Cationic lipid containing macrocyclic polyamine [12]anen3, transgenic carrier and preparation method thereof

A cationic lipid, transgenic vector technology, applied in other methods of inserting foreign genetic materials, gene therapy, carboxylate preparation, etc., can solve the problems of gene size virus mutation, safety concerns, low transfection efficiency, etc. Easy to handle, simple preparation method, good compatibility effect

Active Publication Date: 2018-11-13
BEIJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Viral vector is a highly efficient transfection reagent, but it has disadvantages, such as immunogenicity and carcinogenicity, the size of the gene is easily limited by the size of the virus, and there is a possibility of mutation, so its safety is worrying
Viral vectors are non-immunogenic and convenient for large-scale production, but the transfection efficiency is lower than that of viral vectors

Method used

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  • Cationic lipid containing macrocyclic polyamine [12]anen3, transgenic carrier and preparation method thereof
  • Cationic lipid containing macrocyclic polyamine [12]anen3, transgenic carrier and preparation method thereof
  • Cationic lipid containing macrocyclic polyamine [12]anen3, transgenic carrier and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0044] A cationic lipid of macrocyclic polyamine [12] aneN3, the structural formula of the cationic lipid is as follows:

[0045]

[0046] In formula (I), R is a long-chain fatty acid.

[0047] Where R is

[0048] versus One of them.

[0049] The method of cationic lipid, its synthetic route is as follows:

[0050]

[0051] The specific steps of the method include:

[0052] Step 1. Add 1,3-dibromopropane (98.5mmol) to the 30.0mL acetonitrile solution containing the compound of formula 1 (3.0g, 16.5mmol), stir and reflux for 8-10 hours, until there is a large amount of white solid in the bottle Stop the reaction when it appears. The acetonitrile was removed by rotary evaporation under reduced pressure to obtain a yellow solid-liquid mixture. Add 15.0 mL ethanol and 15.0 mL hydrobromic acid to the obtained yellow solid-liquid mixture, stir and reflux for 7-8 hours; after the reaction is over, wait for the reaction temperature to drop to room temperature, stand still, separate the re...

Embodiment 1

[0124] Preparation of transgenic vector containing cationic lipid compounds of formula 7a-7f in Example 1

[0125] The raw materials include the cationic lipid compounds of formula 7a-7f prepared in Example 1, dioleoylphosphotidylethanolamine (dioleoylphosphotidylethanolamine) and anhydrous chloroform. Dissolve the synthesized cationic lipid formula 1 compound (0.005 mmol) and DOPE in 2.5 mL anhydrous chloroform at a molar ratio of 1:2 in a high-temperature sterilized flask, mix thoroughly and dissolve, and spin-dry the solvent under reduced pressure at room temperature. Transgenic carrier membrane, the mixture is dried in a vacuum drying oven (drying time 12 hours, temperature 25 ℃) to remove residual chloroform, the dried transgenic carrier membrane and preheated to 70 ℃ in advance, tris-hydrochloric acid (Tris -HCl) buffer (10mM, pH 7.4) is mixed, and the amount of buffer solution added makes the final concentration of lipid 1.0mM. Finally, the mixture was sonicated under ult...

Embodiment 2

[0127] The transgenic vector prepared in Example 2 was tested in the following aspects:

[0128] (1) Agarose gel electrophoresis experiment of transgenic vector and pEGFP-N1 plasmid complex Preparation of transgenic vector and pEGFP-N1 plasmid complex

[0129] At room temperature, add the transgenic vector prepared by 7a-7f into the PE tube. The N / P ratio of the transgenic vector to pEGFP-N1 is 0, 2, 4, 8, 16, and the mass of pEGFP-N1 plasmid is 0.125 μg. Use the corresponding volume of ultrapure water to keep the final volume of the reaction solution at 20 μL, and centrifuge to mix evenly. Put it in a constant temperature water bath at 37°C and keep it for 1 hour, then add 2μL of 10×Loading Buffer to stop the reaction.

[0130] Preparation of agarose gel

[0131] Weigh 280mg of agarose, add 40mL 1×TAE buffer solution, microwave heating to completely dissolve the agarose particles, and obtain a colorless transparent liquid. When the temperature drops to about 60°C, add 2μL of Goldvi...

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Abstract

The invention provides a cationic lipid containing macrocyclic polyamine [12]aneN3 and a transgene vector containing the cationic lipid. The invention further discloses preparation methods of the cationic lipid and the transgene vector. Due to the fact that the cationic lipid contains fatty acid chains, the DNA condensation concentration can be effectively lowered, the cationic lipid can form nanoparticles with DNA through the hydrophobic function of the fatty acid chains, the nanoparticles enter cells through aendocytosis, membrane crossing is facilitated, and the transgene vector formed by adopting the cationic lipid has the low cytotoxicity and the higher transfection efficiency. The preparation methods of the cationic lipid and the transgene vector are simple, mature and easy to control.

Description

Technical field [0001] The present invention relates to cationic lipids and genetically modified vectors, in particular to a kind of simultaneous containing macrocyclic polyamines [12]aneN 3 The cationic lipid, the transgenic vector containing the cationic lipid and the preparation method thereof. Background technique [0002] Gene therapy is to introduce foreign normal genes into target cells through a certain method to replace or compensate for some defective or abnormal genes to achieve the purpose of treatment. Gene therapy provides the possibility for the treatment of many diseases, such as: cancer, diabetes, cystic fibrosis, AIDS, cardiovascular disease, etc. [0003] The key to gene therapy is to efficiently introduce therapeutic genes into cells. However, naked DNA generally exists in the form of a stretched linear spiral, which is large in size, and the DNA molecule itself is a negatively charged anion, which will interact with the cell membrane when it enters the cell. T...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D255/02C07C53/18C07C51/41C12N15/87A61K48/00
CPCA61K48/0008C07D255/02C12N15/87
Inventor 卢忠林卢富华高永光张影
Owner BEIJING NORMAL UNIVERSITY
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