A kind of cationic amino lipid and its synthesis method and application

An amino lipid and cationic technology, applied in the field of cell transfection, can solve the problems of limiting the application range of liposome transfection reagents, high production and R&D costs, and ineffective transfection, so as to reduce production costs and difficulty , the effect of increasing productivity

Inactive Publication Date: 2017-07-25
吴国球
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many cell lines, especially primary cells such as stem cells, cannot be effectively transfected with most existing liposome reagents
[0007] 2. The synthesis is complex and expensive
Due to the current mainstream liposome transfection reagents often have extremely complex structures, their production and research and development costs are high, resulting in high prices of liposome transfection reagents, which greatly limits the use of liposome transfection reagents. Application range

Method used

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  • A kind of cationic amino lipid and its synthesis method and application
  • A kind of cationic amino lipid and its synthesis method and application
  • A kind of cationic amino lipid and its synthesis method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Synthesis of N-(2-(dimethylamino)ethyl)-4,5-bis(undecylthio)-4-pentenamide (LW-30)

[0039]

[0040] The synthesis of amino lipids is divided into two steps. The first step is the synthesis of 4,5-bis(undecylthio)-4-pentenoic acid. Dissolve 0.5 mmol 4-pentynoic acid, 0.75 mmol diundecyl disulfide and 5 mg 2,2-dimethoxy-2-phenylacetone phenyl ketone (DMPA) in 3 mL THF, and add 20 mL In a glass vial, the vial was irradiated with 365nm UV light for 1 h after deoxygenating with nitrogen gas for 5 min.

[0041] The second step is the amidation reaction of 4,5-bis(undecylthio)-4-pentenoic acid with N,N-dimethylethylenediamine to generate N-(2-(dimethylamino )ethyl)-4,5-bis(undecylthio)-4-pentenamide. Add 0.6mmol N,N'-diisopropylcarbodiimide (DIC) to the vial of the first step, stir well, then add 0.5mmol N,N-dimethylethylenediamine, stir for half an hour, then add 0.6 mmol dimethylaminopyridine, stirred overnight. Then the solution was poured into a 25 mL o...

Embodiment 2

[0042] Embodiment 2: the screening of the transfection activity of synthetic lipid

[0043] Cell line: HEK293

[0044] Transfection material: pBL-EGFP fluorescent plasmid

[0045] Cytotoxicity test method: MTT method

[0046] The first step; screening of transfection efficiency:

[0047] (1) Dissolving 29 new types of cationic lipid molecules synthesized in ethanol solution to prepare liposomes. The specific steps are to take 2 mg of each lipid molecule and place them in a round-bottomed flask, spin dry on a rotary evaporator overnight, add 1 mL of PBS solution to fully hydrate after the solvent is fully removed, and incubate on a rotary evaporator for 40 minutes. corresponding liposomes;

[0048] (2) Dissolve 50 nanograms of pBL-EGFP in 100 microliters of PBS, add 50 nanograms of liposomes, and incubate at room temperature for 30 minutes;

[0049] (3) HEK293 cells were digested with trypsin and diluted to 10 with serum-free DMEM medium 5 cell suspension per mL;

[0050] ...

Embodiment 3

[0057] Example 3: Rapid Transfection of Primary Mouse Embryonic Stem Cells

[0058] Amino lipids obtained by hydration Prepare 2mg / mL liposome, which is the mother solution of transfection reagent for cell transfection.

[0059] Reagent: Tube A is the mother solution of the transfection reagent of the present invention; Tube B is the 1×PBS dilution

[0060] Cell line: primary mouse embryonic stem cells

[0061] Transfection material: pBL-EGFP fluorescent plasmid

[0062] Quick transfection step

[0063] (1) Shake tube A slightly before use to mix the solution evenly, take 10 microliters of transfection reagent mother solution and add 390 microliters of 1×PBS dilution to make transfection working solution;

[0064] (2) Dissolve 500 nanograms of pBL-EGFP in 400 microliters of 1× diluent to make a DNA suspension;

[0065] (3) Mix the transfection working solution and DNA suspension at a volume ratio of 1:1 to make a transfection solution and incubate at room temperature for...

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Abstract

The invention discloses cation amino lipid and a synthesis method and application thereof. Structure of cation amino lipid is shown as follows. The synthesis method is mild in synthesis reaction condition and high in solvent selectivity, reaction difficulty is lowered greatly, and yield is increased; raw materials are simple and cheap, so that production cost is lowered greatly; more importantly, lipid molecules obtained through screening have the characteristics of high efficiency and low toxicity and can realize good effect in blood serum, so that operation steps of transfection are simplified greatly.

Description

technical field [0001] The invention belongs to the field of cell transfection, and specifically relates to a cationic amino lipid and its synthesis method and application. Background technique [0002] Among all types of transfection reagents currently available, liposome-based transfection reagents are recognized as the most effective. This is because liposome transfection reagents are easy to use and often highly efficient. Liposomes are artificially prepared spherical vesicles with a bilayer structure, which can fuse with other bilayers, such as cell membranes, to transport the molecules it wraps from the outside of the cell membrane to the inside of the cell. [0003] Due to their unique properties, liposomes are widely used for drug delivery. Liposomes can carry both hydrophilic molecules and hydrophobic molecules, which can be well combined with biologically active molecules, such as various drugs, nucleic acids, polypeptides, etc., and can deliver these substances ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07C323/54C07C319/22C12N15/88A61K9/127A61K47/20A61K48/00
CPCA61K9/1272A61K47/20A61K48/00C07C319/22C07C323/54C12N15/88
Inventor 吴国球杜鑫范小波
Owner 吴国球
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