 0.5:1; react at room temperature for 12 hours. After the reaction is completed, rotary evaporation is performed to obtain a thiolated terminal fluorinated polyoxazoline compound C; (4) Add 2-6 mol / L hydrochloric acid to make the concentration of compound C in the hydrochloric acid solution about 50 mg / mL, react at 100° C. for 10 h, and after the reaction is completed, precipitate in methanol precooled in an ice water bath for more than 15 min, wash, and dry in a vacuum oven to obtain a compound of formula I.

4. The gold / fluorinated polyethyleneimine composite according to claim 3, characterized in that: The molar ratio of 2-ethyl-2-oxazoline to fluoride ion in the perfluoroalkyl compound in step (1) is 1:0.1-0.3, and / or the perfluoroalkyl compound is 1-iodo-1H,1H,2H,2H-perfluorodecane or 1,1,2,2-tetrahydroperfluorohexyl iodide.

5. The gold / fluorinated polyethyleneimine composite according to claim 3, characterized in that: In step (2), the molar concentration of potassium ethyl xanthate in acetonitrile is 20-30 mol / L.

6. The gold / fluorinated polyethyleneimine composite according to claim 1, characterized in that: The particle size of the gold nanoparticles is 5-100 nm.

7. The gold / fluorinated polyethyleneimine composite according to claim 7, characterized in that The preparation method of the gold nanoparticles comprises the following steps: (1) boiling a trisodium citrate solution, adding a chloroauric acid solution, reacting for 10 minutes, and terminating the reaction in an ice water bath to obtain a gold nanoparticle solution, wherein the molar ratio of the chloroauric acid to the trisodium citrate is 1:4, and the concentration of the chloroauric acid in the reaction system is 0.25 mol / L; (2) The gold nanoparticle solution prepared in step (1) is used as a gold seed solution, and the particle size of the gold nanoparticles is regulated by adjusting the ratio of trisodium citrate, chloroauric acid, gold seed solution, and hydroquinone in the reaction system.

8. The method for preparing the gold / fluorinated polyethyleneimine composite according to any one of claims 1 to 7, characterized in that: The method comprises the following steps: mixing an aqueous solution of a compound of formula I with a gold nanoparticle solution for reaction, and obtaining the gold / fluorinated polyethyleneimine complex after the reaction is completed.

9. The method for preparing the gold / fluorinated polyethyleneimine composite according to claim 8, characterized in that: The mass ratio of the compound of formula I to the gold nanoparticles is 1 to 5:

1.

10. Use of the gold / fluorinated polyethyleneimine complex according to any one of claims 1 to 7 in gene delivery and cell imaging.

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