Based on tetraphenylethylene containing macrocyclic polyamine [12]anen 3 Cationic lipid, transgene carrier and preparation method thereof

A technology of cationic lipids and transgenic carriers, which is applied in liposome delivery, gene therapy, medical preparations of non-active ingredients, etc., can solve the problems of gene size virus variation, safety concerns, and low transfection efficiency, and achieve Easy to handle, strong transfection efficiency, and low cytotoxicity

Active Publication Date: 2020-06-02
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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  • Based on tetraphenylethylene containing macrocyclic polyamine [12]anen <sub>3</sub> Cationic lipid, transgene carrier and preparation method thereof
  • Based on tetraphenylethylene containing macrocyclic polyamine [12]anen <sub>3</sub> Cationic lipid, transgene carrier and preparation method thereof
  • Based on tetraphenylethylene containing macrocyclic polyamine [12]anen <sub>3</sub> Cationic lipid, transgene carrier and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0045] A cationic lipid containing macrocyclic polyamine [12]aneN3 based on tetraphenylethylene, the structural formula of the cationic lipid is as follows:

[0046]

[0047] Wherein, in formula (I), n is a positive integer, and R is a hydrocarbon group;

[0048] Among them, n is 1 or 2, R is or

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

[0050]

[0051]

[0052] The specific steps of the method include:

[0053] Step 1. The compound of formula 1 was dissolved in bromoethanol (1.24 g, 10 mmol), sodium azide (3.25 g, 50 mmol), and 10 mL of DMF, and reacted at 80° C. for 12 hours. Add 30 mL of saturated NaCl aqueous solution, extract with ethyl acetate (25 mL×3), combine the organic phases, wash with saturated NaCl aqueous solution (50 mL×2), dry the organic phase with anhydrous sodium sulfate powder, filter, and distill off the solvent under reduced pressure to obtain Colorless liquid, in a 50mL single-necked bottle, the above-men...

Embodiment 1

[0104] Preparation of the transgenic vector containing cationic lipid formula 11a-11d compound in embodiment 1

[0105] The raw materials include cationic lipid compounds of formulas 11a-11d prepared in Embodiment 1, dioleoylphosphatidylethanolamine DOPE (dioleoylphosphotidylethanolamine) and anhydrous chloroform. In a high-temperature sterilized flask, the synthetic cationic lipid formula 1 compound (0.005mmol) and DOPE were dissolved in 2.5mL of anhydrous chloroform with a molar ratio of 2:1 or 1:1 or 1:2, and after fully mixing and dissolving, the The solvent was spin-dried under reduced pressure at room temperature to obtain the transgenic carrier membrane, and the mixture was dried in a vacuum drying oven (drying time 12 hours, temperature 25°C) to remove residual chloroform, and the dried transgenic carrier membrane was preheated to 70°C in advance, trihydroxy Methylaminomethane-hydrochloric acid (Tris-HCl) buffer (10 mM, pH 7.4) was mixed, and the amount of buffer solut...

Embodiment 2

[0107] The transgenic carrier prepared in embodiment 2 was tested in the following aspects:

[0108] (1) Agarose gel electrophoresis experiment of the complex of transgenic vector and pUC18 plasmid

[0109] Preparation of the complex of transgenic vector and pUC 18 plasmid

[0110] At room temperature, add the transgenic vectors prepared by 11a-11d into PE tubes respectively, in which the concentrations of the transgenic vectors are 0, 5, 10, 15, 20, 25, and 30 μM, and the concentration of the pUC 18 plasmid is 9 μg / mL. Ultrapure water was used to keep the final volume of the reaction solution at 20 μL, and centrifuged to mix evenly. Put it in a constant temperature water bath at 37°C, and after incubating for 1 hour, add 2 μL of 10×Loading Buffer to terminate the reaction.

[0111] Preparation of agarose gel

[0112] Weigh 280 mg of agarose, add 40 mL of 1×TAE buffer solution, and heat in microwave to completely dissolve the agarose particles to obtain a colorless transpar...

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Abstract

The invention provides a cationic lipid based on tetraphenyl ethylene and containing macrocyclic polyamines [12] ane N3 and a transgenic vector containing the cationic lipid. The invention further discloses a preparation method of the cationic lipid and the transgenic vector. The cationic lipid not only contains a saturated fat chain, but also is introduced with a tetraphenyl ethylene matrix, so that the cationic lipid has a double-head part and a double-tail part; the coagulation concentration of DNA is reduced effectively, the cationic lipid and DNA form nano-particles by a hydrophobicity function of the saturated fat chain, the nano-particles enters cells by endocytosis, membrane-disrupting is facilitated, the transgenic vector which is formed by the cationic lipid has low cytotoxicity, and the transfection efficiency is high; and the preparation method of the cationic lipid and the transgenic vector is simple, mature and easy to control.

Description

technical field [0001] The present invention relates to a cationic lipid and a transgene carrier, in particular to a tetraphenylethylene-containing macrocyclic polyamine [12]aneN 3 The cationic lipid, the transgenic carrier containing the cationic lipid and the preparation method thereof. Background technique [0002] Gene therapy is to introduce exogenous normal genes into target cells through certain methods to replace or compensate 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 and so on. [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 helix, 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. E...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K9/127A61K9/14A61K47/22A61K48/00C07D403/14
CPCA61K9/1277A61K9/145A61K47/22A61K48/0033C07D403/14
Inventor 卢忠林张可欣谭筝丽丁爱祥
Owner BEIJING NORMAL UNIVERSITY
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