CN108395411B - Dendrimer cationic lipid, transgenic vector and preparation method thereof - Google Patents
Dendrimer cationic lipid, transgenic vector and preparation method thereof Download PDFInfo
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Abstract
The invention provides dendrimer cationic lipid and a transgenic vector containing the cationic lipid. The invention also discloses a preparation method of the cationic lipid and the transgenic vector. The cationic lipid provided by the invention consists of hydrocarbyl chains with different lengths, benzene rings, triazole and dendritic molecules, and the cationic liposome formed after the cationic lipid is reacted with the auxiliary lipid dioleoyl phosphatidylethanolamine has good blocking performance on DNA, lower cytotoxicity and higher transfection efficiency, and the preparation methods of the cationic lipid and the transgenic carrier are simple, mature and easy to control.
Description
Technical Field
The invention relates to cationic lipid and a transgenic vector, in particular to cationic lipid simultaneously containing a hydrocarbyl chain, a benzene ring, triazole and dendritic molecules, a transgenic vector containing the cationic lipid and a preparation method thereof.
Background
Gene therapy, one of the leading medical approaches, has shown great power in the treatment of various major diseases such as cancer and aids. In the course of gene therapy, gene vectors play a very important role.
Naked DNA is loose, large in volume, generally exists in a stretched linear spiral form, and has electronegativity, so that the naked DNA is difficult to enter cells through membranes, and even a small amount of DNA enters the cells and is easily degraded by nuclease in the cells before reaching the nucleus for expression. These disadvantages make it difficult to perform efficient gene transfection without the aid of other technical means, and therefore the development of safe and effective gene vectors is a prerequisite for successful gene therapy.
Gene vectors include two broad classes: viral vectors and non-viral vectors. The virus vector has the advantages of high transfection efficiency, good targeting property and the like. But the potential immune response and the small load greatly limit the clinical application. Non-viral vectors can completely overcome the above disadvantages of viral vectors, and have the advantages of large carrying capacity, high safety, easy modification of structure, etc., and thus have received extensive attention from researchers.
The dendrimer PAMAM contains more positive charges and can interact with DNA with negative charges. As a gene vector, it has a strong proton sponge effect, and when the pH value in the lysosome is reduced, PAMAM can absorb a large amount of protons, leading to lysosome rupture and DNA release to the cytoplasm. But the greatest disadvantage is its high toxicity. As the molecular weight increases, the positive charge density increases and the cytotoxicity increases. Low molecular weight PAMAM is less cytotoxic, but transfection efficiency is also low. Therefore, the low molecular weight branch type molecule PAMAM is modified, so that the transfection efficiency can be effectively improved, and the cytotoxicity is reduced.
Disclosure of Invention
As a result of a variety of extensive and intensive studies and experiments, the inventors of the present invention have found that low molecular weight PAMAM, after being modified with different lengths of hydrocarbyl chains, exhibits lower cytotoxicity, and that transfection efficiency is greatly related to the lengths of the hydrocarbyl chains: transfection efficiency increases with increasing hydrocarbyl chains, and vectors containing unsaturated bonds transfect more efficiently than vectors containing saturated bonds. Based on this finding, the present invention has been completed.
An object of the present invention is to solve at least the above problems and/or disadvantages and to provide at least the advantages described hereinafter.
It is still another object of the present invention to provide a transgenic vector with multifunctional dendrimer-like cationic lipids, which not only has low cytotoxicity, but also has high transfection efficiency, so as to promote the development of gene therapy.
The invention also aims to provide a preparation method of the dendrimer-containing cationic lipid and a preparation method of a transgenic carrier containing the cationic lipid.
To achieve these objects and other advantages of the present invention, there is provided a dendrimer-based cationic lipid having the following structural formula (1):
in the formula (1), R1Being a hydrocarbyl chain, R2Is a low generation dendritic polyamide molecule.
Among them, it is preferable that R is1Is CH3(CH2)2CH2，CH3(CH2)6CH2，CH3(CH2)10CH2，CH3(CH2)16CH2，CH3(CH2)7CHCH(CH2)7CH2One of (1), R2Is composed of
The object of the invention can be further achieved by a method for preparing dendrimer-like cationic lipids containing hydrocarbyl chains with different lengths, which comprises the following synthetic route:
the method comprises the following specific steps:
step one, weighing a compound of a formula 2, aliphatic amine, a catalyst, a condensing agent and an organic base in proportion, adding a solvent for dissolving, reacting for 8-10 hours in a nitrogen atmosphere, concentrating under reduced pressure after the reaction is finished, and performing column chromatography separation to obtain a compound of a formula 3;
and step two, dissolving the compound of the formula 3 obtained in the step one and propargylamine protected by Boc in a tetrahydrofuran solvent to obtain a tetrahydrofuran solution, adding an aqueous solution dissolved with copper sulfate and vitamin C into the tetrahydrofuran solution, reacting at room temperature for 15-20 hours, after the reaction is finished, concentrating under reduced pressure, separating by column chromatography, adding the obtained compound into a saturated ethyl acetate solution of hydrogen chloride, stirring at room temperature for reacting for 0.5-1 hour, concentrating the solvent, and drying in vacuum for 18-24 hours to obtain the compound of the formula 1.
Preferably, in the first step, the catalyst is 1-hydroxybenzotriazole, the condensing agent is 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, the organic base is triethylamine, and the compound of formula 2 and the fatty amine R are mixed together1NH2The compound, 1-hydroxybenzotriazole, 1-ethyl- (3-dimethylaminopropyl) carbonyldiimidate hydrochloric acid and triethylamine are in a mass ratio of 1:1:2:2:3, an eluant used in column chromatographic separation is petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to the ethyl acetate is 4: 1.
Preferably, in the second step, the volume ratio of tetrahydrofuran in the tetrahydrofuran solution to water in the aqueous solution is 2:1, the molar ratio of the compound of formula 3 to the compound of formula 4 is 1:2.2, the eluent used in the column chromatography separation is dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 10: 1.
To achieve these objects and other advantages in accordance with the present invention, there is also provided a class of transgenic vectors consisting of a dendrimer-like cationic lipid modified with different length hydrocarbyl chains, dioleoylphosphatidylethanolamine, and a highly pure sterilization buffer.
Preferably, the molar concentration of the cationic lipid is 1.0mM, and the molar ratio of the cationic lipid to the dioleoylphosphatidylethanolamine is 1: 6.
The object of the present invention can be further achieved by a method for preparing a transgenic vector, which specifically comprises the steps of: adding the cationic lipid, dioleoyl phosphatidylethanolamine and anhydrous chloroform into a flask sterilized at high temperature, dissolving, concentrating under reduced pressure to obtain a liposome membrane, vacuum drying the liposome membrane to remove residual chloroform, mixing the dried liposome membrane with a trihydroxymethyl aminomethane-hydrochloric acid buffer solution preheated to 70 ℃ in advance to prepare a solution with required concentration, and performing ultrasonic treatment to obtain the transgenic vector.
The aim of the invention can be further realized by the application of the dendrimer cationic lipid containing hydrocarbyl chains with different lengths in the preparation of transgenic vectors.
The invention at least comprises the following beneficial effects:
1. the structure of the cationic liposome provided by the invention contains secondary amine and tertiary amine, and the cationic liposome can be partially protonated and positively charged under physiological conditions, so that the cationic liposome can interact with negatively charged DNA electrostatically, and the DNA is concentrated into nanoparticles suitable for endocytosis of cells;
2. the transgenic vector has low cytotoxicity, and the survival rate of the vector/DNA compound and cells cultured by the cells is more than 90 percent;
3. the preparation method of the cationic lipid and the transgenic vector is simple, mature and easy to control;
4. the transfection efficiency of the transgenic vector is lower in E1 and HeLa cells, and is lower than that of a commercial transgenic vector Lipofectamine 2000; the transfection efficiency in the cell HEK293 is about 2 times of that of Lipofectamine2000, namely the provided cationic liposome shows certain cell selectivity, the transfection efficiency in different cells is greatly different, and the possibility can be provided for clinical targeted therapy;
additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a schematic diagram showing the result of agarose gel electrophoresis of a transgenic vector and DNA complex of the present invention;
FIG. 2 is a graph of cytotoxicity of complexes of the transgenic vectors of the present invention with plasmid DNA in E1, HeLa, MG63, and HEK293 cells;
FIG. 3 shows the particle size and zeta potential of the complex of the transgenic vector and plasmid DNA according to the present invention;
FIG. 4 is a schematic representation of the transfection of the transgenic vectors 1a-1E of the present invention with pGL-3DNA complexes in E1, HeLa, MG63 and HEK293 cells.
The present invention is described in further detail below with reference to specific embodiments and the attached drawings so that those skilled in the art can practice the invention with reference to the description.
Detailed Description
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
< example 1>
Step one, weighing a compound (0.86mmol) of formula 2, an n-butylamine compound (0.86mmol), a 1-hydroxybenzotriazole catalyst (1.72mmol), a 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride condensing agent (1.72mmol) and triethylamine (2.58mmol) according to a ratio, adding 40mL of a dichloromethane solvent for dissolving, reacting for 8-10 hours under a nitrogen atmosphere, after the reaction is finished, evaporating the solvent under reduced pressure, adding 35mL of water, extracting dichloromethane (40mL of x 2), combining organic phases, washing with saturated saline, drying with anhydrous sodium sulfate, removing the solvent, and separating by silica gel column chromatography (the volume ratio of petroleum ether to ethyl acetate is 4/1) to obtain a white solid to obtain the compound of formula 3a, wherein the yield is as follows: 89 percent;
nuclear magnetism1H NMR(400MHz,CDCl3)δ7.67(s,2H),7.40(s,1H),6.18(brs,1H),4.43(s,4H),3.47(dd,J＝3.0,7.1Hz,2H),1.65-1.60(m,2H),1.47-1.38(m,2H),0.97(t,J＝7.3Hz,3H)；
Nuclear magnetism13C NMR(101MHz,CDCl3)δ166.68,136.75,136.09,130.10,126.46,54.12,39.99,31.61,20.15,13.74；
Mass spectrum ESI-MS: M/z 288.1554([ M + H)]+).
And step two, dissolving the compound (0.1mmol) of the formula 3a obtained in the step one and the propargylamine protected by Boc (0.22mmol) of the compound of the formula 4 in a tetrahydrofuran solvent to obtain a tetrahydrofuran solution, adding an aqueous solution dissolved with copper sulfate and vitamin C into the tetrahydrofuran solution, reacting at room temperature for 15-20 hours, concentrating under reduced pressure after the reaction is finished, and separating by column chromatography (an eluent is dichloromethane and methanol with the volume ratio of 10:1) to obtain a white solid. And then adding the solid into a saturated ethyl acetate solution of hydrogen chloride, stirring at room temperature for reaction for 0.5-1 hour, concentrating the solvent, and drying in vacuum for 18-24 hours to obtain the cationic lipid compound of the formula 1a, wherein the yield is as follows: 45 percent.
Nuclear magnetism1H NMR(400MHz,D2O)δ8.42(s,2H),7.64(s,2H),7.59(s,1H),5.76(s,4H),4.64(s,4H),3.66(brs,8H),3.54-3.50(m,40H),3.42(brs,8H),3.33(brs,2H),3.16(brs,16H),2.93(brs,8H),2.86(brs,16H),1.59-1.51(m,2H),1.36-1.32(m,2H),0.89(t,J＝7.2Hz,3H)；
Nuclear magnetism13C NMR(101MHz,D2O)δ172.69,172.41,136.20,135.96,130.93,128.35,126.98,53.48,52.05,49.82,49.38,46.90,43.60,39.88,39.13,36.90,34.34,30.57,29.20,28.75,19.57,13.15；
Mass spectrum HR-MS:883.5003([1/2M + H)]+).
< example 2>
Step one, weighing a compound (0.86mmol) of formula 2, an n-octylamine compound (0.86mmol), a 1-hydroxybenzotriazole catalyst (1.72mmol), a 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride condensing agent (1.72mmol) and triethylamine (2.58mmol) according to a ratio, adding 40mL of a dichloromethane solvent for dissolving, reacting for 8-10 hours under a nitrogen atmosphere, after the reaction is finished, evaporating the solvent under reduced pressure, adding 35mL of water, extracting dichloromethane (40mL of x 2), combining organic phases, washing with saturated saline, drying with anhydrous sodium sulfate, removing the solvent, and performing silica gel column chromatography separation (the volume ratio of petroleum ether to ethyl acetate is 4/1) to obtain a white solid to obtain a compound of formula 3b, wherein the yield is as follows: 91%;
nuclear magnetism1H NMR(400MHz,CDCl3)δ7.67(s,2H),7.39(s,1H),6.24(brs,1H),4.42(s,4H),3.44(dd,J＝13.2,7.1Hz,2H),1.64-1.60(m,2H),1.37-1.29(m,10H),0.87(t,J＝6.8Hz,3H)；
Nuclear magnetism13C NMR(101MHz,CDCl3)δ166.65,136.75,136.09,130.08,126.45,54.12,40.33,31.78,29.57,29.29,29.19,27.03,22.62,14.05；
Mass spectrum ESI-MS: M/z 344.2196([ M + H)]+).
And step two, dissolving the compound (0.1mmol) of the formula 3b obtained in the step one and the propargylamine protected by Boc (0.22mmol) of the compound of the formula 4 in a tetrahydrofuran solvent to obtain a tetrahydrofuran solution, adding an aqueous solution dissolved with copper sulfate and vitamin C into the tetrahydrofuran solution, reacting at room temperature for 15-20 hours, concentrating under reduced pressure after the reaction is finished, and separating by column chromatography (an eluent is dichloromethane and methanol with a volume ratio of 10:1) to obtain a white solid. And then adding the solid into a saturated ethyl acetate solution of hydrogen chloride, stirring at room temperature for reaction for 0.5-1 hour, concentrating the solvent, and drying in vacuum for 18-24 hours to obtain the compound cationic lipid shown in the formula 1b, wherein the yield is as follows: 66 percent.
Nuclear magnetism1H NMR(400MHz,D2O)δ8.52(s,2H),7.76(s,2H),7.67(s,1H),5.82(s,4H),4.70(s,4H),3.72(brs,8H),3.60-3.56(m,40H),3.48(brs,8H),3.34(brs,2H),3.23-3.20(m,16H),3.00(brs,8H),2.99-2.91(m,16H),1.55(brs,2H),1.26-1.10(m,10H),0.71(brs,3H)；
Nuclear magnetism13C NMR(101MHz,D2O)δ172.73,172.45,136.28,136.01,135.71,131.52,128.41,127.19,53.56,52.18,49.91,49.45,47.01,40.18,39.24,36.96,34.43,31.06,29.27,28.85,28.33,26.15,22.04,13.58；
Mass spectrum HR-MS:912.6323([1/2M + H)]+).
< example 3>
Step one, weighing a compound (0.86mmol) of formula 2, a 1-dodecylamine compound (0.86mmol), a 1-hydroxybenzotriazole catalyst (1.72mmol), a 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride condensing agent (1.72mmol) and triethylamine (2.58mmol) according to a ratio, adding 40mL of a dichloromethane solvent for dissolving, reacting for 8-10 hours under a nitrogen atmosphere, after the reaction is finished, evaporating the solvent under reduced pressure, adding 35mL of water, extracting dichloromethane (40mL of x 2), combining organic phases, washing with saturated saline, drying with anhydrous sodium sulfate, removing the solvent, and performing silica gel column chromatography separation (the volume ratio of petroleum ether to ethyl acetate is 4/1) to obtain a white solid to obtain a compound of formula 3c, wherein the yield is as follows: 89 percent;
nuclear magnetism1H NMR(400MHz,CDCl3)δ7.67(s,2H),7.41(s,1H),6.13(brs,1H),4.44(s,4H),3.46(dd,J＝13.2,7.1Hz,2H),1.64-1.61(m,2H),1.38-1.24(m,18H),0.88(t,J＝6.8Hz,3H)；
Nuclear magnetism13C NMR(101MHz,CDCl3)δ166.60,136.81,136.15,130.10,126.39,54.15,40.34,31.91,29.66,29.62,29.57,29.36,29.34,27.05,22.68,14.10；
Mass spectrum ESI-MS: M/z 400.2814([ M + H)]+)；
And step two, dissolving the compound (0.1mmol) of the formula 3C obtained in the step one and the propargylamine protected by Boc (0.22mmol) of the compound of the formula 4 in a tetrahydrofuran solvent to obtain a tetrahydrofuran solution, adding an aqueous solution dissolved with copper sulfate and vitamin C into the tetrahydrofuran solution, reacting at room temperature for 15-20 hours, concentrating under reduced pressure after the reaction is finished, and separating by column chromatography (an eluent is dichloromethane and methanol with a volume ratio of 10:1) to obtain a white solid. And then adding the solid into a saturated ethyl acetate solution of hydrogen chloride, stirring at room temperature for reaction for 0.5-1 hour, concentrating the solvent, and drying in vacuum for 18-24 hours to obtain the compound cationic lipid shown in the formula 1c, wherein the yield is as follows: and 63 percent.
Nuclear magnetism1H NMR(400MHz,D2O)δ8.53(s,2H),7.81(s,2H),7.62(s,1H),5.78(s,4H),4.69(b,4H),3.72(brs,8H),3.58(brs,40H),3.48(brs,8H),3.33(brs,2H),3.22(brs,16H),2.99(brs,8H),2.92(brs,16H),1.55(brs,2H),1.05(brs,18H),0.71(brs,3H)；
Nuclear magnetism13C NMR(101MHz,D2O)δ207.87,172.74,172.41,168.39,136.28,135.93,135.66,131.57,128.46,127.43,123.05,53.60,52.21,49.92,49.42,47.06,43.62,40.25,39.31,37.02,34.46,31.50,29.36,29.17,28.93,26.45,22.32,13.83；
Mass spectrum HR-MS:940.6643([1/2M + H)]+).
< example 4>
Step one, weighing a compound (0.86mmol) of formula 2, a stearamide compound (0.86mmol), a 1-hydroxybenzotriazole catalyst (1.72mmol), a 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride condensing agent (1.72mmol) and triethylamine (2.58mmol) according to a ratio, adding 40mL of a dichloromethane solvent for dissolving, reacting for 8-10 hours under a nitrogen atmosphere, after the reaction is finished, evaporating the solvent under reduced pressure, adding 35mL of water, extracting dichloromethane (40mL of x 2), combining organic phases, washing with saturated saline, drying with anhydrous sodium sulfate, removing the solvent, and separating by silica gel column chromatography (the volume ratio of petroleum ether to ethyl acetate is 4/1) to obtain a white solid to obtain a compound of formula 3d, wherein the yield is as follows: 91%;
nuclear magnetism1H NMR(400MHz,CDCl3)δ7.67(s,2H),7.41(s,1H),6.13(brs,1H),4.44(s,4H),3.46(dd,J＝13.3,7.0Hz,2H),1.66-1.60(m,2H),1.37-1.18(m,30H),0.88(t,J＝6.8Hz,3H)；
Nuclear magnetism13C NMR(101MHz,CDCl3)δ166.58,136.82,136.17,130.10,126.37,54.16,40.34,31.93,2971,29.68,29.63,29.37,27.05,22.69,14.11；
Mass spectrum ESI-MS: M/z 484.3754([ M + H)]+).
And step two, dissolving the compound (0.1mmol) of the formula 3d obtained in the step one and the propargylamine protected by Boc (0.22mmol) of the compound of the formula 4 in a tetrahydrofuran solvent to obtain a tetrahydrofuran solution, adding an aqueous solution dissolved with copper sulfate and vitamin C into the tetrahydrofuran solution, reacting at room temperature for 15-20 hours, concentrating under reduced pressure after the reaction is finished, and separating by column chromatography (an eluent is dichloromethane and methanol with a volume ratio of 10:1) to obtain a white solid. And then adding the solid into a saturated ethyl acetate solution of hydrogen chloride, stirring at room temperature for reaction for 0.5-1 hour, concentrating the solvent, and drying in vacuum for 18-24 hours to obtain the compound cationic lipid shown in the formula 1d, wherein the yield is as follows: 54 percent.
Nuclear magnetism1H NMR(400MHz,D2O)δ8.52(s,2H),7.83(s,2H),7.54(s,1H),5.77(s,4H),4.68(s,4H),3.71-3.48(m,58H),3.21(brs,16H),2.98-2.92(m,24H),1.58(brs,2H),1.19(brs,30H),0.79(brs,3H)；
Nuclear magnetism13C NMR(101MHz,D2O)δ172.66,172.31,168.32,136.20,135.81,130.92,128.43,127.42,62.63,53.54,52.08,49.82,49.30,47.00,40.27,39.22,36.96,34.36,31.73,29.52,29.31,28.87,26.79,22.49,13.90；
Mass spectrum HR-MS:982.7103([1/2M + H)]+).
< example 5>
Step one, weighing a compound (0.86mmol) of formula 2, an oleylamine compound (0.86mmol), a 1-hydroxybenzotriazole catalyst (1.72mmol), a 1-ethyl- (3-dimethylaminopropyl) carbonyldiimidate hydrochloric acid condensing agent (1.72mmol) and triethylamine (2.58mmol) according to a ratio, adding 40mL of a dichloromethane solvent for dissolving, reacting for 8-10 hours under a nitrogen atmosphere, after the reaction is finished, evaporating the solvent under reduced pressure, adding 35mL of water, extracting dichloromethane (40mL of x 2), combining organic phases, washing with saturated saline, drying with anhydrous sodium sulfate, removing the solvent, and separating by silica gel column chromatography (the volume ratio of petroleum ether to ethyl acetate is 4/1) to obtain a white solid to obtain a compound of formula 3e, wherein the yield is as follows: 71 percent;
nuclear magnetism1H NMR(400MHz,CDCl3)δ7.66(s,2H),7.41(s,1H),6.13(brs,1H),5.45-5.27(m,2H),4.43(s,4H),3.46(dd,J＝13.3,7.0Hz,2H),2.02-2.00(m,4H),1.63-1.61(m,4H),1.35-1.24(m,20H),0.88(t,J＝6.8Hz,3H)；
Nuclear magnetism13C NMR(101MHz,CDCl3)δ166.60,136.83,136.15,130.11,129.98,129.76,126.37,54.15,40.33,31.90,29.76,29.70,29.63,29.52,29.47,29.31,29.25,27.22,27.04,22.68,14.10；
Mass spectrum ESI-MS: M/z 482.3507([ M + H)]+).
And step two, dissolving the compound (0.1mmol) of the formula 3e obtained in the step one and the propargylamine protected by Boc (0.22mmol) of the compound of the formula 4 in a tetrahydrofuran solvent to obtain a tetrahydrofuran solution, adding an aqueous solution dissolved with copper sulfate and vitamin C into the tetrahydrofuran solution, reacting at room temperature for 15-20 hours, concentrating under reduced pressure after the reaction is finished, and separating by column chromatography (an eluent is dichloromethane and methanol with a volume ratio of 10:1) to obtain a white solid. And then adding the solid into a saturated ethyl acetate solution of hydrogen chloride, stirring at room temperature for reaction for 0.5-1 hour, concentrating the solvent, and drying in vacuum for 18-24 hours to obtain the cationic lipid of the compound shown in the formula 1e, wherein the yield is as follows: 48 percent.
Nuclear magnetism1H NMR(400MHz,D2O)δ8.55(s,2H),7.87(s,2H),7.54(s,1H),5.80(s,4H),5.34(s,2H),4.75(s,4H),3.75(brs,8H),3.66-3.56(m,40H),3.51(brs,8H),3.46-3.44(m,2H),3.25(brs,16H),3.02(brs,8H),2.95(brs,16H),1.98(brs,4H),1.61(brs,2H),1.21(brs,22H),0.82(brs,3H)；
Nuclear magnetism13C NMR(101MHz,D2O)δ206.30,172.68,172.34,168.27,136.25,135.51,130.17,130.07,128.51,128.34,127.50,53.56,52.33,52.14,49.85,49.33,47.03,43.66,39.27,36.99,34.41,31.73,30.99,29.53,29.34,28.92,28.82,27.05,22.53,13.96；
Mass spectrum HR-MS:981.7049([1/2M + H)]+).
< example 6>
Preparation of transgenic vectors containing the cationic lipid Compounds of formulae 1a-1e in examples 1-5
The starting materials included the cationic lipid compounds of formula 1a-1e prepared in examples 1-5, dioleoylphosphatidylethanolamine DOPE (dioleylphosphatidylethanolamine), anhydrous chloroform and Tris-hydroxymethyl aminomethane-hydrochloric acid (Tris-HCl) buffer. The synthesized cationic lipid of formula 1 (0.005mmol) and DOPE were dissolved in 2.5mL of anhydrous chloroform at different molar ratios (1:2, 1:4, 1:6, 1:8) in a high-temperature sterilized flask, the solvents were thoroughly mixed and dissolved, the solvents were spin-dried under reduced pressure at room temperature to obtain liposome membranes, the mixture was put into a vacuum drying oven to be dried (drying time 12 hours, temperature 25 ℃) to remove residual chloroform, the dried liposome membranes were mixed with a Tris-HCl buffer (10mM, pH7.4) preheated to 70 ℃ in advance, and a buffer solution was added in an amount such that the final concentration of the cationic lipid was 1.0 mM. Finally, the mixture is subjected to ultrasonic treatment at 60 ℃ for 20 minutes and stored in a refrigerator at 4 ℃ for later use.
< example 7>
Example 6 the obtained transgenic vector was tested in the following ways:
(1) agarose gel electrophoresis experiment of transgenic vector and pUC18-DNA compound
Preparation of a transgenic vector and pUC18-DNA Complex
At room temperature, adding the transgenic vector, pUC18-DNA and ultrapure water with corresponding volume into a PE tube respectively to keep the final volume of the reaction liquid at 20 mu L, mixing uniformly, wherein the concentration of the substance of the transgenic vector in the reaction liquid is 1 mu M, 5 mu M,10 mu M,20 mu M and the concentration of pUC18-DNA is 9 mu g/mL respectively, then placing the uniformly mixed reaction liquid into a 37 ℃ constant temperature water bath, preserving the temperature for 0.5 hour, and adding 2 mu L of 10 × Loading Buffer to terminate the reaction.
Preparation of agarose gel
Weighing 280mg of agarose, adding 40mL of 1 × TAE (Tris-acetic acid) buffer solution, heating by microwave to completely dissolve agarose particles to obtain colorless transparent liquid, cooling to about 60 ℃, adding 2 mu L of Goldview II nucleic acid color developing agent, pouring the mixture into a gel making tank with a comb while the mixture is hot after uniform mixing, cooling for 40 minutes until the gel is completely solidified, carefully pulling out the comb, putting the gel making tank with the gel into an electrophoresis tank, and adding 1 × TAE electrophoresis buffer solution to slightly cover the gel surface.
Agarose gel electrophoresis experiment of the complexes
The transgenic vector and 10. mu.L of pUC18-DNA complex prepared above were added to the wells on the gel. Covering an electrophoresis cover, stopping electrophoresis under the voltage of 80V for 40 minutes, taking out the gel, and exposing and sampling in a gel electrophoresis phase forming system.
As shown in FIG. 1, we observed the effect of the transgenic vector on pUC18-DNA using agarose gel electrophoresis. The five vectors have better DNA agglomeration capacity. At a concentration of 10. mu.L, DNA was blocked in the wells.
(2) Transgenic vector cytotoxicity assay
Preparation of a transgenic vector and pUC18-DNA Complex
At room temperature, adding the transgenic vector, pUC18-DNA and a culture medium with a corresponding volume into a PE tube respectively to keep the final volume of the reaction liquid at 500 mu L, mixing uniformly, wherein the concentrations of the transgenic vector in the reaction liquid are respectively 1 mu M,10 mu M,15 mu M and 20 mu M, and the concentration of pUC18-DNA is 9 mu g/mL, then placing the uniformly mixed reaction liquid into a thermostatic water bath at 37 ℃, and preserving the temperature for 0.5 hour.
Cytotoxicity test
Trypsinization of log phase E1, HeLa, MG63 and HEK293 cells approximately 7000 cells per well were seeded into 96-well plates containing 5% CO at 37 deg.C2The culture box is cultured for 24 hours, so that the cells grow to 70-80% of fusion. Discarding the original culture medium in the holes, washing with PBS buffer solution for 1-2 times, adding 100 μ L of the prepared transgenic vector and pUC18-DNA compound with different concentrations into each hole, and setting five parallel holes for each concentration; DMEM medium without transgene vector was used as a blank, commercial Lipofectamine2000 was used as a positive control, and DMEM without cells was used as a blank. After 4 hours all the medium was aspirated, 200. mu.L of DMEM containing 10% fetal bovine serum was added to each well, after 24 hours incubation in an incubator, 10. mu.L of MTT solution was added to each well, after 4 hours all the addition was aspirated, and 150. mu.L of dimethyl sulfoxide was added, and after 10 minutes shaking on a shaker, the absorbance value at 490nm of each well was measured with a microplate reader. The cell survival rate (%) - [ A490 test-blank was calculated as follows]/[ A490 control-blank]×100。
As shown in fig. 2, the cytotoxicity of the transgenic vectors was determined in E1, HeLa, MG63 and HEK293 cells, and the toxicity was found to be low in all of the four cells. The survival rate of the cells is basically over 90 percent.
(3) Particle size and surface potential experiments of transgenic vector and pUC18-DNA complex
Preparation of a transgenic vector and pUC18-DNA Complex
At room temperature, the transgenic vector, pUC18-DNA and the corresponding volume of ultrapure water were added to a PE tube, and the final volume of the reaction solution was kept at 50. mu.L, and the mixture was mixed uniformly, wherein the concentrations of the transgenic vector, pUC18-DNA and pUC18-DNA were 10. mu.M, 20. mu.M, 30. mu.M and 40. mu.M, respectively. Then, the mixture was placed in a thermostatic water bath at 37 ℃ and diluted to 500. mu.L after 30 minutes of heat preservation.
Particle size and surface potential
As shown in FIG. 3, we investigated the particle size of the condensed transgene vector and DNA using dynamic laser light scattering technique. The formed condensing reagents can form particles with the size of 100-700nm with DNA, and the surface potential is-30-10 mV.
(4) In vitro transfection experiment of transgenic vector and pGL-3DNA complex
Preparation of transgenic vector and pGL-3DNA Complex
Adding a transgenic vector, pGL-3DNA and transgenic vectors with different concentrations into a PE tube at room temperature, wherein the concentration of the pGL-3DNA is 9 mug/mL, adding DMEM with corresponding volume to keep the final volume of the reaction liquid at 200 mug/mL, gently blowing, uniformly mixing, putting into a constant-temperature water bath at 37 ℃, and preserving heat for 30 minutes.
In vitro transfection assay
Pancreatin digestion of logarithmic growth phase HepG2 cells will be harvested approximately 7.0 × 10 per well4-9.0×104The individual cells were seeded in 24-well cell culture plates containing 5% CO at 37 deg.C2The culture box is cultured for 24 hours, so that the cells grow to 70-80% of fusion. The original culture medium in the wells is discarded, washed 1-2 times with PBS buffer, and 200. mu.L of the prepared transgenic vector and pGL-3DNA plasmid complex with different concentrations is added into each well. After 4 hours, all the media were aspirated, 200. mu.L of DMEM containing 10% fetal bovine serum was added to each well, and after 24 hours of incubation in an incubator, the 24-well plate was removed and washed twice with PBS wash. Adding 120 mu L of cell lysate, shaking at normal temperature for 15min, transferring the fully-lysed product into a 1.5mL EP tube, centrifuging at 12000rpm for 30s, and transferring the centrifuged supernatant into a new EP tube for subsequent detection. Adding 20 mu L of the lysis sample into a 96-hole white board, adding 100 mu L of Luciferase Assay Reagent, blowing and beating for two to three times, mixing uniformly, and placing on an enzyme-linked immunosorbent Assay for determination.
A complex of the transgenic vector Lipofectamine2000 and pGL-3DNA was prepared as described above. The mass ratio of the transgenic vector 1a-1E to the DOPE is 1:6 to form a cationic liposome, and the cationic liposome and the pGL-3DNA compound are added into E1 cells to carry out in vitro transfection experiments.
As shown in FIG. 4, transfection efficiencies of other transgenic vectors in E1, HeLa, MG63 and HEK293 cells were investigated at a transgenic vector concentration of 10. mu.M. In transgenic vectors 1a-1e, transfection efficiency increased with increasing length of the hydrocarbyl chain. In addition, compared with the transgenic vector Lipofectamine2000, the transgenic vector 1e has higher transfection efficiency in MG63 and HEK293 cells, and particularly in HEK293 cells, the transfection efficiency can reach about 2 times of Lipofectamine 2000.
In conclusion, the transgenic vector 1a-1e has low cytotoxicity, high transfection efficiency in MG63 and HEK293 cells, simple and mature preparation method and easy control.
While embodiments of the invention have been disclosed above, it is not intended to be limited to the uses set forth in the specification and examples. It can be applied to all kinds of fields suitable for the present invention. Additional modifications will readily occur to those skilled in the art. It is therefore intended that the invention not be limited to the exact details and illustrations described and illustrated herein, but fall within the scope of the appended claims and equivalents thereof.
Claims (9)
1. A dendrimer-based cationic lipid, wherein the cationic lipid has the following structural formula (1):
in the formula (1), R1Is CH3(CH2)2CH2，CH3(CH2)6CH2，CH3(CH2)10CH2，CH3(CH2)16CH2，CH3(CH2)7CHCH(CH2)7CH2One of (1), R2Is a low generation dendritic polyamide molecule.
2. The dendrimer cationic lipid of claim 1, wherein R is2Is composed of
3. A method for preparing a dendrimer-like cationic lipid according to claim 1 or 2, wherein the cationic lipid is synthesized by the following route:
the method specifically comprises the following steps:
step one, weighing the compound in the formula 2 and the fatty amine R in proportion1NH2Adding a solvent into the mixture to dissolve the catalyst, the condensing agent and the organic base, reacting for 8-10 hours in a nitrogen atmosphere, concentrating under reduced pressure after the reaction is finished, and carrying out column chromatography separation to obtain a compound shown in the formula 3;
and step two, dissolving the compound of the formula 3 obtained in the step one and the propargylamine protected by Boc (propargyl amine), the compound of the formula 4 in a tetrahydrofuran solvent to obtain a tetrahydrofuran solution, adding an aqueous solution in which copper sulfate and vitamin C are dissolved into the tetrahydrofuran solution, reacting at room temperature for 15-20 hours, concentrating under reduced pressure after the reaction is finished, separating by column chromatography to obtain a compound, adding the compound into a saturated ethyl acetate solution of hydrogen chloride, stirring at room temperature for reacting for 0.5-1 hour, concentrating the solvent, and drying in vacuum for 18-24 hours to obtain the compound of the formula 1.
4. The method according to claim 3, wherein in step one, the catalyst is 1-hydroxybenzotriazole, the condensing agent is 1-ethyl- (3-dimethylaminopropyl) carbonyldiimidate hydrochloride, the organic base is triethylamine, the compound of formula 2, the compound of formula (I) and the compound of formula (II) are mixed, and the mixture is heated to a temperature at which the reaction is carried out,Aliphatic amines R1NH2The compound, 1-hydroxybenzotriazole, 1-ethyl- (3-dimethylaminopropyl) carbonyldiimidate hydrochloric acid and triethylamine are in a mass ratio of 1:1:2:2:3, an eluant used in column chromatographic separation is petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to the ethyl acetate is 4: 1.
5. The method according to claim 3, wherein in the second step, the volume ratio of tetrahydrofuran in the tetrahydrofuran solution to water in the aqueous solution is 2:1, the molar ratio of the compound of formula 3 to the compound of formula 4 is 1:2.2, the eluent used in the column chromatography separation is dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 10: 1.
6. A transgenic vector comprising dioleoylphosphatidylethanolamine, a highly purified sterilization buffer, and the cationic lipid of claim 1.
7. The transgenic vector according to claim 6, wherein the molar concentration of the cationic lipid is 1.0mM and the molar ratio of the cationic lipid to the dioleoylphosphatidylethanolamine is 1: 6.
8. A method for preparing a transgenic vector according to claim 7, the method comprising in particular: adding the cationic lipid, dioleoyl phosphatidylethanolamine and anhydrous chloroform into a flask sterilized at high temperature, dissolving, concentrating under reduced pressure to obtain a liposome membrane, vacuum drying the liposome membrane to remove residual chloroform, mixing the dried liposome membrane with a trihydroxymethyl aminomethane-hydrochloric acid buffer solution preheated to 70 ℃ in advance to prepare a solution with required concentration, and performing ultrasonic treatment to obtain the transgenic vector.
9. Use of the cationic lipid of claim 1 for the preparation of a transgenic vector.
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