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

Abstract

The application provides a preparation method and application of a gold / fluorinated PEI composite gene carrier. The gold / fluorinated PEI composite gene carrier is a thiolated fluorine-containing polyethylene imine modified by gold nanoparticles, and is referred to as F-LPEI-SH-Au. Fluorine groups are modified on one end of the linear polyethylene imine, thiol groups are modified on the other end of the linear polyethylene imine, and the gold nanoparticles are connected with the polyethylene imine through the thiol groups. The gold nanoparticles are prepared by reducing chloroauric acid with citric acid. The gold nanoparticles are introduced into the thiolated end group fluorinated linear polyethylene imine to synthesize a gene delivery carrier which has superior transfection performance and small cytotoxicity. The synthesis process is simple, has high repeatability, and is helpful to construct a high-efficiency low-toxicity cationic polymer gene delivery carrier.

Description

Technical Field

[0001] The present 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, which involves the transfer of nucleic acids into target cells, is considered an advanced treatment option for otherwise difficult-to-treat or incurable diseases, such as cancer. Cationic polymers are emerging as the most promising non-viral vectors for nucleic acid delivery due to their stability, ease of synthesis, chemical modification, and high transfection efficiency. Polyethyleneimine (PEI) is one of the most widely studied cationic polymers for transfection. Commercially available PEIs are categorized by structure as linear polyethyleneimine (LPEI) and branched polyethyleneimine (BPEI). Branched polyethyleneimine 25k (BPEI 25k) has become the "gold standard" for transfection due to its high transfection efficiency. However, the high cytotoxicity of BPEI 25k has severely limited its clinical application. Following the discovery of BPEI as an effective transfection agent, LPEI, derived from the hydrolysis of poly(2-ethyl-2-oxazoline), was found to be an even safer and more efficient transfection reagent.

[0003] Low molecular weight LPEI has almost no cytotoxicity, but has low transfection efficiency; high molecular weight LPEI has 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 functionality of PEI.

[0004] Gold nanoparticles have the outstanding advantages of being bioinert, biocompatible, low immunogenicity and simple to synthesize. In addition, the controllable surface chemical properties make gold nanoparticles highly versatile, as they are easy to functionalize, bind targeting ligands and construct complex conjugated nanostructures. Cationic capping (such as LPEI) facilitates electrostatic interactions with DNA, thereby ensuring effective gene delivery into cells. Gold nanoparticles chemically functionalized with LPEI exhibit 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 gene delivery efficiency under near-ultraviolet irradiation. Therefore, the choice of gold nanoparticles combined with LPEI for modification is very appropriate.

[0005] Most existing technologies are based on BPEI-modified gold nanoparticles, involving direct complexation of BPEI with chloroauric acid or coating of BPEI with chloroauric acid. BPEI's molecular weight and molecular weight distribution (PDI) are uncontrollable, and BPEI-SH is a side-chain modification, which is random and uncontrollable, resulting in very low process reproducibility and batch stability. Therefore, the development of cationic polymer gene vectors with high transfection efficiency and low cytotoxicity is urgently needed. 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 carrier, so as to provide a cationic polymer gene carrier with low cytotoxicity and high transfection efficiency, which can safely and efficiently perform cell transfection.

[0007] In a first aspect, the present invention provides a gold / fluorinated polyethyleneimine complex, namely, 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 synthetic 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 the mixture, and evaporate the mixture to obtain compound A.

[0015] (2) Potassium ethyl xanthate and acetonitrile were added at 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 the intermediate compound B.

[0016] (3) Tetrahydrofuran was added at a mass ratio of 0.05 to 0.025:1, and after dissolving, n-butylamine was added at a molar ratio of 1 to 0.5:1. The reaction was allowed to proceed at room temperature for 12 hours. After the reaction was completed, rotary evaporation was performed to obtain a thiolated end-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 pre-cooled in an ice water bath for more than 15 min, wash, and dry in a vacuum oven to obtain the 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 gold nanoparticles of the present invention comprises the following steps:

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

[0024] The 5 nm gold nanoparticle solution prepared by the above method served as the gold seed solution. The gold nanoparticle size was controlled 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 using 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 and stir at room temperature overnight to obtain 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: blending an F-LPEI-SH aqueous solution with a gold nanoparticle solution to react, and obtaining an 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 to 10,000.

[0032] Preferably, the gold nanoparticles have a particle size of 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 a photothermal effect and biological inertness to prepare a F-LPEI-SH-Au cationic polymer. 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 the gold nanoparticles, they have low cytotoxicity, thereby preparing a safe and efficient F-LPEI-SH-Au cationic polymer gene carrier.

[0035] In some exemplary 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 proposes a cationic polymer gene carrier formed by the co-blending reaction of F-LPEI-SH and gold nanoparticles. This co-blended gene carrier has the advantages of both F-LPEI-SH and gold nanoparticles. It not only utilizes the high transfection efficiency of FPEI as a cationic polymer gene delivery carrier, but also utilizes the photothermal effect of gold nanoparticles to promote cell transfection. Its biological inertness also makes it biocompatible and weakly cytotoxic. Therefore, it is a safe and efficient cationic polymer gene carrier.

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

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

[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, 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 centrifugation 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 this invention are produced by reducing chloroauric acid with sodium citrate. They are non-cytotoxic, require no centrifugation, and can be directly compounded with PEI-SH. The complex requires no post-treatment and can be directly applied to cells. The gold nanoparticles modified with trisodium citrate in this invention have a high conversion rate, are simple and convenient to prepare, have low capital and equipment costs, can be prepared in large quantities, and are suitable for commercial production.

[0042] The terminally fluorinated linear polyethyleneimine (F-LPEI) prepared in this invention imparts excellent self-assembly properties, anti-fouling capabilities, and low cytotoxicity to the polymer. Combining these two with the biocompatibility and low cytotoxicity of gold nanoparticles enables the preparation of a cationic polymer gene vector with high transfection efficiency and low cytotoxicity. Furthermore, the gold / fluorinated PEI composite gene vector is readily available, the synthesis method is simple, and the design is highly reproducible, making it a commercially viable cationic polymer gene vector. 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 These are product photos of gold nanoparticles and Au-FPEI-SH prepared in the present invention.

[0045] Figure 3 This is a diagram showing the particle size 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 to form a complex.

[0048] Figure 6 This is a fluorescence image 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 This is a flow chart showing the transfection of GFP (green fluorescent protein) DNA using the Au-FPEI-SH cationic polymer gene vector prepared in 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 using the Au-FPEI-SH cationic polymer gene vector prepared in the present invention in the presence of serum.

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

[0052] Figure 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 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.

[0054] The present invention blends FPEI-SH with bioinert gold nanoparticles, which exhibit photothermal effects. This creates the Au-FPEI-SH cationic polymer gene vector. The introduced gold nanoparticles significantly enhance cell transfection due to their inherent photothermal effects. Furthermore, their bioinertness contributes to low cytotoxicity, resulting in a safe and efficient Au-FPEI-SH cationic polymer gene vector.

[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 in 10 mL of anhydrous chlorobenzene in a sealed container and react at 100° C. for 24 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 remove the residual solvent to obtain terminal fluorinated poly-2-ethyl-2-oxazoline (Compound A).

[0063] (2) Compound A was reacted with potassium ethylxanthate in a molar ratio of 5:1 to 2-ethyl-2-oxazoline, and 20 mL of acetonitrile was added. The reaction was allowed to proceed at room temperature for 24 h. After the reaction, the acetonitrile was removed by rotary evaporation, dissolved in dichloromethane, and transferred to a separatory funnel. An equal volume of saturated sodium chloride solution was added. The organic phase was extracted five times with dichloromethane, dried over anhydrous magnesium sulfate, filtered, and rotary 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, and the molar ratio of compound B to n-butylamine was 0.5:1. The mixture was reacted at room temperature for 12 hours. After the reaction was completed, rotary evaporation was performed to obtain a thiolated end-fluorinated polyoxazoline compound C.

[0065] (4) 6 mol / L hydrochloric acid was added to make the concentration of compound C in the hydrochloric acid solution about 50 mg / mL, and the reaction was carried out at 100°C for 10 h. After the reaction was completed, the compound was precipitated in methanol pre-cooled in an ice water bath for more than 15 min, washed, and dried in a vacuum oven to obtain the product as 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 in the synthesized compound A is 50; when the molar ratio is 1:0.1-0.15, p in the synthesized compound A is 100.

[0067] The compound of formula I was 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 preparation method of 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 minutes, and terminate in an ice water bath to obtain a 5 nm gold nanoparticle solution.

[0076] (2) Preparation of 50nm 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 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 was a fresh solution prepared according to the method of Example 2, and water was added to adjust its concentration to 0.05 mg / mL.

[0083] To 500 μL of 0.1 mg / mL F-LPEI-SH aqueous solution, 500 μL of 0.05 mg / mL 5 nm gold nanoparticle aqueous solution (Au 5), 50 nm gold nanoparticle aqueous solution (Au 50), or 100 nm gold nanoparticle aqueous solution (Au100) was added and stirred at room temperature overnight to obtain a 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] To 250 μL of 0.1 mg / mL F-LPEI-SH aqueous solution, 500 μL of 0.05 mg / mL 5 nm gold nanoparticle aqueous solution (Au 5), 50 nm gold nanoparticle aqueous solution (Au 50), or 100 nm gold nanoparticle aqueous solution (Au100) was added and stirred at room temperature overnight to obtain Au-FPEI-SH at a ratio of 1:0.5.

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

[0094] F-LPEI-SH is 17F100-SH, namely Au 5-17F100-SH, Au 50-17F100-SH, Au 100-17F100-SH with 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 characterized for particle size and potential using a particle size analyzer.

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

[0099] The potential of the Au-FPEI-SH cationic polymer gene carrier prepared in the above examples was determined using a particle size analyzer. Figure 4 The potential characterization results of Au-FPEI-SH cationic polymer gene carrier are shown in the figure. Figure 4 It can be seen that the potential of gold nanoparticles is negative, and the potential of Au-FPEI-SH cationic polymer gene carrier is positive, which can be endocytosed by cells, and meets the cell transfection conditions.

[0100] Example 6

[0101] Au-FPEI-SH cationic polymer gene carrier and plasmid complexed agarose gel electrophoresis, using green fluorescent protein plasmid (GFP) as an example.

[0102] Prepare 20 ng / μL of GFP plasmid aqueous solution, and prepare Au-FPEI-SH cationic polymer aqueous solution of different concentrations. Mix 5 μL of GFP plasmid aqueous solution with 5 μL of Au-FPEI-SH cationic polymer aqueous solution and vortex, and then stand at room temperature for 15 min. Add 2 μL of 6×DNA Loading Buffer loading buffer, mix well, and then add the prepared 1% agarose gel into the gel well to test the complexing ability of Au-FPEI-SH cationic polymer to plasmid.

[0103] Figure 5 The agarose gel electrophoresis results of Au-FPEI-SH cationic polymer gene carrier prepared in Example 3 are shown in the figure. Figure 5 It can be seen that the Au-FPEI-SH cationic polymer can completely complex the plasmids, which is specifically manifested as the complex remaining in the gel pores.

[0104] Example 7

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

[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 under an inverted fluorescence microscope using a cell counting plate. After calculation, 2×10 cells per well were used. 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 volume fraction 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 it stand at room temperature for 15 minutes, then add 0.8 μg pDNA per well to a 24-well plate, incubate in a constant temperature incubator for 24 hours for transfection.

[0108] Remove the cells from the incubator 24 hours after transfection, aspirate the culture medium from the well plate, add 500uL of PBS to each well, and observe the expressed green fluorescent protein signal under a fluorescence microscope. Transfected cells can produce green fluorescence, while 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 cytometry to detect the percentage of transfected positive cells to determine the transfection efficiency.

[0110] Figure 6 、 Figure 7 、 Figure 8 This is the result of 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 was used as a negative control, and BPEI 10k and BPEI 25k were 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 currently commonly used method of polyethyleneimine modification of gold nanoparticles, the terminal fluorinated polyethyleneimine was directly complexed with chloroauric acid.

[0113] Specific steps: Compound A was prepared according to the preparation method of 9F50-SH in Example 1, and compound A was directly hydrolyzed with hydrochloric acid without thiolation to obtain fluorinated polyethyleneimine, which was labeled as 9F50. 0.25 mL of 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, and 2.5 mg / mL 9F50 were added to 4.75 mL of 50 mg / L HAuCl4·3H2O aqueous solution, and the mixture was reacted at 37°C for 24 h or at 100°C for 20 min. The FPEI prepared in this example was reacted with chloroauric acid at different ratios at 37°C for 24 h or at 100°C for 20 min. The transfection efficiency was as follows: Figure 9 As shown in the figure, 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: To 15 mL of a 1 mM HAuCl4·3H2O aqueous solution, add 2 mL of a 10 mM CTAB solution and stir at room temperature for 15 minutes. The solution turns orange-yellow. Then, add 2 mL of a 100 mM NaBH4 solution dropwise, and the solution turns orange-red. Stirring at room temperature for 15 minutes without discoloration is performed. CTAB-modified gold nanoparticles are prepared and labeled Au15. Following the method of Example 3, a 1:1 ratio of Au15-17F50-SH was prepared.

[0116] from Figure 10 As can be seen, Au 5 is a gold nanoparticle modified with sodium citrate, and Au 15 is a gold nanoparticle modified with CTAB. After complexing with F-LPEI-SH, the transfection efficiency of the gold nanoparticles modified with sodium citrate was significantly improved, while the transfection efficiency of F-LPEI-SH modified with CTAB gold nanoparticles was extremely low.

[0117] 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 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 the thiol group. 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: ， 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, n=3 or 7, m=1, 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) Dissolve 2-ethyl-2-oxazoline and a perfluoroalkyl compound in anhydrous chlorobenzene, react at 100°C in a sealed container for 24 hours, add dichloromethane to dissolve the mixture after the reaction, and evaporate the mixture to obtain compound A; the perfluoroalkyl compound is 1-iodo-1H,1H,2H,2H-perfluorodecane or 1,1,2,2-tetrahydroperfluorohexyl iodide; (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, 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 the intermediate compound B. (3) Tetrahydrofuran was added at a mass ratio of 0.05 to 0.025:1, and after dissolution, n-butylamine was added, with a molar ratio of 1 to 0.5:1; the reaction was carried out at room temperature for 12 hours; after the reaction was completed, the polyoxazoline compound C with a thiolated terminal fluorinated compound was obtained by rotary evaporation; (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 pre-cooled in an ice water bath for more than 15 min, wash, and dry in a vacuum oven to obtain the 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 the fluorine atoms in the perfluoroalkyl compound in step (1) is 1:0.1-0.

3.

5. The gold / fluorinated polyethyleneimine composite according to claim 3, characterized in that In step (2), the molar concentration of potassium ethylxanthate 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 6, characterized in that The preparation method of the gold nanoparticles comprises the following steps: (1) After boiling the trisodium citrate solution, a chloroauric acid solution was added, and the reaction was carried out for 10 minutes. The reaction was terminated in an ice-water bath to obtain a gold nanoparticle solution. The molar ratio of the chloroauric acid to the trisodium citrate was 1:4, and the concentration of the chloroauric acid in the reaction system was 0.25 mol / L. (2) The gold nanoparticle solution prepared in step (1) is used as the gold seed solution, and the particle size of the gold nanoparticles is controlled 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 the 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 1-5:

1.

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

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