Sulfonium salt-modified polyethyleneimine, preparation method thereof, and use of gene carrier

Abstract

The present invention relates to the field of medical technology, and in particular to a polymer of low molecular weight polyethyleneimine modified by a sulfonium salt containing a hydrophobic group, having a general formula of SP, a preparation method thereof and use as a gene carrier. The preparation method of SP is simple, a hydrophobic alkane substituent and a carboxylic acid substituent are connected to the sulfonium salt, and then the sulfonium salt is modified to the low molecular weight polyethyleneimine with different coupling degrees through an amide bond to obtain a polymer. Gene carrier capacity test shows that SP has a strong plasmid gene loading capacity, embodies low toxicity and high delivery capacity.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a low-molecular-weight polyethyleneimine polymer modified by a sulfonium salt containing a hydrophobic group, a preparation method thereof and use thereof as a gene carrier. Background Art

[0002] Gene therapy has become an important method for treating many serious diseases. Non-viral gene vectors are a promising way to transfer genes into cells. Polyethyleneimine (PEI) is one of the most promising polymers. The gene carrier capacity of PEI has been widely studied. High molecular weight PEI has good transfection efficiency but high toxicity, while low molecular weight PEI has less toxicity but low transfection rate. Modification of low molecular weight PEI is an effective method to obtain high transfection and low toxicity vectors. The hydrophobic modification of PEI is to increase the hydrophobic interaction of the polymer, promote the compression of the polymer on the gene, and increase the lipophilicity of the nanoparticles, which is beneficial to cell membrane interaction and endosomal escape by modification. Through modification, the charge density of PEI is reduced and the cytotoxicity is changed.There are many reports on the modification methods of PEI: some studies have coupled farnesylthiosalicylic acid (FTS) with PEI to obtain amphiphilic FTS-PEI polymers as effective gene delivery vectors for tumor vaccination (Chen Y, Huang Y, Huang H, et al. Farnesylthiosalicylic acid-derivatized PEI-based nanocomplex for improved tumor vaccination. Mol Ther Nucleic Acids. 2021; 26: 594-602.); Cho KS et al.'s study combined low molecular weight polyethyleneimine (LMW PEI) with D-sorbitol and folic acid FA to form a cancer-targeted gene delivery system (FPS), which can enhance the efficacy of ligand targeting and effectively deliver nanocomplexes to cancer cells at a greater ratio than normal cells (Cho KS, Kim S, Chun HB, et al. Efficient gene transfection to lung cancer cells via Folate-PEI-Sorbitol gene transporter. PLoS One.2022;17(5):e0266181.); A series of fluorinated polymer gene carriers were synthesized based on low molecular weight polyethyleneimine (PEI) and fluorobenzoic acid (FBAs), which can achieve more efficient cell uptake and exhibit lower cytotoxicity, and have the potential for practical application (Xiao YP, Zhang J, Liu YH, et al. Low molecular weight PEI-based fluorinated polymers for efficient gene delivery. Eur J MedChem.2019;162:602-611.); Arginine-modified polyethyleneimine was coupled to chitosan (CS-PEI-Arg), and the results showed that CS-PEI-Arg is a promising candidate gene carrier (Zhang X, Duan Y, Wang D, et al. Preparation of arginine modified PEI-conjugated chitosan copolymer for DNA delivery. Carbohydr Polym.2015;122:53-9.). The various modification methods mentioned above have greatly improved the carrier performance of PEI and achieved the reprinting of various types of genes (DNA, RNA), but the modification of PEI still needs continuous exploration. Summary of the invention

[0003] The present invention provides a polymer based on PEI, synthesizing sulfonium salt-modified low molecular weight polyethyleneimine (SP). The present invention provides a method for synthesizing SP, firstly connecting two alkane substituents and a carboxylic acid substituent to the sulfonium salt, and then modifying the sulfonium salt to the low molecular weight PEI through an amide bond. The present invention explores the use of SP for gene transfection, the length and coupling degree of the hydrophobic alkane on the polymer can adjust the balance between the charge density and lipophilicity of the polymer, the positive charge on the polymer and the phosphate anion in the plasmid have an electrostatic effect, the hydrophobic long chain group can wrap the plasmid and then exert a hydrophobic effect to form a nanocomposite, according to the composite principle of polymer and gene, the genes that can be acted on by SP are: DNA including reporter genes, anticancer genes or cytokine genes, and RNA including siRNA, miRNA or piRNA.

[0004] The polymer SP of the present invention has the following general formula:

[0005]

[0006] In the general formula, R is a straight chain alkane group C8H 17 , C 14 H 29 , C 16 H 33 ;X - Represents anion Cl - Br - ,I - 、NO3 - OH - 、BF4 - 、HSO4 - 、HCO3 - 、H2PO3 - ; m is the number of polyethyleneimine units that are not coupled with the sulfonium salt containing a hydrophobic group; n is the number of units that form a coupled structure between the sulfonium salt containing a hydrophobic group and polyethyleneimine.

[0007] In the general formula, the polyethyleneimine in SP is a branched low molecular weight polyethyleneimine with a molecular weight ranging from greater than or equal to 300 Daltons to less than or equal to 5000 Daltons; the coupling degree of the sulfonium salt and the polyethyleneimine is the ratio of the molar number of the sulfonium salt to the molar number of the structural unit C2H5N of the polyethyleneimine, and the value is 0.05:1 to 0.25:1.

[0008] The preparation method of SP in the general formula of the present invention and the preferred synthesis route are shown in Scheme 1.

[0009] Scheme 1

[0010]

[0011] The main steps of the synthetic route of the above SP are:

[0012] Ethyl 5-bromovalerate and n-octyl mercaptan / n-tetradecyl mercaptan / n-hexadecyl mercaptan are dissolved in DMF and reacted in the presence of a base. The crude product is separated by column chromatography to obtain sulfide A; then sulfide A is dissolved in dry acetonitrile, n-butyl iodide and AgBF4 are added, refluxed for 24 hours, cooled, filtered, the filtrate is treated with an ion exchange resin for 2 hours, filtered, and separated by column chromatography to obtain sulfonium ester B; sulfonium ester B is dissolved in ethanol, and a 2.5 mmol / mL NaOH aqueous solution is added dropwise, and sulfonium ester B is hydrolyzed to obtain a sulfonium salt C containing a hydrophobic group; sulfonium salt C is activated by EDC and NHS, and then reacted with PEI at room temperature for 48 hours, the product is purified by dialysis for 48 hours, and freeze-dried to obtain polyethyleneimine SP modified with a sulfonium salt containing a hydrophobic group.

[0013] In the above synthetic route, as a preferred embodiment, the compound can be:

[0014] In A1, B1, and C1, R=C8H 17

[0015] In A2, B2, and C2, R = C 14 H 29

[0016] In A3, B3, and C3, R = C 16 H 33

[0017] In the above general formula, the polymer, as a preferred embodiment, SP can be:

[0018] SP1: PEI in SP structure is 1.8k Da, X - Cl - 、R is C8H 17 , the coupling degree is 0.1:1;

[0019] SP2: PEI in SP structure is 1.8k Da, X - Cl - 、R is C 14 H 29 , the coupling degree is 0.1:1;

[0020] SP3: PEI in SP structure is 1.8k Da, X - Cl - 、R is C 16 H 33 , the coupling degree is 0.1:1.

[0021] The polymer of the present invention has the use of gene carrier, and the experiment selects the widely used green fluorescent protein gene (eGFP) as the test gene. The gel electrophoresis blocking experiment shows that SP has good plasmid DNA loading capacity, and as the alkane chain in the polymer increases, the loading capacity of plasmid DNA is also enhanced; the particle size of the SP and DNA complex is in the range of 150 to 320nm, the Zeta potential of the complex is in the range of +16 to +45mV, and the particle size and Zeta potential of the complex have the ability to penetrate the cell membrane; the MTT experiment is used to measure the cytotoxicity of SP on HelpG2 cells, showing that the cytotoxicity is small, and the IC50 value of SP is about 1 / 5 of PEI 25k; the cell uptake experiment of SP and gene complex shows that SP can efficiently transport gene plasmids into cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 .Infrared spectra of compounds PEI 1.8k, C2, and SP2.

[0023] Figure 2 . Implement H NMR spectra of compounds PEI 1.8k, C2, and SP2.

[0024] Figure 3 . Gel electrophoresis diagram of the complexes of compounds PEI 1.8k, SP1, SP2, SP3 and eGFP at mass ratios of 1 / 3, 1 / 1, 2 / 1, 4 / 1, 6 / 1, and 10 / 1.

[0025] Figure 4 . The particle size of the complexes of compounds SP1, SP2, SP3 and eGFP at mass ratios of 2 / 1 and 4 / 1.

[0026] Figure 5 . Zeta potential diagrams of the complexes of compounds SP1, SP2, SP3 and eGFP at mass ratios of 2 / 1 and 4 / 1.

[0027] Figure 6 .The cytotoxicity results of HepG2 cells treated with compounds PEI 25k, PEI 1.8k, SP1, SP2, and SP3 for 24 hours.

[0028] Figure 7 .The cytotoxicity results of HepG2 cells treated with compounds PEI 25k, PEI 1.8k, SP1, SP2, and SP3 for 48 hours.

[0029] Figure 8 . A graph showing the results of HepG2 cell uptake of the complex of compounds PEI 25k, PEI 1.8k, SP1, SP2, SP3 and Cy5-eGFP plasmid at a w / w ratio of 2 / 1. DETAILED DESCRIPTION

[0030] 1. Main materials and instruments for the experiment

[0031] All chemical reagents were purchased from commercial sources, the solvents used were dried over calcium hydride and distilled, and all aqueous solutions were prepared from deionized water. The green fluorescent protein gene (eGFP) was purchased from Aldevron and cloned in Escherichia coli-DH5α (TIANGEN), and then extracted using the QIAGEN plasmid kit (QiagenEndoFree plasmid, Sigma-Aldrich). Label IT Cy5 fluorescent labeling kit (Mirus Bio) was used to label eGFP to provide Cy5-eGFP. The HepG2 cell line was obtained from the American Type Culture Collection (ATCC, Manassas, Virginia, USA) and stored in the laboratory. Fetal bovine serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Dulbecco's modified medium (DMEM) were purchased from Solarbio. Bruker Ascend 600M AVANCE III HD nuclear magnetic resonance instrument; Nicolet 380 infrared spectrometer (KBr pellet); Thermo Finnigan mass spectrometer; Thermo Nanodrop2000C spectrophotometer; Sub-Cell electrophoresis instrument; Universal Hood II gel imager (BIO-RAD); Nano-ZS90 nanoparticle size and zeta potential instrument (Malvern, UK); laser confocal microscope TCS SP8 (Leica).

[0032] 2. Synthesis and structural characterization of polymer SP

[0033] General method for the synthesis of A: Use a syringe to add n-octyl mercaptan / n-tetradecapryl alcohol / n-hexadecyl mercaptan (1.2 eq) to a vacuum-dried round-bottom flask, add 10 mL of N,N-dimethylformamide (DMF) as a solvent (the amount of the solvent is 0.1-0.5 mol / L), add a saturated NaOH solution (1.2 eq), and stir at room temperature for about 1 hour. Then, add 5-bromovalerate (1 eq) to the reaction system and stir at room temperature for 16 hours. Use thin layer chromatography to track the progress of the product synthesis. When the reaction reaches the end point, add 10 mL of distilled water. Use ether as an extractant for extraction (20 mL×3 times), and collect the ether phase. Add saturated brine to the ether phase for washing, then dry with anhydrous sodium sulfate, filter, and concentrate the product with a rotary evaporator, and then separate and purify it by column chromatography to obtain compound A, which is directly used in the next step reaction.

[0034] General method for the synthesis of B: Under argon protection, take the above-synthesized pure compound A (1 eq) and add 10 mL of acetonitrile after anhydrous treatment, then add n-butyl iodide (1 eq), and then add AgBF4 (1 eq), and reflux the reaction in the dark for 24 hours. When the reaction reaches the end point, cool it, filter it, and add a strong base anion exchange resin (Cl - The mixture was placed in a shaker and stirred for about 2 h, filtered, and the filtrate was concentrated. Compound B was then separated and purified by column chromatography.

[0035] General method for the synthesis of C: Place the above-synthesized pure compound B (1 eq) in a clean and dry round-bottom flask, add 2-3 mL of anhydrous ethanol as a solvent, then drop a 2.5 mmol / mL NaOH aqueous solution (1.5 eq), and react at 50°C for about 2 hours to hydrolyze the ester bond on compound B into an acid. After the reaction is completed, add an appropriate amount of 1 M HCl, adjust the pH value to weak acidity, concentrate the product using a rotary evaporator, and then separate and purify it by column chromatography to obtain compound C.

[0036] General method for the synthesis of SP: Compound C (0.1 eq) and PEI 1.8k (1 eq, according to the structural unit C2H5N) were placed in a clean and dry round-bottom flask and vacuum dried for 4 hours. Anhydrous DMF (1-2 mL) was added to the round-bottom flask of the dried compound C as a solvent, and then EDC·HCl (0.15 eq) and NHS (0.15 eq) were added and stirred at room temperature for 2 hours for activation. DMF (1-2 mL) was added to the round-bottom flask containing PEI 1.8k and stirred to dissolve, and the activated C was slowly added dropwise to the bottle of PEI 1.8k, and stirred at room temperature for 48 hours in the dark. After the reaction was completed, a dialysis bag (MWCO 1000) was used to perform dialyzation purification in a 50% ethanol dialysate system for 48 hours, and the dialysate was changed every 8 hours, and a total of 6 changes were required. The polymer SP after dialysis purification needs to be removed by a rotary evaporator to remove ethanol, and then freeze-dried to obtain a solid polymer SP.

[0037] S-(4-Ethoxycarbonylbutyl)-butyl-octylsulfonium chloride (B1)

[0038] Using the general synthesis method B, A1 (1.24 g, 4.5 mmol) was reacted with n-butyl iodide (0.77 mL, 6.8 mmol, 1.5 eq) and AgBF4 (1.31 g, 6.8 mmol, 1.5 eq) to obtain a light yellow oily product B1 (0.94 g, 2.84 mmol, yield 63%); R f =0.6 (MeOH-CH2Cl2=1:19). 1HNMR (CDCl3, δ): δ4.12 (q, J=7.2Hz, 2H), 3.27-3.18 (m, 4H), 3.21-3.16 (m, 2H), 2.37 (t, J=7.6Hz, 2H), 1 .76-1.57(m, 8H), 1.45-1.36(m, 4H), 1.40-1.21(m, 11H), 0.92(t, J=7.1Hz, 3H), 0.89(t, J=6.8Hz, 3H); 13 CNMR (CDCl3, δ): 173.72, 60.72, 36.52, 36.29, 36.04, 33.98, 31.78, 29.33, 29.32, 28.37 , 25.78, 25.64, 25.13, 22.71, 21.58, 19.02, 14.19, 14.09, 13.96; HR-MS (ESI) m / z: Calcd for C 19 H 39 O2S{[M-Cl - ]}331.2665, found: 331.2789.

[0039] S-(4-Ethoxycarbonylbutyl)-butyl-tetradecylsulfonium chloride (B2)

[0040] Using the general synthesis method B, A2 (0.67 g, 1.87 mmol) was reacted with n-butyl iodide (0.32 mL, 2.81 mmol, 1.5 eq) and AgBF4 (0.55 g, 2.81 mmol, 1.5 eq) to give a light yellow oily product B2 (0.65 g, 1.67 mmol, yield 89%); R f =0.5(MeOH-CH2Cl2=1:19). 1 HNMR (CDCl3, δ): 4.12 (q, J=7.1Hz, 2H), 3.45-3.30 (m, 6H), 2.41 (t, J=6.8Hz, 2H), 1.94-1.75 (m, 8H), 1.55-1.45 (m, 4H), 1.36-1.25 (m, 23H), 1.00 (t, J=7.3Hz, 3H), 0.88 (t, J=6.9Hz, 3H); 13CNMR (CDCl3, δ): 172.91, 60.62, 40.16, 40.04, 39.87, 32.90, 31.92, 29.88, 29.69, 29.65, 29.61, 29.49, 29.36 , 29.28, 28.94, 28.41, 26.64, 24.81, 24.04, 23.26, 22.69, 21.68, 14.20, 14.12, 13.34; HR-MS (ESI) m / z: Calcd for C 25 H 51 O2S{[M-Cl - ]}415.3604, found: 415.3563.

[0041] S-(4-Ethoxycarbonylbutyl)-butyl-hexadecylsulfonium chloride (B3)

[0042] Using the general synthesis method B, A3 (0.72 g, 1.87 mmol) was reacted with n-butyl iodide (0.32 mL, 2.81 mmol, 1.5 eq) and AgBF4 (0.55 g, 2.81 mmol, 1.5 eq) to give a light yellow oily product B3 (0.38 g, 0.86 mmol, yield 46%); R f =0.5(MeOH-CH2Cl2=1:19). 1 HNMR (CDCl3, δ): 4.12 (q, J=7.1Hz, 2H), 3.44-3.33 (m, 6H), 2.41 (t, J=6.8Hz, 2H), 1.92-1.76 (m, 8H), 1.55-1.45 (m, 4H), 1.37-1.25 (m, 27H), 1.00 (t, J=7.3Hz, 3H), 0.88 (t, J=7.0Hz, 3H); 13 CNMR (CDCl3, δ): 172.90, 60.61, 40.15, 40.04, 39.91, 39.87, 32.90, 31.93, 29.70, 29.67, 29.61, 29.50, 29.37, 29.29 , 28.94, 28.41, 26.63, 24.80, 24.04, 23.98, 23.26, 22.69, 21.68, 14.23, 14.20, 14.12, 13.33; HR-MS (ESI) m / z: Calcd for C 27 H 55 O2S{[M-Cl - ]}443.3917, found: 443.4059.

[0043] S-(4-Carboxybutyl)-butyl-octylsulfonium chloride (C1)

[0044] Using the general synthesis method C, B1 (0.46 g) was hydrolyzed under alkaline conditions to give a pale yellow oily product C1 (0.21 g, 0.68 mmol); R f =0.4(MeOH-CH2Cl2=1:9). 1 HNMR (CD3OD, δ): 3.34 (dt, J=14.9, 6.6Hz, 6H), 2.43 (q, J=5.1, 2.9Hz, 2H), 1.85 (ddq, J=41.8, 16.1, 8.0 Hz, 8H), 1.54 (dp, J=23.8, 7.8Hz, 4H), 1.42-1.34 (m, 8H), 1.03 (t, J=7.4Hz, 3H), 0.92 (t, J=6.9Hz, 3H); 13 CNMR (CD3OD, δ): 175.21, 38.94, 38.69, 32.26, 31.45, 28.92, 28.71, 28.54, 28.10 , 25.74, 23.82, 23.25, 23.14, 22.25, 21.33, 12.99, 12.25; HR-MS (ESI) m / z: Calcd for C 17 H 35 O2S{[M-Cl - ]}303.2352, found: 303.2466.

[0045] S-(4-Carboxybutyl)-butyl-tetradecylsulfonium chloride (C2)

[0046] Using the general method C, B2 (0.73 g) was hydrolyzed under alkaline conditions to give a pale yellow oily product C2 (0.36 g, 0.93 mmol); R f =0.4(MeOH-CH2Cl2=1:9). 1 HNMR (CD3OD, δ): 3.34 (dt, J=15.5, 6.8Hz, 4H), 3.16 (t, J=7.4Hz, 2H), 2.43 (t, J=7.2Hz, 2H), 1.95- 1.76 (m, 8H), 1.58-1.47 (m, 4H), 1.43-1.30 (m, 20H), 1.03 (t, J=7.5Hz, 3H), 0.91 (t, J=7.3Hz, 3H); 13CNMR (CD3OD, δ): 175.18, 38.95, 38.70, 32.27, 31.67, 31.06, 29.36, 29.32, 29.22, 29.07, 29.04, 28.58 , 28.09, 25.75, 23.83, 23.26, 23.14, 22.33, 31.11, 21.34, 19.67, 13.04, 12.27; HR-MS (ESI) m / z: Calcd for C 23 H 47 O2S{[M-Cl - ])387.3291, found: 387.3139.

[0047] S-(4-Carboxybutyl)-butyl-hexadecylsulfonium chloride (C3)

[0048] Using the general synthesis method C, B3 (0.38 g) was hydrolyzed under alkaline conditions to give a pale yellow oily product C3 (0.17 g, 0.41 mmol); R f =0.4(MeOH-CH2Cl2=1:9). 1 HNMR (CD3OD, δ): 3.37-3.33 (m, 4H), 3.17 (t, J=7.2Hz, 2H), 2.44 (t, J=7.1Hz, 2H), 1.95-1.86 (m, 4H), 1.82 (dq, J=1613, 8.0Hz, 4H), 1.55 (dt, J =18.0, 8.8Hz, 2H), 1.49 (q, J=7.8, 7.3Hz, 2H), 1.39 (dq, J=31.4, 7.8Hz, 6H), 1.38-1.26 (m, 18H), 1.04 (t, J=7.5Hz, 3H), 0.94 (t, J=7.3Hz, 3H); 13 CNMR (CD3OD, δ): 171.79, 38.93, 38.82, 38.69, 38.67, 31.67, 31.07, 29.38, 29.35, 29.31, 29.21, 29.06, 29.04 , 28.58, 28.10, 25.73, 23.81, 23.25, 23.15, 22.32, 21.35, 21.09, 19.67, 13.02, 12.27; HR-MS (ESI) m / z: Calcd for C 25 H 51 O2S{[M-Cl - ]}415.3604, found: 415.3601.

[0049] SP1

[0050] Using the general SP synthesis method, C1 (0.08 g, 0.25 mmol, 0.1 eq) was activated with EDC·HCl (0.07 g, 0.38 mmol, 0.38 eq) and NHS (0.08 g, 0.38 mmol, 0.38 eq), and then added to PEI 1.8k (0.11 g, 2.5 mmol) to react for 48 h. After dialyzed and freeze-dried, a light yellow gel product SP1 (0.09 g, yield 50%) was obtained.

[0051] SP2

[0052] Using the general SP synthesis method, C2 (0.07 g, 0.18 mmol, 0.1 eq) was activated with EDC·HCl (0.05 g, 0.28 mmol, 0.15 eq) and NHS (0.06 g, 0.28 mmol, 0.15 eq), and then added to PEI 1.8k (0.08 g, 1.8 mmol) to react for 48 h. After dialyzed and freeze-dried, a light yellow gel product SP2 (0.11 g, yield 77%) was obtained.

[0053] SP3

[0054] Using the general SP synthesis method, C3 (0.05 g, 0.13 mmol, 0.1 eq) was activated with EDC·HCl (0.04 g, 0.20 mmol, 0.15 eq) and NHS (0.04 g, 0.20 mmol, 0.15 eq), and then added to PEI 1.8k (0.06 g, 1.3 mmol) to react for 48 h. After dialyzed and freeze-dried, a light yellow gel product SP3 (0.05 g, yield 45%) was obtained.

[0055] The structure of the synthesized SP was characterized by infrared spectroscopy and nuclear magnetic resonance hydrogen spectroscopy. Figure 1 Medium, 3358.79cm -1 It is the NH vibration absorption peak of PEI 1.8k, 2945.37cm -1 and 2833.33cm -1 It is the symmetric and asymmetric absorption peak of CH. In the spectrum of C2, 1725.11cm -1 It is the stretching vibration of C=O, 1063.43cm -1 is the vibration of SC. In the spectrum of SP2, C=O in the new amide bond appears at 1634.20 cm -1 SC appears at 1058.98cm -1 . It is proved that SP2 is a coupling compound of PEI and sulfonium salt. Figure 2In the figure, the peak of PEI 1.8k appears at 2.8-2.5ppm, the chemical shift of hydrogen on C2 is annotated in the figure, and the spectrum of SP2 is the combination of PEI 1.8k and C2. The peak integration of the H-NMR spectrum calculates that on average each PEI 1.8k molecule carries about 4 sulfonium salt molecules, proving that the coupling degree of sulfonium salt with polyethyleneimine is 0.1:1.

[0056] 3. Experimental methods for testing the performance of SP gene vectors

[0057] The polymer of the present invention has the use of a gene carrier. The widely used green fluorescent protein gene (eGFP) was selected as a test gene in the main experiment, and the following technical scheme was adopted.

[0058] 1. Preparation of SP / DNA complex and gel retardation experiment

[0059] To study the loading capacity of SP for eGFP, gel electrophoresis blocking experiment was used to test SP / eGFP at mass ratios of 1 / 3, 1 / 1, 2 / 1, 4 / 1, 6 / 1 and 10 / 1, with PEI 1.8k as reference. SPs polymer was diluted with HEPES (hydroxyethylpiperazine ethanesulfonic acid pH = 7.5, 5mmol / L) to form a group of SP sample solutions with concentrations of 20ng / μL, 60ng / μL, 120ng / μL, 240ng / μL, 360ng / μL, and 600ng / μL. This group of SP samples (5uL) with different concentrations were added to eGFP solutions (3uL, 300ng), and the mixture was gently shaken and incubated at 37°C for 30min to prepare SP / eGFP complexes with mass ratios of 1 / 3, 1 / 1, 2 / 1, 4 / 1, 6 / 1 and 10 / 1. The complex was mixed with a loading buffer (1 μL) containing bromophenol blue, then loaded onto a 1% (w / v) agarose gel containing ethidium bromide (EB) and tris acetate (TAE), and electrophoresed at 120 V for 40 min in a Sub-Cell electrophoresis system. The experimental results were observed using a UV lamp from a Bio-Rad Universal Hood II.

[0060] The results are attached Figure 3 , polymers SP1, SP2, and SP3 showed retardation at a mass ratio of 2 / 1, and as the alkane chain in the polymer increased, the condensation ability with plasmid DNA also increased; while PEI 1.8k showed retardation at a mass ratio of 6 / 1. SP showed condensation ability for plasmid genes, and the results showed that SP could better condense plasmid DNA and had better gene loading capacity.

[0061] 2. SP / DNA complex particle size and Zeta potential detection

[0062] The eGFP complexes of SP1, SP2, and SP3 were prepared at a mass ratio of 2 / 1 and 4 / 1, and the particle size and Zeta potential of the complexes were determined. SPs were prepared into SP (PEI) sample solutions with a concentration of 100 ng / μL and 200 ng / μL with deionized water for standby use. The eGFP aqueous solution (8 μL, 800 ng) was added dropwise to the SP sample (16 μL) to form SP / DNA composite nanoparticles at a w / w ratio of 2 / 1 and 4 / 1, respectively. The complex was then gently shaken and incubated at 37°C for 30 min, diluted to 1 mL with deionized water, and the particle size and Zeta potential were measured with a Nano-ZS90 laser particle size and Zeta potential analyzer.

[0063] The particle size results are shown in the attached Figure 4 It was found that the size of the complexes formed by SP1, SP2, SP3 and DNA was in the range of 150-320 nm; the Zeta potential Figure 5 In the range of +16 to +45 mV, the complex has a suitable particle size and Zeta potential and has the ability to penetrate the cell membrane.

[0064] 3. Cell culture and cytotoxicity experiments of SP polymers

[0065] The cytotoxicity of SP1, SP2, and SP3 was determined on HelpG2 cells by MTT assay and compared with PEI 25k and PEI1.8k. HepG2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with fetal bovine serum (FBS, 10%, v / v) and penicillin-streptomycin (100 μg / mL) in a humidified 5% CO2 incubator at 37°C. The cells were seeded in a 96-well plate with 8000 cells and 150 μL of culture medium per well, and cultured for 8 h to ensure their adherent growth. The culture medium was replaced with 150 μL of DMEM medium without FBS, and the cells were treated with 1 μL of SP polymer solution at concentrations of 24.16 μg / μL, 12.08 μg / μL, 6.04 μg / μL, 3.02 μg / μL, 0.604 μg / μL, 0.0604 μg / μL, and 0.00604 μg / μL, respectively, and further incubated for 24 h and 48 h. After incubation, the culture medium was removed, 90 μL of DMEM medium without FBS and 10 μL of MTT (5 mg / mL) were added to the wells, and the cells were incubated for another 4 h. The culture medium was discarded, 150 μL of DMSO was added, and the cells were shaken for 30 min. The absorbance of each well was measured at 490 nm using a microplate reader, and the relative viability of the cells was calculated based on the data of five parallel tests by comparison with the control.

[0066] The experimental results are as attached Figure 6 With attached Figure 7 The trends of cell viability at 24h and 48h are roughly the same, showing that the cytotoxicity of these polymers is dose-dependent. At a concentration of 4μg / mL, SP1, SP2, and SP3 have lower cytotoxicity than PEI 25k, and the cell viability is about 85% to 80%. At a concentration of 20μg / mL, the cell viability of PEI 1.8k and SP1 is about 80%, the cell viability of SP2 and SP3 is about 30% to 40%, and that of PEI 25k is 20%. Overall, the IC50 value of SP is about 1 / 5 of that of PEI 25k.

[0067] 4. Cellular uptake experiment of SP / Cy5-eGFP complex

[0068] Using the Label IT Cy5 labeling kit, according to the operating instructions, the eGFP plasmid was labeled with Cy5 to form Cy5-eGFP. The SP / Cy5-eGFP complex prepared at a mass ratio of 2 / 1 was subjected to a cell uptake experiment on HepG2 cells, with PEI 25k and PEI 1.8k as controls. The SP solution (8 μL, 200 ng / μL) was diluted to 15 μL with DMEM, and Cy5-eGFP (800 ng, 15 μL) DMEM solution was added to the diluted SP solution, and then the mixed complex was gently shaken and cultured at 37°C for 30 min to form a SP / Cy5-eGFP complex with a mass ratio of 2 / 1. HepG2 cells were seeded into a 24-well plate with a cell slide, 40,000 cells / well, and 500 μL of DMEM medium containing 10% FBS was added. The cells were cultured at 37°C, 5% CO2 until the cell confluence reached 70-80%, the culture medium was poured out and the cells were washed with PBS. The culture medium without FBS (500 μL) was used instead, and the SP / Cy5-eGFP complex solution (40uL) was added. The cells were continued to be cultured at 37°C, 5% CO2 for 4h, and then the cell culture medium was removed. The cells were washed with PBS (300 μL×3) and fixed with 4% paraformaldehyde (400 μL) for 30min, after which the paraformaldehyde solution was removed, the cells were washed three times with PBS, and then 400 μL 0.1% Triton (30min) was added. After removing Triton, the cells were washed three times with PBS, and the cell slides were sealed on a slide with DAPI stain. The fluorescence results of the cells were observed on a laser confocal microscope TCS SP8. The excitation and emission wavelengths of Cy5 (red) were 649 / 670nm and DAPI (blue) were 364 / 454nm.

[0069] The results of electron microscopic observation are shown in the attached Figure 8All samples showed obvious red fluorescence. The red fluorescence of SP1 was weaker than that of PEI 25k, while the fluorescence intensities of SP2 and SP3 were higher than that of PEI 25k, indicating that SP2 and SP3 had better cell uptake ability than PEI 25k and had better gene delivery ability.

Claims

1. A low molecular weight polyethyleneimine polymer modified by a sulfonium salt containing a hydrophobic group, characterized in that: A conjugate formed by a sulfonium salt containing a hydrophobic group and polyethyleneimine; the polyethyleneimine is a branched low molecular weight polyethyleneimine with a molecular weight ranging from greater than or equal to 300 Daltons to less than or equal to 5000 Daltons; the coupling degree of the sulfonium salt and the polyethyleneimine is the ratio of the molar number of the sulfonium salt to the molar number of the structural unit C2H5N of the polyethyleneimine, and the value is 0.05:1 to 0.25:1; its general formula SP is as follows: Wherein: m is the number of polyethyleneimine units that are not coupled with the sulfonium salt containing a hydrophobic group; n is the number of units of the sulfonium salt containing a hydrophobic group and polyethyleneimine that form a coupled structure; R is a linear alkane group C8H 17 , C 14 H 29 , C 16 H 33 ;X - Represents anion Cl - Br - ,I - 、NO3 - OH - 、BF4 - 、HSO4 - 、HCO3 - 、H2PO3 - .

2. The polymer according to claim 1, characterized in that The molecular weight of polyethyleneimine is 1.8k Daltons.

3. The polymer according to claim 1, characterized in that The coupling degree of sulfonium salt and polyethyleneimine is 0.1:

1.

4. The method for preparing the polymer according to claim 1, wherein the synthetic route is as follows: The main operation steps are as follows: ethyl 5-bromovalerate and n-octyl mercaptan / n-tetradecyl mercaptan / n-hexadecyl mercaptan are dissolved in DMF for reaction, and the crude product is separated by column chromatography to obtain sulfide A; then sulfide A is dissolved in dry acetonitrile, n-butyl iodide and AgBF4 are added, refluxed for 24 hours, cooled, filtered, the filtrate is treated with ion exchange resin for 2 hours, filtered, and separated by column chromatography to obtain sulfonium ester B; sulfonium ester B is dissolved in anhydrous ethanol, and a 2.5 mmol / mL NaOH aqueous solution is added dropwise, and sulfonium ester B is hydrolyzed to obtain sulfonium salt C containing a hydrophobic group; sulfonium salt C is activated by EDC and NHS, and then reacted with PEI at room temperature for 48 hours, the product is purified by dialysis for 48 hours, and freeze-dried to obtain polyethyleneimine SP modified with sulfonium salt containing a hydrophobic group.

5. The nanocomposite of polymer and gene according to claim 1, characterized in that: Used as a gene carrier.

6. The nanocomplex formed with a gene according to claim 5, characterized in that: For use as a gene vector, wherein the gene is: DNA including a reporter gene, an anti-cancer gene or a cytokine gene, and RNA including siRNA, miRNA or piRNA.

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