CN117843541A - Lipid compound containing sulfonium cations, preparation method thereof and application of gene vector - Google Patents

Abstract

The invention relates to the technical field of chemical synthesis and application, in particular to a cationic lipid compound with a formula I, a formula II or a formula III, and discloses a preparation method of the compound, wherein the compound can be polycondensed with various genes to form a nano-composite, the particle size of the composite is 160-350 nm, the surface potential is +35- +52mV, and the composite has higher cell uptake effect and transfection effect. The compound is convenient to synthesize and has wide application of gene transfer.

Description

Lipid compound containing sulfonium cations, preparation method thereof and application of gene vector

Technical Field

The invention relates to the technical field of medicines, in particular to a cationic lipid compound containing sulfonium positive ions, an organic synthesis preparation method thereof and application of the cationic lipid compound serving as a gene vector.

Background

Cationic lipid compounds have been widely developed and can be used in a variety of fields, for example, multifunctional cations with labile amide linkages developed by Paluch et al as antifungal agents (Appl Microbiol Biotechnol.2021;105 (3): 1237-1251); bouraoui et al reported for the first time that double-stranded cationic amphiphiles with two thioether functionalities were used as novel transfection reagents (Chemphyschem.2019; 20 (17): 2187-2194); miller JB et al developed a panel of cationic Ji Anhuang amide amino lipids (CSAL) and evaluated them for small interfering RNA (siRNA) delivery (ACS Appl Mater interfaces.2018;10 (3): 2302-2311). Among them, the function of cationic lipids as a gene vector has been paid attention to and widely studied, and it can exhibit strong gene-loading ability, high safety and practicality, and easy preparation. The structure of the cationic lipid consists of a positive charge head part, a hydrophobic tail part and a connecting group, wherein the connecting group is mainly ester bonds, amides, ethers and the like, and the hydrophobic long chain is usually saturated or unsaturated hydrocarbon. Cationic lipid carriers rely on the interaction of cations with the negative charge of nucleic acids, and hydrophobic moieties combine to form nanoparticles of lipids with nucleic acids. The cationic lipid has various types, but most of the cationic lipid still has the problems of complex synthetic route, low synthetic yield, high cost, difficulty in mass production and the like, and has important significance in developing the low-cost and high-efficiency cationic lipid compound. Most cationic lipid compounds form positive charge groups by nitrogen atoms, including amines, ammonium salts, imidazoles, guanidine groups and the like, sulfonium compounds with cationic structures are also widely studied, sulfonium compounds are composed of three substituents linked by sulfur atoms, the structures of the sulfonium compounds are similar to quaternary ammonium salts, and gene vectors constructed by sulfonium as the positive charge groups are also gradually reported. The study of Hemp et al combined traditional free radical polymerization techniques with alkylation to synthesize sulfur homopolymers and sulfur diblock copolymers revealed for the first time that elemental sulfur provides another pathway for designing cationic macromolecules for non-viral nucleic acid delivery (ACS Macro Lett.2013;2 (8): 731-735). Sulfonium compounds as gene vectors deserve further development in terms of reduced cytotoxicity and increased transfection efficiency, and future work will be aimed at increasing functionality to create more efficient delivery vehicles.

Disclosure of Invention

The invention provides a lipid compound containing sulfonium ions, which takes methionine as a core, changes carboxyl and amino of methionine into ester bonds or amide bonds through chemical structural modification, links one or two oleoyl groups, and completes the synthesis of cationic lipid compounds through sulfonium positive ions formed by thioether of methionine. The compound generates electrostatic attraction through sulfonium positive ions and phosphate negative ions in nucleic acid, and the substituent group contains a hydrophobic long-chain group, so that a nano-composite is formed, and the hydrophobic chain is constructed through ester bonds and amide bonds and has degradability. The invention uses gel blocking experiment, nano particle diameter and zeta potential detection to prove that the compound can polycondense nucleic acid to form nano compound, the size of the compound is 160-350 nm and the surface potential is +35- +52mV, and the compound has higher uptake effect and transfection effect by using uptake experiment and transfection experiment. The invention has the advantages of convenient and quick combination preparation, high effect of the gene vector and wider application prospect.

A lipid compound containing sulfonium cations, which is characterized by having a structure shown in a formula I or a formula II or a formula III.

Wherein the amino acid chirality is L configuration and D configuration;

R ₁ ＝C ₁₀ H ₁₉ 、C ₁₂ H ₂₃ 、C ₁₄ H ₂₇ 、C ₁₆ H ₃₁ 、C ₁₈ H ₃₅ ；R ₂ ＝CH ₃ 、C ₂ H ₅ 、C ₃ H ₇ 、C ₄ H ₉ ；R ₃ ＝C ₉ H ₁₉ 、C ₁₁ H ₂₃ 、C ₁₃ H ₂₇ 、C ₁₅ H ₃₁ 、C ₁₇ H ₃₅ 。

the preparation method of the compound is shown in Scheme 1, and corresponding reaction compounds are selected according to different substituents to generate the product.

Scheme 1

The main operation steps are as follows

(1) Fmoc-methionine (1 eq) and direct alkane alcohol (1.2 eq) were dissolved in dry N, N-Dimethylformamide (DMF), DCC (1.5 eq), DMAP (0.1 eq) were added, stirred overnight at room temperature, the insoluble material was removed by filtration, the filtrate evaporated to dryness and the crude product was column chromatographed on silica gel to give Fmoc-methionine alkyl ester. Fmoc-methionine alkyl ester is dissolved in DMF and piperidine is added, the mixture is shaken for 15min, the solvent is evaporated, and the obtained product is subjected to column chromatography separation by silica gel to obtain the compound a. Compound a (1 eq) is dissolved in dry dichloromethane (5 mL) solution under the protection of argon, triethylamine (1.5 eq) is added, oleoyl chloride (1.2 eq) is slowly dripped into the solution at 0 ℃ for reaction for 30min, stirring is carried out for 1h at room temperature, water quenching is carried out, dichloromethane extraction is carried out, a dichloromethane layer is reserved, the solution is washed by saturated saline solution, anhydrous sodium sulfate is dried, the solvent is evaporated under reduced pressure to obtain a crude product, and column chromatography separation is carried out by silica gel to obtain compound b. Compound b (1 eq) and a different iodo alkane (1 eq) were dissolved in dry acetonitrile solution, silver tetrafluoroborate (1.2 eq) was added, magnetically stirred in an oil bath at 65 ℃ for 24h, and then cooled to room temperature. Filtering to remove precipitate, exchanging with strong alkali chloride ion exchange resin for 2h, filtering, evaporating solvent under reduced pressure to obtain crude product, and separating with silica gel column chromatography to obtain compound with formula I.

(2) Fmoc-methionine (1 eq) and oleoylethanolamide (1.2 eq) were dissolved in dry N, N-Dimethylformamide (DMF), DCC (1.5 eq), DMAP (0.1 eq) were added, stirred overnight at room temperature, the insoluble material was removed by filtration, the filtrate evaporated to dryness and the crude product was separated by column chromatography on silica gel to give Fmoc-methionine alkyl ester. Fmoc-methionine alkyl ester is dissolved in DMF and piperidine is added, the mixture is shaken for 15min, the solvent is evaporated, and the obtained product is subjected to column chromatography separation by silica gel to obtain the compound c. Under the protection of argon, the compound c (1 eq) is dissolved in a dry dichloromethane (5 mL) solution, triethylamine (1.5 eq) is added, acyl chloride or oleoyl chloride (1.2 eq) with different chain lengths is slowly dripped at 0 ℃ to react for 30min, the mixture is stirred for 1h at room temperature, water is added to quench, dichloromethane is extracted, a dichloromethane layer is reserved, the mixture is washed with saturated saline water, anhydrous sodium sulfate is dried, the solvent is evaporated under reduced pressure to obtain a crude product, and column chromatography separation is carried out on silica gel to obtain the compound d or e. Compound d or e (1 eq) and the different iodo alkane (1 eq) were dissolved in dry acetonitrile solution, silver tetrafluoroborate (1.2 eq) was added, magnetically stirred in an oil bath at 65 ℃ for 24h, and then cooled to room temperature. Filtering to remove precipitate, exchanging with strong alkali chloride ion exchange resin for 2h, evaporating solvent under reduced pressure to obtain crude product, and separating with silica gel column chromatography to obtain compound of formula II or III.

Further, the structure of the compounds of the above general formula may be:

further, the above compounds are named as follows:

S-methyl-N-oleoyl-D-methionine dodecyl chloride (MO 1),

S-ethyl-N-oleoyl-D-methionine dodecyl chloride (MO 2),

S-propyl-N-oleoyl-D-methionine dodecyl chloride (MO 3),

S-butyl-N-oleoyl-D-methionine dodecyl chloride (MO 4),

S-methyl-N-dodecanoyl-D-methionine oleoylaminoethyl ester chloride (MO 5),

S-ethyl-N-lauroyl-D-methionine oleoylaminoethyl chloride (MO 6),

S-propyl-N-dodecanoyl-D-methionine oleoylaminoethyl ester chloride (MO 7),

S-butyl-N-dodecanoyl-D-methionine oleoylaminoethyl ester chloride (MO 8),

S-methyl-N-oleoyl-D-methionine oleoylaminoethyl chloride (MO 9),

S-ethyl-N-oleoyl-D-methionine oleoylaminoethyl chloride (MO 10),

S-propyl-N-oleoyl-D-methionine oleoylaminoethyl chloride (MO 11),

S-butyl-N-oleoyl-D-methionine oleoylaminoethyl chloride (MO 12),

the compounds are characterized by mass spectrum and nuclear magnetism, represented by compounds MO2, MO6 and MO10, and are shown in figures 1-7.

The cationic lipid compound has the application of a gene vector, and green fluorescent protein genes (gWiz-GFP, 5757bp, alvetron company) and short-chain herring sperm DNA (hsDNA, 20bp, shanghai Yingxin laboratory equipment Co., ltd.) are selected as test genes in experiments.

The invention detects the compound ability of the compound MO1-MO12 to the two nucleic acids gWiz-GFP plasmid and hsDNA through gel blocking experiment. The gel electrophoresis blocking experimental results of the compounds MO1-MO12 and gWiz-GF after being compounded according to different S/P ratios are shown in the accompanying drawings 8, 9 and 10, and the experiments show that the compounds MO1-MO12 can effectively block gWiz-GFP plasmids. The gel electrophoresis blocking experimental results of the compounds MO1-MO12 and hsDNA after being compounded according to different S/P ratios are shown in the accompanying drawings 11, 12 and 13, and the experiments show that the compounds MO1-MO12 can effectively block hsDNA.

The invention uses a nanometer particle size analyzer and a zeta potentiometer to detect the particle size and zeta potential of a compound formed by the compounds MO1-MO12, gWiz-GFP and hsDNA nucleic acid in a complete compounding proportion. The detection data of the nanocomposite formed by the compounds MO1-MO12 with the two nucleic acids in a ratio of complete complexing are shown in Table 1. Particle size is 150-300 nm, and electromotive force is +8- +50 mV. The data indicate that the compounds form nanoparticles with nucleic acids that have cell penetrating ability.

TABLE 1 particle size and zeta potential results of nanocomposites of Compounds MO1-MO12 with plasmid gWiz-GFP and hsDNA

According to the invention, the cell uptake condition of the complex formed by MO1-MO12, cy5-gWiz-GFP and Cy5-siRNA is detected by using an uptake experiment, and the cell transfection condition of the complex formed by MO1-MO12 and gWiz-GFP plasmid is detected by using a transfection experiment, as shown in fig. 14, 15 and 16, the complex shows better cell uptake and transfection effects.

Drawings

FIG. 1 is a mass spectrum of an embodiment of the present invention, MO 2;

FIG. 2 is a chart showing the hydrogen nuclear magnetic resonance spectrum of MO2, a compound embodying the present invention;

FIG. 3 is a nuclear magnetic resonance carbon spectrum of an embodiment of the compound MO 2;

FIG. 4 is a mass spectrum of MO6, a compound embodying the present invention;

FIG. 5 is a chart showing the hydrogen nuclear magnetic resonance spectrum of MO6, a compound embodying the present invention;

FIG. 6 is a nuclear magnetic resonance carbon spectrum of an embodiment of the compound MO 6;

FIG. 7 is a mass spectrum of MO10, a compound embodying the present invention;

FIG. 8 shows gel electrophoresis patterns of compounds MO1-MO12 and gWiz-GFP plasmid after complexing in different ratios;

FIG. 9 shows gel electrophoresis patterns of compounds MO1-MO12 and gWiz-GFP plasmid after complexing in different ratios;

FIG. 10 shows gel electrophoresis patterns of compounds MO1-MO12 and gWiz-GFP plasmid after complexing in different ratios;

FIG. 11 shows gel electrophoresis patterns of compounds MO1-MO12 in different ratios with hsDNA plasmids;

FIG. 12 shows gel electrophoresis patterns of compounds MO1-MO12 in different ratios with hsDNA plasmids;

FIG. 13 shows gel electrophoresis patterns of compounds MO1-MO12 in different ratios with hsDNA plasmids;

FIG. 14 is a graph showing the effect of partial compounds and Cy5-gWiz-GFP plasmid complexes on uptake in HepG2 cells under serum-free conditions;

FIG. 15 is a graph showing the effect of partial compounds and Cy5-siRNA plasmid complexes on uptake in serum-free conditions in HepG2 cells;

FIG. 16 is a graph showing the effect of a portion of the compound and gWiz-GFP plasmid complex on transfection of green fluorescent protein genes in HepG2 cells under serum-free conditions;

Detailed Description

The synthesis, structural characterization and nucleic acid complex transfer ability experiments of the compounds of the present invention are described below by way of specific examples.

Experimental main materials and instruments

All chemical reagents were purchased from commercial sources and the chemical synthesis reactions were performed using GF254 TLC plates, using potassium permanganate staining, and all aqueous solutions were prepared from deionized water. The green fluorescent protein gene (gWiz-GFP) was purchased from Aldriron and cloned in E.coli-DH 5. Alpha (TIANGEN) and then extracted with the QIAGEN plasmid kit (QiagenEndoFree plasmid, sigma-Aldrich); short-chain herring sperm DNA (hsDNA, 20bp, shanghai glume laboratories equipment limited); pro-apoptotic genes (siRNA, 21bp, shanghai Biotechnology Co., ltd.); lipo2000 (Invitrogen) ^TM Lipofectamine ^TM 2000 transfection reagent, thermo Fisher Scientific company); agarose (Biowest Agarose Co.); bruker Assend 600M AVANCE III HD nuclear magnetic resonance apparatus; a Thermo Finnigan mass spectrometer; thermo Nanodrop 2000C spectrophotometer; sub-Cell electrophoresis apparatus; universal Hood II gel imager (BIO-RAD Co.); nano-ZS90 nanometer particle size and Zeta potentiometer (Markov UK).

Example 1

S-methyl-N-oleoyl-D-methionine dodecyl chloride (MO 1)

Fmoc-D-methionine (0.250 g,0.673 mmol) was reacted with n-dodecanol (0.151 g,0.258 mmol) in the presence of DCC (0.208 g, 1.010mmol) and DMAP (0.008 g,0.067 mmol) to give Fmoc-D-methionine dodecyl ester, which was then removed in a mixed solvent of piperidine and DMF to give methionine dodecyl ester a (0.189 g,0.596 mmol). Dodecyl methionine (0.432 g, 1.292 mmol) was reacted with oleoyl chloride (0.675 mL,2.043 mmol) in the presence of triethylamine to give dodecyl N-oleoyl-D methionine b (0.534 g,0.925 mmol). Product b (0.150 g,0.258 mmol) was then reacted with methyl iodide (0.016 mL,0.258 mmol) and silver tetrafluoroborate (0.060 g,0.310 mmol) to give product MO1 (0.104 g,0.175mmol, yield 67.89%) as a yellow oil; r is R _f =0.35 (methanol-dichloromethane 1:10) HR-MS (ESI) m/z: calcd for C ₂₈ H ₅₆ NO ₃ S ⁺ {[M-Cl-]+}486.3975，found：486.4064。

Example 2

S-ethyl-N-oleoyl-D-methionine dodecyl chloride (MO 2)

Product b (0.202 g, 0.277 mmol) was reacted with iodoethane (0.028 mL, 0.277 mmol), silver tetrafluoroborate (0.082 g,0.420 mmol) to give product MO2 (0.147 g,0.241mmol, 69.45%) as a yellow oil; r is R _f =0.35 (methanol-dichloromethane 1:10). ¹ H NMR(CD ₃ Cl ₃ ，δ)：6.87(dd，J＝7.6，7.6Hz，1H)，5.38-5.36(m，1H)，5.35-5.31(m，1H)，4.60(m，1H)，4.18-4.09(m，2H)，3.54-3.45(m，2H)，3.43-3.32(m，2H)，2.91(s，3H)，2.41-2.33(m，1H)，2.31-2.24(m，3H)，2.02-1.99(m，3H)，1.97-1.95(m，1H)，1.66-1.58(m，4H)，1.50(t，J＝7.4Hz，3H)，1.35-1.22(m，38H)，0.88(d，J＝6.9Hz，6H)； ¹³ C NMR(CD ₃ Cl ₃ ，δ)：174.71，170.54，129.99，129.77，66.43，50.70，38.72，38.57，36.49，36.05，32.63，31.92，29.79，29.73，29.68，29.66，29.64，29.56，29.54，29.37，29.34，29.32，29.27(2C)，29.24，28.43，27.23，26.71，25.80，25.52，25.49，22.70，22.02，21.77，14.12，8.96，8.81。HR-MS(ESI)m/z：Calcd for C ₃₇ H ₇₂ NO ₃ S ⁺ {[M-Cl-]+}610.5227，found：610.5436。

Example 3

S-propyl-N-oleoyl-D-methionine dodecyl chloride (MO 3)

Product b (0.133 g,0.229 mmol) was reacted with iodopropane (0.020mL, 0.229 mmol) and silver tetrafluoroborate (0.053 g,0.274 mmol) to give product MO3 (0.107 g,0.171mmol, 74.67% yield) as a yellow oil; r is R _f =0.35 (methanol-dichloromethane 1:20). HR-MS (ESI) m/z: calcd for C ₃₈ H ₇₄ NO ₃ S ⁺ {[M-Cl-]+}624.5384，found：624.5402。

Example 4

S-butyl-N-oleoyl-D-methionine dodecyl chloride (MO 4)

Product b (0.058 g,0.0997 mmol) was reacted with n-butyl iodide (0.01 mL,0.099 mmol) and silver tetrafluoroborate (0.023 g,0.119 mmol) to give product MO4 (0.04)3g,0.068mmol, 68.21% yield) as yellow oil; r is R _f =0.35 (methanol-dichloromethane 1:20). HR-MS (ESI) m/z: calcd for C ₃₉ H ₇₆ NO ₃ S ⁺ {[M-Cl-]+}638.5540，found：638.5599。

Example 5

S-methyl-N-lauroyl-D-methionine oleoylaminoethyl ester chloride (MO 5)

Product d (0.278 g,0.404 mmol) was reacted with methyl iodide (0.025 mL,0.404 mmol) and silver tetrafluoroborate (0.094 g, 0.480 mmol) to give product MO5 (0.191 g,0.292mmol, yield 72.34%) as a milky white solid; r is R _f =0.30 (methanol-dichloromethane 1:9). HR-MS (ESI) m/z: calcd for C ₃₈ H ₇₃ N ₂ O ₄ S ⁺ {[M-Cl-]+}653.5286，found：653.5338。

Example 6

S-ethyl-N-lauroyl-D-methionine oleoylaminoethyl chloride (MO 6)

Product d (0.284 g, 0.418 mmol) was reacted with iodoethane (0.034 ml,0.6415 mmol) and silver tetrafluoroborate (0.097 g,0.498 mmol) to give product MO6 (0.246 g,0.369mmol, yield 88.80%) as a milky solid; r is R _f =0.30 (methanol-dichloromethane 1:9). ¹ H NMR(CD ₃ Cl ₃ ，δ)：7.19(dd，J＝11.1，7.4Hz，1H)，6.07(dd，J＝6.1，6.0Hz，1H)，5.37-5.30(m，2H)，4.57-4.53(m，1H)，4.33-4.27(m，1H)，4.20-4.16(m，1H)，3.54-3.30(m，6H)，2.91(s，3H)，2.46-2.41(m，1H)，2.30-2.24(m，3H)，2.20-2.18(m，2H)，2.02-1.99(m，4H)，1.62-1.57(m，4H)，1.50(t，J＝7.4Hz，3H)，1.34-1.25(m，36H)，0.88(t，J＝6.9Hz，6H)； ¹³ C NMR(CD ₃ Cl ₃ ，δ)：174.81，174.28，170.44，129.97，129.22，65.07，51.07，51.00，38.46，38.19，36.42，36.35，36.29，31.91，29.80，29.77，29.69，29.65，29.60，29.53，29.47，29.43，29.36(2C)，29.33，29.31，29.26，27.23，26.28，26.23，25.77，25.54，25.52，22.68，21.72，21.69，14.08，8.76，8.67.HR-MS(ESI)m/z：Calcd for C ₃₉ H ₇₅ N ₂ O ₄ S ⁺ {[M-Cl-]+}：667.5442，found：667.5717。

Example 7

S-propyl-N-lauroyl-D-methionine oleoylaminoethyl ester chloride (MO 7)

Product d (0.263 g,0.382 mmol) was reacted with iodopropane (0.034 mL,0.382 mmol), silver tetrafluoroborate (0.089 g,0.458 mmol) to give product MO7 (0.187 g,0.274mmol, 71.82% yield) as a milky white solid; r is R _f =0.30 (methanol-dichloromethane 1:9). HR-MS (ESI) m/z: calcd for C ₄₀ H ₇₇ N ₂ O ₄ S ⁺ {[M-Cl-]+}681.5599，found：681.5866。

Example 8

S-butyl-N-lauroyl-D-methionine oleoylaminoethyl ester chloride (MO 8)

Product d (0.222 g,0.322 mmol) was reacted with n-butyl iodide (0.056 mL,0.322 mmol), silver tetrafluoroborate (0.075 g, 0.3836 mmol) to give product MO8 (0.165 g,0.237mmol, 73.67%) as a milky solid; r is R _f =0.30 (methanol-dichloromethane 1:9). HR-MS (ESI) m/z: calcd for C ₄₁ H ₇₉ N ₂ O ₄ S ⁺ {[M-Cl-]+}695.5755，found：695.6006。

Example 9

S-methyl-N-oleoyl-D-methionine oleoylaminoethyl ester chloride (MO 9)

Product e (0.248 g,0.344 mmol) was reacted with methyl iodide (0.021 mL,0.344 mmol) and silver tetrafluoroborate (0.080 g,413 mmol) to give product MO9 (0.119 g,0.162mmol, 47.03%) as a milky white solid; r is R _f =0.30 (methanol-dichloromethane 1:9). HR-MS (ESI) m/z: calcd for C ₄₄ H ₈₃ N ₂ O ₄ S ⁺ {[M-Cl-]+}735.6068，found：735.6308。

Example 10

S-ethyl-N-oleoyl-D-methionine oleoylaminoethyl chloride (MO 10)

Product e (0.214 g,0.300 mmol) was taken with iodoethane (0.024 mL,0.300 mmol), silver tetrafluoroborate @0.070g,0.360 mmol) to give product MO10 (0.099 g,0.132mmol, 44.02%) as a milky solid; r is R _f =0.30 (methanol-dichloromethane 1:9). HR-MS (ESI) m/z: calcd for C ₄₅ H ₈₅ N ₂ O ₄ S ⁺ {[M-Cl-]+}749.6225，found：749.6464。

Example 11

S-propyl-N-oleoyl-D-methionine oleoylaminoethyl ester chloride (MO 11)

Product e (0.210 g,0.290 mmol) was reacted with iodopropane (0.026 mL,0.290 mmol), silver tetrafluoroborate (0.068 g,0.348 mmol) to give product MO11 (0.106 g,0.139mmol, 47.87%) as a milky white solid; r is R _f =0.30 (methanol-dichloromethane 1:9). HR-MS (ESI) m/z: calcd for C ₄₆ H ₈₇ N ₂ O ₄ S ⁺ {[M-Cl-]+}：763.6381，found：763.6433。

Example 12

S-butyl-N-oleoyl-D-methionine oleoylaminoethyl ester chloride (MO 12)

Product e (0.575 g,0.798 mmol) was reacted with n-butyl iodide (0.091 mL,0.800 mmol) and silver tetrafluoroborate (0.186 g,0.958 mmol) to give product A12 (0.720 g,1.12mmol, 97.1% yield) as a milky white solid; r is R _f =0.30 (methanol-dichloromethane 1:9). HR-MS (ESI) m/z: calcd for C ₄₇ H ₈₉ N ₂ O ₄ S ⁺ {[M-Cl-]+}777.6538，found：777，6617。

Gel electrophoresis blocking experiment

MO1-MO12 was prepared as a solution of 0.231 nmol/. Mu.L, 0.462 nmol/. Mu.L, 0.924 nmol/. Mu.L, 1.848 nmol/. Mu.L, 2.769 nmol/. Mu.L, 3.696 nmol/. Mu.L in DMSO in 2. Mu.L, and each solution was mixed with deionized water (4. Mu.L) to prepare a total volume of 6. Mu.L. gWiz-GFP is prepared into 100 ng/. Mu.L by deionized water, 3uL (300 ng) is added into prepared MO1-MO12 samples with different concentrations, vortex shaking is carried out for 1S, then standing culture is carried out for 15min at 37 ℃, and MO/gWiz-GFP complexes with S/P ratios of 1/2, 1/1, 2/1, 4/1, 6/1, 8/1 and the like are prepared for standby.

MO1-MO12 was prepared as solutions of 0.173 nmol/. Mu.L, 0.346 nmol/. Mu.L, 0.692 nmol/. Mu.L, 1.384 nmol/. Mu.L, 2.768 nmol/. Mu.L, and 5.536 nmol/. Mu.L using DMSO, and each solution was mixed with deionized water (4. Mu.L) to prepare a total volume of 6. Mu.L solution. The hsDNA is prepared into 300 ng/. Mu.L by deionized water, 3uL (900 ng) is added into prepared MO1-MO12 samples with different concentrations, vortex shaking is carried out for 1S, then standing culture is carried out for 15min at 37 ℃, and MO/hsDNA compound with S/P ratio of 1/8, 1/4, 1/2, 1/1, 2/1, 4/1 and the like is prepared for standby.

0.35g of agarose was weighed and placed in a 100mL flask, and 35mL of tris (hydroxymethyl) aminomethane acetate buffer (TAE) was added to prepare a 1% agarose gel. After the liquid is heated, 1 mu L of ethidium bromide (EB 1 mg/mL) is added, the mixture is poured into an organic glass tank, a comb is inserted, the comb is taken off when the gel is completely solidified, and agarose gel is put into an electrophoresis tank for standby.

Each MO/gWiz-GFP, MO/hsDNA complex sample was added with 1 μl of bromophenol blue mixture solution, respectively, to form a volume of 10 μl, and then added into gel wells, and the gel wells were electrophoresed under 120V and 110V voltage respectively for 20min with naked DNA as a control, and the experimental results were analyzed by a gel imager.

Detection of composite nano particle size and Zeta potential

The nano-particle size and Zeta potential of the compound MO1-MO12 and gWiz-GFP are detected under the complete compounding proportion. Preparing the sulfonium compound to be detected into 1 nmol/mu L solution by using DMSO respectively for standby. At the complete compounding ratio of each sample and DNA, a corresponding amount of compound is diluted to 16 mu L in volume by ionized water, mixed with 8 mu L of DNA (containing 800ng of DNA) and vibrated for 1s, and then kept stand at room temperature for 20min, the compound is diluted to 1mL by deionized water, and the particle size and Zeta potential of the compound are measured by a Nano-ZS ZEN instrument.

The nano-particle size and Zeta potential of the compound MO1-MO12 and hsDNA are detected under the complete compounding proportion. Preparing the sulfonium compound to be detected into 1 nmol/mu L solution by using DMSO respectively for standby. At the complete compounding ratio of each sample and DNA, a corresponding amount of compound is diluted to 16 mu L in volume by ionized water, mixed with 8 mu L of DNA (containing 800ng of DNA) and vibrated for 1s, and then kept stand at room temperature for 20min, the compound is diluted to 1mL by deionized water, and the particle size and Zeta potential of the compound are measured by a Nano-ZS ZEN instrument.

Uptake experiments

Plasmid labelling was performed according to the procedure using a Cy5 labelling kit, and after labelling, the plasmid was collected in an enzyme-free EP tube, diluted to 100 ng/. Mu.L with sterile water and stored at-20℃under light-shielding for further use. The compounds MO1-MO12 were dissolved in DMSO to prepare a1 nmol/. Mu.L solution for use.

HepG2 cells in the logarithmic growth phase were selected for stable growth, and 150. Mu.L of the cell suspension was seeded at 8000 cells/well in 96-well plates. At 37 ℃,5% CO ₂ After culturing in the incubator for 12 hours until the cells adhere to the wall, the culture solution is replaced with a serum-free culture solution. Mixing Cy5-siRNA or Cy5-gWiz-GFP plasmid (2 μL,100 ng/. Mu.L) with solution of compound MO1-MO12 with corresponding S/P ratio, slightly vibrating, incubating for 20min under dark condition, adding the mixed solution into HepG2 cell, standing at 37deg.C, and adding 5% CO ₂ Culturing in a concentration incubator for 4h, removing culture solution, washing with PBS solution for 5min×3 times, adding 4% paraformaldehyde solution, standing for 30min, fixing cells, washing with PBS solution for 15min×3 times, adding 0.1% triton solution, standing for 20min, perforating cell membranes, washing with PBS solution for 20min×3 times again, fixing cell climbing sheet on a glass slide with DAPI-containing sealing tablet, staining cell nuclei, observing fluorescence distribution under a Carl Zeiss lnverted Microscope (Axiovert 5 digital) fluorescence microscope, wherein Cy5 excitation and emission wavelengths are 649/670 respectively, and DAPI excitation and emission wavelengths are 364/454 respectively.

Transfection experiments

HepG2 cells in the logarithmic growth phase were selected for stable growth, and 150. Mu.L of the cell suspension was seeded at 8000 cells/well in 96-well plates. At 37 ℃,5% CO ₂ After culturing in the incubator for 12 hours until the cells adhere to the wall, the culture solution is replaced with a serum-free culture solution. Mixing gWiz-GFP (2. Mu.L, 100 ng/. Mu.L) with corresponding solution of compound MO1-MO12 at S/P ratio, vibrating slightly, incubating for 20min, adding the mixture into HepG2 cells (with Lipo 2000/gWiz-GFP complex as control), standing at 37deg.C, 5% CO ₂ Culturing in a concentration incubator for 4 hr, removing culture solution, washing with PBS solution for 2 times, adding 10% FBS culture solution, standing at 37deg.C, and 5% CO ₂ After 48h incubation in incubator, the plates were removed and observed under a Carl Zeiss lnverted Microscope (Axiovert 5 digital) fluorescence microscope, with gWiz-GFP excitation and emission wavelengths of 488nm/507nm, respectively.

Claims (5)

1. A cationic lipid compound, which is characterized by having a structure shown in formula I or formula II or formula III.

Wherein the amino acid chirality is L configuration and D configuration; r is R ₁ ＝C ₁₀ H ₁₉ 、C ₁₂ H ₂₃ 、C ₁₄ H ₂₇ 、C ₁₆ H ₃₁ 、C ₁₈ H ₃₅ ；R ₂ ＝CH ₃ 、C ₂ H ₅ 、C ₃ H ₇ 、C ₄ H ₉ ；R ₃ ＝C ₉ H ₁₉ 、C ₁₁ H ₂₃ 、C ₁₃ H ₂₇ 、C ₁₅ H ₃₁ 、C ₁₇ H ₃₅ 。

2. The process for preparing a lipid compound according to claim 1, which comprises the following synthetic route:

the main operation steps are as follows

(1) Fmoc-methionine (1 eq) and direct alkane alcohol (1.2 eq) were dissolved in dry N, N-Dimethylformamide (DMF), DIC (1.5 eq), DMAP (0.1 eq) were added, stirred overnight at room temperature, the insoluble material was removed by filtration, the filtrate evaporated to dryness and the crude product was column chromatographed on silica gel to give Fmoc-methionine alkyl ester. Fmoc-methionine alkyl ester is dissolved in DMF and piperidine is added, the mixture is shaken for 15min, the solvent is evaporated, and the obtained product is subjected to column chromatography separation by silica gel to obtain the compound a. Compound a (1 eq) is dissolved in dry dichloromethane (5 mL) solution under the protection of argon, triethylamine (1.5 eq) is added, oleoyl chloride (1.2 eq) is slowly dripped into the solution at 0 ℃ for reaction for 30min, stirring is carried out for 1h at room temperature, water quenching is carried out, dichloromethane extraction is carried out, a dichloromethane layer is reserved, the solution is washed by saturated saline solution, anhydrous sodium sulfate is dried, the solvent is evaporated under reduced pressure to obtain a crude product, and column chromatography separation is carried out by silica gel to obtain compound b. Compound b (1 eq) and a different iodo alkane (1 eq) were dissolved in dry acetonitrile solution, silver tetrafluoroborate (1.2 eq) was added, magnetically stirred in an oil bath at 65 ℃ for 24h, and then cooled to room temperature. Filtering to remove precipitate, exchanging with strong alkali chloride ion exchange resin for 2h, evaporating solvent under reduced pressure to obtain crude product, and separating with silica gel column chromatography to obtain compound with formula I.

(2) Fmoc-methionine (1 eq) and oleoylethanolamide (1.2 eq) were dissolved in dry N, N-Dimethylformamide (DMF), DIC (1.5 eq), DMAP (0.1 eq) were added, stirred overnight at room temperature, the insoluble material was removed by filtration, the filtrate evaporated to dryness and the crude product was column chromatographed on silica gel to give Fmoc-methionine alkyl ester. Fmoc-methionine alkyl ester was dissolved in DMF and piperidine was added, shaking for 15min, solvent was evaporated and the resulting product was column chromatographed on silica gel to give compound c. Under the protection of argon, the compound c (1 eq) is dissolved in a dry dichloromethane (5 mL) solution, triethylamine (1.5 eq) is added, acyl chloride or oleoyl chloride (1.2 eq) with different chain lengths is slowly dripped at 0 ℃ to react for 30min, the mixture is stirred for 1h at room temperature, water is added to quench, dichloromethane is extracted, a dichloromethane layer is reserved, the mixture is washed with saturated saline water, anhydrous sodium sulfate is dried, the solvent is evaporated under reduced pressure to obtain a crude product, and column chromatography separation is carried out on silica gel to obtain the compound d or e. Compound d or e (1 eq) and the different iodo alkane (1 eq) were dissolved in dry acetonitrile solution, silver tetrafluoroborate (1.2 eq) was added, magnetically stirred in an oil bath at 65 ℃ for 24h, and then cooled to room temperature. Filtering to remove precipitate, exchanging with strong alkali chloride ion exchange resin for 2h, evaporating solvent under reduced pressure to obtain crude product, and separating with silica gel column chromatography to obtain compound of formula II or III.

3. The cationic lipid compound according to claim 1, wherein is a compound of any one of the following structures:

compounds of the formula are designated

S-methyl-N-oleoyl-D-methionine dodecyl chloride (MO 1),

S-ethyl-N-oleoyl-D-methionine dodecyl chloride (MO 2),

S-propyl-N-oleoyl-D-methionine dodecyl chloride (MO 3),

S-butyl-N-oleoyl-D-methionine dodecyl chloride (MO 4),

S-methyl-N-dodecanoyl-D-methionine oleoylaminoethyl ester chloride (MO 5),

S-ethyl-N-lauroyl-D-methionine oleoylaminoethyl chloride (MO 6),

S-propyl-N-dodecanoyl-D-methionine oleoylaminoethyl ester chloride (MO 7),

S-butyl-N-dodecanoyl-D-methionine oleoylaminoethyl ester chloride (MO 8),

S-methyl-N-oleoyl-D-methionine oleoylaminoethyl chloride (MO 9),

S-ethyl-N-oleoyl-D-methionine oleoylaminoethyl chloride (MO 10),

S-propyl-N-oleoyl-D-methionine oleoylaminoethyl chloride (MO 11),

S-butyl-N-oleoyl-D-methionine oleoylaminoethyl chloride (MO 12).

4. Use of a compound according to claims 1-3 for the preparation of a vector for use as a gene. Wherein the genes are: DNA selected from a reporter gene, an anti-cancer gene or a cytokine gene; and RNA selected from siRNA, miRNA, piRNA, dsRNA or shRNA.

5. The use according to claim 4, wherein the compound is useful for delivering a gene by forming a nanocomposite with the gene.