CN114181200B - Cationic liposome with efficient gene transfection efficiency and preparation and application thereof - Google Patents
Cationic liposome with efficient gene transfection efficiency and preparation and application thereof Download PDFInfo
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- CN114181200B CN114181200B CN202111403326.5A CN202111403326A CN114181200B CN 114181200 B CN114181200 B CN 114181200B CN 202111403326 A CN202111403326 A CN 202111403326A CN 114181200 B CN114181200 B CN 114181200B
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Abstract
The invention relates to a cationic liposome with high-efficiency gene transfection efficiency, and preparation and application thereof, and belongs to the technical field of chemical medicines. The cationic lipid material comprises a hydrophobic end, a chemically sensitive region, a connecting arm and a hydrophilic end; the hydrophobic end, the chemical sensitive area, the connecting arm and the hydrophilic end are sequentially connected; the chemically sensitive region includes a chemically sensitive bond that is a bond that is cleavable under redox conditions, enzymatic conditions, or acidic pH conditions. The cationic lipid material can be self-assembled to obtain the cationic liposome with a bilayer structure, has small particle size and positive charge, can form a stable nano-composite with the gene medicine with negative charge, can be effectively taken in cells to enter the cells, is stably transfected in the cells, and has high transfection efficiency.
Description
Technical Field
The invention belongs to the technical field of chemical medicines, and particularly relates to a cationic liposome with high-efficiency gene transfection efficiency, and preparation and application thereof.
Background
With the growing understanding of disease pathogenesis at the cellular level, gene therapy has become a hotspot for scientists to study. There is also increasing interest in finding safe and effective gene delivery vehicles.
Current studies show that gene delivery vectors fall into two main categories: viral vectors and non-viral vectors. Viral vectors have certain advantages in gene delivery due to their high transfection efficiency and targeting to most cells. However, the viral vector may cause a series of immune reactions in vivo due to its immunogenicity, and contains a transcribable viral gene, which may undergo genetic recombination or complementation in vivo, further causing injury to the human body.
In recent years, research on non-viral vectors for gene drug delivery has also achieved a certain amount of research effort. The research directions of non-viral vectors mainly comprise cationic polymers (such as polyacetylimine, polylysine and the like), biological macromolecules (such as nucleic acid aptamers, membrane penetrating peptides and the like) and cationic liposomes. The currently commonly used cationic polymers and biomacromolecules have certain limitations, such as systemic toxicity, low transfection efficiency and the like, so that the cationic polymers and biomacromolecules are difficult to be used for intravenous system administration of gene drugs and are commonly used for local administration (such as intratumoral injection). In 1987, felgner et al first prepared 10mg of N- [1- (2, 3) -dioleyloxy ] propyl-N, N, N-trimethylammonium chloride (DOTMA) and dioleoyl phosphatidylethanolamine (DOPE) into small unilamellar liposomes (transfection reagent, trade name Lipofectin, transformed lipid), and experiments demonstrated that this transfection reagent could be used for DNA transfection. Although the transfection efficiency in vitro is conventional chemical transfection methods such as DEAE dextran method and calcium phosphate coprecipitation method, the transfection efficiency in vivo is still low and the cost is high.
Therefore, in summary, the development of the vector material with high gene transfection efficiency, simple preparation method and low price has a very high application prospect.
Disclosure of Invention
The invention solves the key technical problems of immunogenicity, systemic toxicity, complex preparation method, low transfection efficiency, instability, high price and the like of the existing gene delivery vector (including viral vectors and non-viral vectors). The invention provides a cationic liposome with high-efficiency gene transfection efficiency, and preparation and application thereof, wherein the cationic liposome material comprises a hydrophobic end, a chemical sensitive area, a connecting arm and a hydrophilic end; the hydrophobic end, the chemical sensitive area, the connecting arm and the hydrophilic end are sequentially connected; the chemically sensitive region includes a chemically sensitive bond. The cationic liposome has a lipoid structure and is positively charged, so that the cationic liposome can enter cells in an endocytosis or membrane fusion mode, and the transfection efficiency is improved.
According to a first aspect of the present invention there is provided a cationic lipid material comprising a hydrophobic end, a chemically sensitive region, a linker arm and a hydrophilic end; the hydrophobic end, the chemical sensitive area, the connecting arm and the hydrophilic end are sequentially connected; the chemically sensitive region includes a chemically sensitive bond that is a bond that is cleavable under redox conditions, enzymatic conditions, or acidic pH conditions.
Preferably, the hydrophobic end is vitamin E, cholesterol, fatty alcohol, fatty amine, fatty acid, dodecyl isocyanate, octadecyl isocyanate or cis-9-octadecenol; the hydrophilic end is a hydrophilic compound containing at least one amino group;
preferably, the fatty alcohol is a long chain fatty alcohol, a long chain fatty amine or a long chain fatty acid; the carbon atoms of the long-chain fatty alcohol, the long-chain fatty amine and the long-chain fatty acid are at least 14; the hydrophilic end is lysine, arginine, histidine, dicyandiamide, amino hydrochloride or quaternary ammonium salt;
preferably, the long-chain fatty alcohol is a saturated long-chain fatty alcohol or an unsaturated long-chain fatty alcohol; the long-chain fatty amine is saturated long-chain fatty amine or unsaturated long-chain fatty amine; the long-chain fatty acid is saturated long-chain fatty acid or unsaturated long-chain fatty acid.
Preferably, the chemical bond capable of cleavage under redox conditions is a covalent bond containing a sulfur atom or a selenium atom; the chemical bond which can be broken under the acidic pH condition is a hydrazone bond, a phenylhydrazine bond and a hydrazide bond or a pH sensitive bond containing a methyl maleic anhydride bond structure;
preferably, the chemical bond that can be broken under redox conditions is a monosulfide bond, a disulfide bond, a diselenide bond, a monoselene bond, or a diselenide bond; the pH sensitive bond containing the methyl maleic anhydride bond structure is a 2-propionic acid-3-methyl maleic anhydride bond; the connecting arm is 3-amino-1, 2-propanediol or glutamic acid.
According to another aspect of the present invention, there is provided a method for preparing an amphiphilic cationic lipid material, comprising the steps of:
(1) Refluxing a compound containing a monosulfide bond, a disulfide bond, a diselenide bond, a monoselene bond or a diselenide bond in acetyl chloride to obtain an intermediate product A;
(2) Dissolving a hydrophobic end, a catalyst 4-dimethylaminopyridine and the intermediate product A obtained in the step (1) in an organic solvent, and reacting to obtain an intermediate product B; the hydrophobic end is vitamin E, cholesterol or cis-9-octadecenol;
(3) Dissolving the intermediate product B, 3-amino-1, 2-propylene glycol, a catalyst 4-dimethylaminopyridine and a catalyst 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride obtained in the step (2) in an organic solvent, and reacting to obtain an intermediate product C;
(4) Dissolving the intermediate product C obtained in the step (3) in an organic solvent, adding trifluoroacetic acid, and reacting to obtain an intermediate product D;
(5) Dissolving a hydrophilic end, a catalyst 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and a catalyst N-hydroxysuccinimide in an aprotic organic solvent, wherein the hydrophilic end is N-Boc-protected histidine, arginine or lysine, and removing the organic solvent by rotary evaporation after reaction to obtain an active intermediate; dissolving the obtained active intermediate, the catalyst triethylamine and the intermediate D obtained in the step (4) in an organic solvent, and reacting to obtain an intermediate E;
(6) And (3) dissolving the intermediate product E obtained in the step (5) in an organic solvent, adding trifluoroacetic acid, and reacting to obtain the amphiphilic cationic lipid material.
According to another aspect of the present invention, there is provided a method for preparing an amphiphilic cationic lipid material, comprising the steps of:
(1) Refluxing a compound containing a monosulfide bond, a disulfide bond, a diselenide bond, a monoselene bond or a diselenide bond in acetyl chloride to obtain an intermediate product A;
(2) Dissolving a hydrophobic end, a catalyst 4-dimethylaminopyridine and the intermediate product A obtained in the step (1) in an organic solvent, and reacting to obtain an intermediate product B; the hydrophobic end is vitamin E, cholesterol or cis-9-octadecenol;
(3) Dissolving the intermediate product B, 3-amino-1, 2-propylene glycol, a catalyst 4-dimethylaminopyridine and a catalyst 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride obtained in the step (2) in an organic solvent, and reacting to obtain an intermediate product C;
(4) Dissolving the intermediate product C obtained in the step (3) in an organic solvent which can be mixed with water, adding concentrated hydrochloric acid, and reacting to obtain an intermediate product D;
(5) Dissolving dicyandiamide and the intermediate product D obtained in the step (4) in an organic solvent with the boiling point of 150-200 ℃, and carrying out reflux reaction at 120-160 ℃ to obtain the amphiphilic cationic lipid material.
According to another aspect of the present invention, there is provided a method for preparing an amphiphilic cationic lipid material, comprising the steps of:
(1) Dissolving a compound containing 2-propionic acid-3-methyl maleic anhydride bond and an activator oxalyl chloride in N, N-dimethylformamide, and reacting to obtain an intermediate product A;
(2) Dissolving a hydrophobic end, a catalyst triethylamine and the intermediate product A obtained in the step (1) in an anhydrous organic solvent, and reacting to obtain an intermediate product B; the hydrophobic end is vitamin E, cholesterol or cis-9-octadecenol;
(3) Dissolving the intermediate product B, 3-amino-1, 2-propylene glycol and a catalyst 4-dimethylaminopyridine obtained in the step (2) in an anhydrous organic solvent, and reacting to obtain an intermediate product C;
(4) Dissolving the intermediate product C obtained in the step (3) in an organic solvent, adding trifluoroacetic acid, and reacting to obtain an intermediate product D;
(5) Dissolving a hydrophilic end, a catalyst 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and a catalyst N-hydroxysuccinimide in an aprotic organic solvent, wherein the hydrophilic end is N-Boc-protected histidine, arginine or lysine, and removing the organic solvent by rotary evaporation after reaction to obtain an active intermediate; dissolving the obtained active intermediate, the catalyst triethylamine and the intermediate D obtained in the step (4) in an organic solvent, and reacting to obtain an intermediate E;
(6) And (3) dissolving the intermediate product E obtained in the step (5) in an organic solvent, adding trifluoroacetic acid, and reacting to obtain the amphiphilic cationic lipid material.
According to another aspect of the present invention, there is provided a method for preparing an amphiphilic cationic lipid material, comprising the steps of:
(1) Dissolving a compound containing 2-propionic acid-3-methyl maleic anhydride bond and an activator oxalyl chloride in N, N-dimethylformamide, and reacting to obtain an intermediate product A;
(2) Dissolving a hydrophobic end, a catalyst triethylamine and the intermediate product A obtained in the step (1) in an anhydrous organic solvent, and reacting to obtain an intermediate product B; the hydrophobic end is vitamin E, cholesterol or cis-9-octadecenol;
(3) Dissolving the intermediate product B, 3-amino-1, 2-propylene glycol and a catalyst 4-dimethylaminopyridine obtained in the step (2) in an anhydrous organic solvent, and reacting to obtain an intermediate product C;
(4) Dissolving the intermediate product C obtained in the step (3) in an organic solvent which can be mixed with water, adding concentrated hydrochloric acid, and reacting to obtain an intermediate product D;
(5) Dissolving dicyandiamide and the intermediate product D obtained in the step (4) in an organic solvent with the boiling point of 150-200 ℃, and carrying out reflux reaction at 120-160 ℃ to obtain the amphiphilic cationic lipid material.
According to another aspect of the present invention there is provided a cationic liposome derived from any of said cationic lipid materials by self-assembly.
According to another aspect of the present invention, there is provided a method for preparing the cationic liposome, the method comprising: dissolving any cationic lipid material in ethanol or methanol, adding into deionized water, uniformly mixing, and dialyzing with an isotonic solution as an external phase to self-assemble the cationic lipid material to obtain the cationic liposome;
or the preparation method comprises the following steps: dissolving any cationic lipid material in chloroform, ethanol or methanol, removing the organic solvent by rotary evaporation to obtain a film, adding an aqueous solution, and performing ultrasonic treatment to self-assemble the cationic lipid material to obtain the cationic liposome.
According to another aspect of the invention there is provided the use of said cationic liposomes for the preparation of a transfection reagent for delivery of a genetic drug into a cell.
In general, compared with the prior art, the above technical solution conceived by the present invention mainly has the following technical advantages:
(1) The invention uses safe and nontoxic hydrophobic molecules as the hydrophobic end of cationic liposome, preferably vitamin E or cholesterol, and amino acids with a plurality of free amino groups as the hydrophilic end of the cationic liposome, and forms a novel cationic lipid material through a connecting arm. The preparation method is simple, the material sources are convenient, and the prepared novel cationic lipid material is good in safety and low in toxicity. In addition, the cationic liposome has a lipoid structure and is positively charged, so that the cationic liposome is beneficial to entering cells through endocytosis or membrane fusion, and the transfection efficiency is improved.
(2) The novel cationic lipid material provided by the invention comprises a chemical sensitive area, wherein chemical sensitive bonds are arranged in the chemical sensitive area. Under the oxidation or reduction condition and under the low pH condition of lysosomes or endosomes, the chemical sensitive bond can be rapidly broken, so that the liposome structure is damaged, and the escape of the lysosomes and the release of free gene medicines are facilitated.
(3) The novel cationic lipid material provided by the invention can rapidly form the cationic liposome with a bilayer structure through simple preparation methods, such as a film dispersion method, an ethanol injection method and the like, has small particle size and positive charge, can form a stable nano-composite with negatively charged gene drugs (such as pDNA, RNA and the like), can be effectively taken into cells by the cells, is stably transfected in the cells, and has high transfection efficiency. The novel cationic liposome provides a brand new strategy for high-efficiency and low-toxicity delivery of gene drugs.
Drawings
FIG. 1 is a block diagram of an amphiphilic cationic lipid material of the present invention; wherein: 1-hydrophobic end, 2-chemosensitive region, 3-linker arm, 4-hydrophilic end.
FIG. 2 is a hydrogen nuclear magnetic resonance spectrum of VE-COOH.
FIG. 3 is t-Boc-NH 2 Hydrogen nuclear magnetic resonance spectrum of (2).
FIG. 4 is VE-NH 2 Hydrogen nuclear magnetic resonance spectrum of (2).
FIG. 5 is a hydrogen nuclear magnetic resonance spectrum of VE-S-COOH.
FIG. 6 is a graph showing the effect of concentrated cationic liposome transfection.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The invention relates to a cationic lipid material, which comprises a hydrophobic end, a chemical sensitive area, a connecting arm and a hydrophilic end; the hydrophobic end, the chemical sensitive area, the connecting arm and the hydrophilic end are sequentially connected; the chemical sensitive area comprises chemical sensitive bonds which can be broken under the condition of oxidation reduction, a certain pH value or enzyme catalysis. The structural formula of the cationic lipid material in the invention is shown in figure 1, wherein: the hydrophobic end 1 is a vitamin E hydrophobic tail, the chemical sensitive area 2 is a carbon chain structure containing sensitive elements or sensitive bonds, the connecting arm 3 is a branched saturated carbon chain structure, and the hydrophilic end 4 is a cation head containing nitrogen atoms.
The invention discloses a preparation method of an amphiphilic cationic lipid material, which comprises the following steps:
(1) Reflux the compound containing monosulfide bond, disulfide bond, diselenide bond, monoselene bond or diselenide bond in acetyl chloride to obtain product A;
(2) Dissolving vitamin E, cholesterol, cis-9-octadecenol, catalyst 4-dimethylaminopyridine and the product A obtained in the step (1) in an organic solvent, and stirring and reacting for 12-36 h at 50-80 ℃ to obtain a product B;
(3) Dissolving the product B obtained in the step (2), 3-amino-1, 2-propylene glycol, a catalyst 4-dimethylaminopyridine and a catalyst 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in an organic solvent, and stirring and reacting for 16-24 hours at 25-37 ℃ to obtain a product C;
(4) Dissolving the product C obtained in the step (3) in an organic solvent, adding trifluoroacetic acid with the same volume as the organic solvent, and stirring and reacting for 4-8 hours at the temperature of 25-37 ℃ to obtain a product D;
(5) Dissolving N-Boc protected histidine, arginine or lysine, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in an aprotic organic solvent, stirring and reacting for 12-24 hours at 25-37 ℃, and removing the organic solvent by rotary evaporation to obtain an active intermediate; dissolving the obtained active intermediate, triethylamine and the product D obtained in the step (4) in an organic solvent, and stirring and reacting for 16-36 h at 25-37 ℃ to obtain a product E;
(6) And (3) dissolving the product E obtained in the step (5) in an organic solvent, adding trifluoroacetic acid with the same volume as the organic solvent, and stirring and reacting for 4-8 hours at the temperature of 25-37 ℃ to obtain the amphiphilic cationic lipid material.
The invention discloses a preparation method of an amphiphilic cationic lipid material, which comprises the following steps:
(1) Reflux the compound containing monosulfide bond, disulfide bond, diselenide bond, monoselene bond or diselenide bond in acetyl chloride to obtain product A;
(2) Dissolving vitamin E, cholesterol, cis-9-octadecenol, catalyst 4-dimethylaminopyridine and the product A obtained in the step (1) in an organic solvent, and stirring and reacting for 12-36 h at 50-80 ℃ to obtain a product B;
(3) Dissolving the product B obtained in the step (2), 3-amino-1, 2-propylene glycol, a catalyst 4-dimethylaminopyridine and a catalyst 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in an organic solvent, and stirring and reacting for 16-24 hours at 25-37 ℃ to obtain a product C;
(4) Dissolving the product C obtained in the step (3) in an organic solvent which can be mixed with water, adding concentrated hydrochloric acid with the same volume as the organic solvent, and stirring and reacting for 4-8 hours at the temperature of 25-37 ℃ to obtain a product D;
(5) And (3) dissolving dicyandiamide and the product D obtained in the step (4) in an organic solvent with the boiling point of 150-200 ℃, and refluxing for 12-14 h at 120-160 ℃ to obtain the amphiphilic cationic lipid material.
The invention discloses a preparation method of an amphiphilic cationic lipid material, which comprises the following steps:
(1) Dissolving a compound containing 2-propionic acid-3-methyl maleic anhydride bond and an activator oxalyl chloride in N, N-dimethylformamide, and stirring and reacting for 12-36 h at 25-37 ℃ to obtain a product A;
(2) Dissolving vitamin E, cholesterol, cis-9-octadecenol and triethylamine in the product A obtained in the step (1) in an anhydrous organic solvent, and stirring and reacting for 12-36 h at 25-37 ℃ to obtain a product B;
(3) Dissolving the product B, 3-amino-1, 2-propylene glycol and a catalyst 4-dimethylaminopyridine obtained in the step (2) in an anhydrous organic solvent, and stirring and reacting for 16-24 h at 25-37 ℃ to obtain a product C;
(4) Dissolving the product C obtained in the step (3) in an organic solvent, adding trifluoroacetic acid with the same volume as the organic solvent, and stirring and reacting for 4-8 hours at the temperature of 25-37 ℃ to obtain a product D;
(5) Dissolving N-Boc protected histidine, arginine or lysine, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in an aprotic organic solvent, stirring and reacting for 12-24 hours at 25-37 ℃, and removing the organic solvent by rotary evaporation to obtain an active intermediate; dissolving the obtained active intermediate, triethylamine and the product D obtained in the step (4) in an organic solvent, and stirring and reacting for 16-36 h at 25-37 ℃ to obtain a product E;
(6) And (3) dissolving the product E obtained in the step (5) in an organic solvent, adding trifluoroacetic acid with the same volume as the organic solvent, and stirring and reacting for 4-8 hours at the temperature of 25-37 ℃ to obtain the amphiphilic cationic lipid material.
The invention discloses a preparation method of an amphiphilic cationic lipid material, which comprises the following steps:
(1) Dissolving a compound containing 2-propionic acid-3-methyl maleic anhydride bond and an activator oxalyl chloride in N, N-dimethylformamide, and stirring and reacting for 12-36 h at 25-37 ℃ to obtain a product A;
(2) Dissolving vitamin E, cholesterol, cis-9-octadecenol and triethylamine in the product A obtained in the step (1) in an anhydrous organic solvent, and stirring and reacting for 12-36 h at 25-37 ℃ to obtain a product B;
(3) Dissolving the product B, 3-amino-1, 2-propylene glycol and a catalyst 4-dimethylaminopyridine obtained in the step (2) in an anhydrous organic solvent, and stirring and reacting for 16-24 h at 25-37 ℃ to obtain a product C;
(4) Dissolving the product C obtained in the step (3) in an organic solvent which can be mixed with water, adding concentrated hydrochloric acid with the same volume as the organic solvent, and stirring and reacting for 4-8 hours at the temperature of 25-37 ℃ to obtain a product D;
(5) And (3) dissolving dicyandiamide and the product D obtained in the step (4) in an organic solvent with the boiling point of 150-200 ℃, and refluxing for 12-14 h at 120-160 ℃ to obtain the amphiphilic cationic lipid material.
Example 1
The embodiment provides a preparation method of a novel amphipathic cationic lipid material with esterase sensitivity, which is synthesized through the following steps:
(1) Synthesis of VE-COOH: 4.3g Vitamin E (VE), 1.5g Succinic Anhydride (SA) and 3.15g triethylamine were weighed into a 100mL round bottom flask and 50mL methylene chloride was added. After stirring and dissolving at 70 ℃, the reflux reaction is continued for 24 hours. After the reaction was completed, 50mL of deionized water was added to extract three times, and the organic layer was dried over anhydrous sodium sulfate overnight, filtered, and the organic solvent was removed by spin evaporation, and the obtained oil was dissolved with three times of n-hexane at 60 ℃, gradually cooled to room temperature, and then recrystallized at 4 ℃ overnight. Filtering to obtain white powdery solid, and vacuum drying the obtained white powder at 40 ℃ overnight to remove residual organic solvent to obtain vitamin E succinate (VE-COOH) with the yield of 60.6%.
(2) N- (2, 3-dihydroxypropyl) carbamic acid tert-butyl ester (t-Boc-NH) 2 ) Is synthesized by the following steps: 4.2g of 3-amino-1, 2-propanediol and 12.1g of di-tert-butyl dicarbonate (Boc) 2 O) is placed in a 100mL round bottom flask, 50mL of N, N-dimethylformamide is added for dissolution, 7.4mL of triethylamine is continuously added, and the mixture is reacted with 1 at 25 ℃ under the protection of nitrogenAnd 6h. After the reaction, N-dimethylformamide and triethylamine were removed by rotary evaporation to give a transparent oily substance. The product was purified by silica gel column chromatography with dichloromethane as eluent: methanol=20: 1. combining the organic solutions containing the desired product, removing the organic solvent by spin evaporation, and continuously drying at 40deg.C under vacuum overnight to remove the residual organic solvent to obtain tert-butyl N- (2, 3-dihydroxypropyl) carbamate (t-Boc-NH) 2 ) The yield was 80.7%.
(3)VE-NH 2 Synthesis of (Boc): 6.21g of VE-COOH, 2.28g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl), 0.777. 0.777g t-Boc-NH were weighed out 2 1.44g of 4-dimethylaminopyridine was dissolved in 250mL of methylene chloride with the addition of 100mL of methylene chloride in a 250mL round bottom flask. Under the protection of nitrogen, the reaction is carried out for 24 hours at 25 ℃. After the reaction is finished, the organic solvent is removed by rotary evaporation, the obtained oily substance is further purified by silica gel column chromatography, and the eluent is petroleum ether: ethyl acetate = 4:1. combining the organic solutions containing the required products, removing the organic solvent by rotary evaporation, and continuously drying at 40 ℃ under vacuum overnight to remove the residual organic solvent to obtain VE-NH 2 (Boc) in a yield of 56.7%.
(4)VE-NH 2 Is synthesized by the following steps: the VE-NH obtained in the step (3) 2 (Boc) after dissolving with 40mL of dichloromethane, an equal volume of trifluoroacetic acid was added and reacted at 25℃for 4h. After the reaction, the organic solvent is removed by rotary evaporation, a small amount of dichloromethane is continuously added for repeated rotary evaporation for three times, and the residual trifluoroacetic acid is removed. After dissolving the resulting oil with 50mL of dichloromethane, it was extracted 3-5 times with 5% aqueous sodium bicarbonate until the organic layer had a pH of 8-9. The organic layer was dried over anhydrous sodium sulfate overnight, filtered, the organic solvent was removed by rotary evaporation, and the residual organic solvent was removed by further vacuum drying overnight at 40 ℃ to give VE-NH2 in 88.7% yield.
(5)VE-NH 2 -lys(Boc) 2 Is synthesized by the following steps: 3.46g of 2, 6-di-tert-butoxycarbonylaminohexanoic acid, 3.8g of 1- (3-dimethylpropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 2.3. 2.3g N-hydroxysuccinimide (NHS) were weighed into a 50mL round bottom flask, dissolved in 30mL of acetonitrile, and reacted at 25℃for 24 hours under nitrogen protection. After the reaction is finished, acetonitrile is removed by rotary evaporation, and the 2, 6-di-tert-butoxycarbonyl amino is obtainedReactive intermediates of caproic acid. Weigh 5.5g VE-NH 2 After 20mL of methylene chloride was dissolved in a 100mL round bottom flask, 1.5mL of triethylamine was added. The active intermediate of 2, 6-di-tert-butoxycarbonyl aminocaproic acid obtained before was dissolved in 20mL of methylene chloride, and then was added dropwise to the above mixed solution to react at 25℃for 24 hours. After the reaction was completed, the reaction mixture was extracted with deionized water for 3-5 times, and the organic layer was dried over anhydrous sodium sulfate, and then dichloromethane was removed by rotary evaporation to give a pale yellow oily substance. Recrystallizing the obtained oily matter with methanol at 4deg.C overnight, washing the solid with methanol for 3 times, vacuum drying at 40deg.C overnight, and removing residual organic solvent to obtain VE-NH 2 -lys(Boc) 2 The yield was 67.8%.
(6)VE-NH 2 -synthesis of lys: the VE-NH obtained in the step (5) 2 -lys(Boc) 2 After dissolution with an appropriate amount of dichloromethane, an equal volume of trifluoroacetic acid was added and reacted for 6h at 25 ℃. After the reaction, the organic solvent is removed by rotary evaporation, a small amount of dichloromethane is continuously added for repeated rotary evaporation for three times, and the residual trifluoroacetic acid is removed. After dissolving the resulting oil with 50mL of dichloromethane, it was extracted 3-5 times with 5% aqueous sodium bicarbonate until the organic layer had a pH of 8-9. Drying the organic layer with anhydrous sodium sulfate overnight, filtering, steaming to remove organic solvent, and vacuum drying at 40deg.C overnight to remove residual organic solvent to obtain VE-NH 2 Lys, yield 89.8%. VE-NH 2 -lys has the formula:
the hydrophobic end in this embodiment is vitamin E, the sensitive bond is esterase sensitive bond (ester bond), the connecting arm is 3-amino-1, 2-propanediol, the hydrophilic end is lysine, and the structural general formula of the cationic lipid material is shown as follows:
wherein L1 represents a portion of the hydrophobic end; r represents CH 2 The esterase-sensitive bond is an ester bond between R and L1; l2 represents a hydrophilic end; the connecting arm is 3-amino-1, 2-propylene glycol.
The reaction formula in this example is shown below:
FIGS. 2,3 and 4 are VE-COOH, t-Boc-NH, respectively 2 And VE-NH 2 The hydrogen nuclear magnetic resonance spectrum of the above structure confirms the successful synthesis of the cationic paper material.
Example 2
The embodiment provides a preparation method of a novel amphiphilic cationic lipid material with redox sensitivity, which is synthesized through the following steps:
(1) Synthesis of 2,2' -thiodiacetic anhydride: 3.0g of 2,2' -thiodiacetic acid was weighed into a 50mL round bottom flask and 25mL of acetic anhydride was added. Reflux reaction is carried out for 4 hours at 65 ℃, and acetic anhydride, acetic acid and the like are removed by reduced pressure distillation; adding proper amount of diethyl ether, repeatedly steaming for 2-3 times to obtain white solid powder, and vacuum drying at 40 ℃ overnight to obtain 2,2' -thiodiacetic anhydride with the yield of 98.7%.
(2) Synthesis of VE-S-COOH: 4.3g Vitamin E (VE), 1.98g 2, 2-thiodiacetic anhydride and 1.12g 4-dimethylaminopyridine were weighed into a 100mL round bottom flask and 50mL methylene chloride was added. After stirring and dissolving at 70 ℃, the reflux reaction is continued for 36h. After the reaction, 50mL of deionized water is added for extraction for three times, the organic layer is dried over night by anhydrous sodium sulfate, filtered, the organic solvent is removed by rotary evaporation, the obtained oily matter is dried over night at 40 ℃ in vacuum to remove the residual organic solvent, and the cake VE-S-COOH is obtained, and the yield is 80.6%.
(3) N- (2, 3-dihydroxypropyl) carbamic acid tert-butyl ester (t-Boc-NH) 2 ) Is synthesized by the following steps: the same synthesis as in step (2) of example 1 gave t-Boc-NH 2 。
(4)VE-S-NH 2 Synthesis of (Boc): the same synthesis as in example 1 gave VE-S-NH 2 (Boc) except for replacing VE-COOH with VE-S-COOHThe synthesis and product purification procedure were the same as in step (3) of example 1.
(5)VE-S-NH 2 Is synthesized by the following steps: the same synthesis as in example 1 gave VE-S-NH 2 Only VE-NH 2 (Boc) substitution with VE-S-NH 2 (Boc), the synthesis and purification of the product were identical to those of step (4) in example 1.
(6)VE-S-NH 2 Synthesis of His: by two-step synthesis of VE-S-NH 2 His, which is synthesized in the same manner as in step (5) and step (6) of example 1, gives VE-S-NH 2 -His。VE-S-NH 2 -His formula:
the hydrophobic end in this embodiment is vitamin E, the sensitive bond is a redox sensitive bond (a single thioether bond), the connecting arm is 3-amino-1, 2-propanediol, the hydrophilic end is histidine, and the structural general formula of the cationic lipid material is shown as follows:
wherein L1 represents a portion of the hydrophobic end; r represents a sulfur atom (S) or a selenium atom (Se); l2 represents a hydrophilic end; the connecting arm is 3-amino-1, 2-propylene glycol.
The reaction formula in this example is shown below:
FIG. 5 is a hydrogen nuclear magnetic resonance spectrum of VE-S-COOH, confirming successful synthesis of cationic lipid materials of the above structure.
Example 3
The embodiment provides a preparation method of a novel amphiphilic cationic lipid material with redox sensitivity, which is synthesized through the following steps:
(1) Synthesis of (ethylene dithio) diacetic anhydride: 2.0g of (ethylenedithio) diacetic acid was weighed into a 50mL round bottom flask and 20mL of anhydrous acetyl chloride was added. Reflux reaction is carried out for 4 hours at 65 ℃, and then acetyl chloride, acetic acid and the like are removed by reduced pressure distillation; adding proper amount of diethyl ether, repeatedly steaming for 2-3 times to obtain white solid powder, and vacuum drying at 40deg.C overnight to obtain (ethylene dithio) diacetic anhydride with yield of 95.6%.
(2) Synthesis of CH-SCCS-COOH: 7.73g of cholesterol, 7.64g of ethylene dithio) diacetic anhydride and 1.22g of 4-dimethylaminopyridine were weighed into a 250mL round bottom flask, 100mL of methylene chloride was added, and after dissolution under reflux and stirring at 65℃the reaction was continued for 48 hours. After the reaction, the reaction solution was extracted three times with a hydrochloric acid solution of 0.1mmoL, the organic layer was dried over night with anhydrous sodium sulfate, the organic solvent was removed by spin evaporation, and the obtained white solid was further dried under vacuum at 40℃over night to remove the residual organic solvent, to obtain CH-SCCS-COOH, with a yield of 86.7%.
(3)CH-SCCS-NH 2 Is synthesized by the following steps: CH-SCCS-NH synthesized by two-step synthesis method 2 The synthesis and product purification procedures were the same as in synthesis steps (3) and (4) of example 1, respectively, except that VE-COOH in step (3) was replaced with CH-SCCS-COOH.
(4)CH-SCCS-NH 2 -synthesis of Arg: CH-SCCS-NH synthesized by two-step synthesis method 2 Arg, method of synthesis and method of purification of product are the same as the synthesis steps (5) and (6) in example 1, respectively, except that 2, 6-di-t-butoxycarbonylaminohexanoic acid is replaced with tri-t-butoxycarbonylarginine, VE-NH 2 Replaced by CH-SCCS-NH 2 。CH-SCCS-NH 2 -Arg has the structural formula:
the hydrophobic end in this embodiment is vitamin E, the sensitive bond is a redox sensitive bond (a single thioether bond), the connecting arm is 3-amino-1, 2-propanediol, the hydrophilic end is histidine, and the structural general formula of the cationic lipid material is shown as follows:
wherein L1 represents a portion of the hydrophobic end; r represents a sulfur atom (S) or a selenium atom (Se); l2 represents a hydrophilic end; the connecting arm is 3-amino-1, 2-propylene glycol.
The reaction formula in this example is shown below:
example 4
The embodiment provides a preparation method of a novel amphiphilic cationic lipid material with redox sensitivity, which is synthesized through the following steps:
(1) VE-S-NH was synthesized in the same manner as in example 2 2 (Boc)。
(2)VE-S-NH 2 Synthesis of HCl: weighing 3.0g of VE-S-NH 2 (Boc) in a 100mL round bottom flask, 30mL dioxane was added to dissolve, and then an equal volume of concentrated hydrochloric acid was added. After the reaction is carried out for 6 to 8 hours at the temperature of 25 ℃, the organic solvent and part of water are removed by reduced pressure rotary evaporation, ethyl acetate is added for repeated rotary evaporation for 5 to 8 times, and the residual water is removed. Drying the oily matter at 40deg.C under vacuum overnight to remove residual organic solvent to obtain cake VE-S-NH 2 HCl, yield 89.7%.
(3)VE-S-NH 2 Synthesis of DCD & HCl: 1.25g of VE-S-NH was weighed out 2 HCl and 0.168g of dicyandiamide are placed in a 10mL round-bottomed flask, 3mL of isopropanol are added, after refluxing at 140 ℃ for 24 hours, the organic solvent is removed by rotary evaporation under reduced pressure, the obtained oily substance is purified by silica gel column chromatography, and the eluent is methanol: dichloromethane = 30:1. collecting the eluent containing the required product, performing rotary evaporation under reduced pressure, and vacuum drying at 40deg.C overnight to remove residual organic solvent to obtain VE-S-NH 2 DCD. HCl, yield 47.4%. VE-S-NH 2 The structural formula of DCD & HCl is as follows:
the hydrophobic end in this embodiment is vitamin E, the sensitive bond is oxidation-reduction sensitive bond (single thioether bond), the connecting arm is 3-amino-1, 2-propanediol, the hydrophilic end is dicyan diamine hydrochloride, and the structural general formula of the cationic lipid material is shown as follows:
wherein L1 represents a portion of the hydrophobic end; r represents a sulfur atom (S) or a selenium atom (Se); l2 represents a hydrophilic end; the connecting arm is 3-amino-1, 2-propylene glycol.
The reaction formula in this example is shown below:
example 5
The embodiment provides a synthesis method of a novel amphipathic cationic lipid material with esterase sensitivity, which is synthesized through the following steps:
(1) Synthesis of GA-C18: 11.8g of glutamic acid and 18.3g of 4-methylbenzenesulfonic acid (p-Tos) were weighed out and placed in a 500mL round bottom flask, and 350mL of toluene was added for dissolution, followed by stirring at 25℃for 1 hour. 47.8g of cis-9-octadecenol was added, and the reaction was continued at 25℃with stirring for 24 hours. After the reaction, removing the organic solvent by rotary evaporation, adding 100mL of dichloromethane for dissolution, extracting with 5% sodium bicarbonate solution with equal volume for three times, drying the organic layer with anhydrous sodium sulfate overnight, filtering, removing the organic solvent by rotary evaporation, and continuously drying at 40 ℃ in vacuum overnight to remove the residual organic solvent, thereby obtaining GA-C18 with the yield of 65.7%.
(2) Synthesis of N-Boc-Lys-GA-C18: 3.46g of 2, 6-di-tert-butoxycarbonylaminohexanoic acid, 3.8g of 1- (3-dimethylpropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 2.3. 2.3g N-hydroxysuccinimide (NHS) were weighed into a 50mL round bottom flask, dissolved in 30mL of acetonitrile, and reacted at 25℃for 24 hours under nitrogen protection. After the reaction is finished, acetonitrile is removed by rotary evaporation, and the active intermediate of the 2, 6-di-tert-butoxycarbonyl amino caproic acid is obtained. 2.7-gGA-C18 was weighed and placed in the 100mL round bottom flask as described above, and after 30mL of methylene chloride was dissolved, 1.5mL of triethylamine was added. The active intermediate of 2, 6-di-tert-butoxycarbonyl aminocaproic acid obtained before was dissolved in 20mL of methylene chloride, and then was added dropwise to the above mixed solution to react at 25℃for 24 hours. After the reaction was completed, the reaction mixture was extracted with deionized water for 3-5 times, and the organic layer was dried over anhydrous sodium sulfate, and then dichloromethane was removed by rotary evaporation to give a pale yellow oily substance. Recrystallizing the obtained oily matter with methanol at 4deg.C overnight, washing the solid with methanol for 3 times, vacuum drying at 40deg.C overnight, and removing residual organic solvent to obtain N-Boc-Lys-GA-C18 with a yield of 77.9%.
(3) Synthesis of Lys-GA-C18: after dissolving the N-Boc-Lys-GA-C18 obtained in the step (2) with a proper amount of dichloromethane, adding an equal volume of trifluoroacetic acid, and reacting for 4 hours at 25 ℃. After the reaction, the organic solvent is removed by rotary evaporation, a small amount of dichloromethane is continuously added for repeated rotary evaporation for three times, and the residual trifluoroacetic acid is removed. After dissolving the resulting oil with 50mL of dichloromethane, it was extracted 3-5 times with 5% aqueous sodium bicarbonate until the organic layer had a pH of 8-9. The organic layer was dried over anhydrous sodium sulfate overnight, filtered, the organic solvent was removed by rotary evaporation, and the residual organic solvent was removed by further vacuum drying overnight at 40℃to give Lys-GA-C18 in 88.7% yield. The structural formula of Lys-GA-C18 is as follows:
the hydrophobic end in this embodiment is cis-9-octadecenol, the sensitive bond is esterase sensitive bond (ester bond), the connecting arm is glutamic acid, the hydrophilic end is lysine, and the structural general formula of the cationic lipid material is shown as follows:
wherein L1 represents a portion of the hydrophobic end; l2 represents a hydrophilic end; the connecting arm is glutamic acid.
The reaction formula in this example is shown below:
example 6
The embodiment provides a preparation method of a novel amphiphilic cationic liposome, which comprises the following steps: weighing 50mg of the cationic lipid material, dissolving in 1mL of absolute ethyl alcohol, rapidly injecting an ethanol solution into deionized water, stirring for 2 hours at 800 rotation speed, taking 5% glucose solution as an external phase, and dialyzing for 12-36 hours to obtain the cationic liposome. The particle size of the obtained cationic liposome is 122.9+/-4.5 nm, the polydispersity PDI is 0.161+/-0.008, and the zeta potential is 46.26+/-3.19.
Example 7
The embodiment provides a preparation method of a liposome and gene drug compound, which comprises the following steps: 10mg of various cationic liposome freeze-dried powder is weighed, and 1mL of PBS is added for dispersion for later use. And uniformly mixing the prepared blank cationic liposome with the concentration of 10mg/mL and the EGFP plasmid according to the mass ratio of 60:1 (the mass of the plasmid is fixed to be 2 micrograms), and incubating at 27 ℃ for 30 minutes to obtain the liposome and gene complex. The resulting complex exhibited higher transfection efficiency for B16F10 cells, and the results are shown in fig. 6. As can be seen from fig. 6, all three cationic liposomes have a certain transfection capacity, and VOBP liposomes showed the best transfection effect compared to PEI 25K (positive control).
It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the invention and is not intended to limit the invention, but any modifications, equivalents, improvements or alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (5)
1. A cationic lipid material, characterized in that the cationic lipid material comprises a hydrophobic end, a chemically sensitive region, a linker arm and a hydrophilic end; the hydrophobic end, the chemical sensitive area, the connecting arm and the hydrophilic end are sequentially connected; the chemical sensitive area comprises a chemical sensitive bond, and the chemical sensitive bond is a single thioether bond;
the hydrophobic end is vitamin E, and the hydrophilic end is dicyandiamide; the connecting arm is 3-amino-1, 2-propylene glycol;
the structural formula of the cationic lipid material is shown as follows:
2. the method of preparing a cationic lipid material according to claim 1, comprising the steps of:
(1) Refluxing 2,2' -thiodiacetic acid in acetyl chloride to obtain an intermediate product A;
(2) Dissolving a hydrophobic end, a catalyst 4-dimethylaminopyridine and the intermediate product A obtained in the step (1) in an organic solvent, and reacting to obtain an intermediate product B; the hydrophobic end is vitamin E;
(3) Dissolving an intermediate product B, N- (2, 3-dihydroxypropyl) carbamic acid tert-butyl ester obtained in the step (2), a catalyst 4-dimethylaminopyridine and a catalyst 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in an organic solvent, and reacting to obtain an intermediate product C;
(4) Dissolving the intermediate product C obtained in the step (3) in an organic solvent which can be mixed with water, adding concentrated hydrochloric acid, and reacting to obtain an intermediate product D;
(5) Dissolving dicyandiamide and the intermediate product D obtained in the step (4) in an organic solvent with the boiling point of 150-200 ℃, and carrying out reflux reaction at 120-160 ℃ to obtain the cationic lipid material.
3. A cationic liposome obtained by self-assembly from the cationic lipid material of claim 1.
4. A method of preparing a cationic liposome according to claim 3, wherein the method of preparing is: dissolving the cationic lipid material of claim 1 in ethanol or methanol, adding into deionized water, uniformly mixing, and dialyzing with an isotonic solution as an external phase to self-assemble the cationic lipid material to obtain the cationic liposome;
or the preparation method comprises the following steps: dissolving the cationic lipid material according to claim 1 in chloroform, ethanol or methanol, removing an organic solvent by rotary evaporation, wherein the organic solvent is chloroform, ethanol or methanol to obtain a film, adding an aqueous solution, and performing ultrasonic treatment to self-assemble the cationic lipid material to obtain the cationic liposome.
5. Use of a cationic liposome according to claim 3 for the preparation of a transfection reagent for delivery of a genetic drug into a cell.
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