Cationic lipid containing peptide dendrimer, transgenic carrier and preparation method and application of transgenic carrier

A peptide dendrimer, cationic lipid technology, applied in the introduction of foreign genetic material, peptides, recombinant DNA technology using vectors, etc., can solve the problems of carcinogenicity, low transfection efficiency, insertion and integration of host DNA, etc., and achieve encapsulation. The effect of strong ability, low cytotoxicity, and good transfection ability

Active Publication Date: 2014-07-16
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the transfection efficiency of viral vectors is high, there are disadvantages such as immunogenicity, carcinogenicity, and host DNA insertion and integration, which limit their application.
Non-viral vectors (such as cationic liposomes) are non-immunogenic and convenient for large-scale production, but transfection efficiency is lower than that of viral vectors

Method used

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  • Cationic lipid containing peptide dendrimer, transgenic carrier and preparation method and application of transgenic carrier
  • Cationic lipid containing peptide dendrimer, transgenic carrier and preparation method and application of transgenic carrier
  • Cationic lipid containing peptide dendrimer, transgenic carrier and preparation method and application of transgenic carrier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037]

[0038] The raw materials include 1 mmol, 1.20 g of compound 1 (peptide dendrimer protected by the second-generation amino Pbf / Boc, R1=arginine), 1 mmol, 0.66 g of compound 2 (fatty alkane amide of glutamic acid), condensing agent (such as: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC, 2mmol, 0.38g, catalyst 1-hydroxybenzotriazole (HOBt, 2mmol, 0.26g), base (N , N-diisopropylethylamine DIPEA, 4mmol, 0.5g), the solvent is anhydrous dichloromethane, compound 1: compound 2: condensing agent: catalyst: base (molar ratio) =1: 1: 2: 2: 4. The ratio is a molar ratio, and the amount of solvent is limited to the complete dissolution of compound 1, compound 2, condensing agent, catalyst, and alkali;

[0039] Weigh compound 1, compound 2, condensing agent, 1-hydroxybenzotriazole (HOBt) and base in proportion, at 0 o C, under nitrogen protection conditions, add dichloromethane solvent, react for 0.5 hours; then react at room temperature for 24 hours, after the...

Embodiment 2

[0046]

[0047] The raw materials include compound 1 (second-generation amino Pbf / Boc protected peptide dendrimer, R1=arginine) 1mmol, 1.20 g, compound 4 (N1-cholesterolamide-1,2-ethylenediamine) 1mmol, 0.47 g , condensing agent (such as: 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride EDC·HCl, 2mmol, 0.38g, catalyst 1-hydroxybenzotriazole (HOBt, 2mmol , 0.26g), base (N, N-diisopropylethylamine DIPEA, 4mmol, 0.5g), solvent is anhydrous dichloromethane, compound 1: compound 4: condensing agent: catalyst: base=1: 1: 2: 2: 4, described ratio is molar ratio, and the amount of solvent is limited with compound 1, compound 4, condensing agent, catalyzer, alkali completely dissolving;

[0048] Weigh compound 1, compound 4, condensing agent, catalyst (1-hydroxybenzotriazole, HOBt) and base in proportion, at 0 o C, under nitrogen protection conditions, add dichloromethane solvent, react for 0.5 hours; then react at room temperature for 24 hours, after the reaction is over...

Embodiment 3

[0051] Example 3. The preparation of the transgenic carrier containing cationic lipid in example 1

[0052] The raw materials include the cationic lipid containing peptide dendrimers prepared in Example 1, cholesterol and anhydrous chloroform, 0.01 mmol of cationic lipid, 0.01 mmol of cholesterol, and 0.5 mL of anhydrous chloroform. Add the cationic lipid, cholesterol and anhydrous chloroform into the eggplant-shaped bottle at room temperature (25°C) and normal pressure. After the cationic lipid and cholesterol are completely dissolved, use a rotary evaporator (100 r / min) The chloroform was distilled off to obtain a dry lipid film, which was then dried in vacuo overnight to remove residual chloroform. Add high-purity sterilized water to the dried lipid film to make a 4 mmol / L solution, stir for 1 hour, ultrasonicate the probe for 1 hour, and finally use liposoFast TM The transgenic vector containing cationic lipids was prepared by liposome extruder, and stored in a refrige...

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PUM

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Abstract

The invention discloses a cationic lipid containing peptide dendrimers, a transgene carrier, a preparation method and application thereof. The transgenic carrier of the present invention has very low cytotoxicity, can effectively block and wrap pEGFP-N1 plasmid or pGL3 plasmid, and has strong wrapping ability, and the complex of the transgenic carrier of the present invention and pEGFP-N1 plasmid or pGL3 plasmid averages The particle size is between 80-200nm, and the Zeta potential is between 5-50mV, which is very suitable for gene transfection. Transfection of HepG2, MCF7, and B16F10 cells with the complexes formed by the transgenic vectors of the present invention and pEGFP-N1 plasmids or pGL3 plasmids all showed good transfection ability, and the transfection ability exceeded commercially available under optimized transfection conditions. PEI25K has a more prominent transfection advantage in the presence of serum.

Description

technical field [0001] The invention relates to a class of cationic lipids, a transgenic carrier containing cationic lipids, a preparation method and application thereof. Background technique [0002] With the continuous development of biochemistry and molecular biology, people's understanding of the functions of nucleic acids (DNA, RNA) has been deepening, and at the same time, the molecular mechanisms of various diseases have been further understood. Among the known human diseases, thousands of diseases are related to genes, including congenital genetic diseases related to single genes and acquired diseases related to polygenes. Most acquired diseases are caused by various internal and external factors acting on the genetic material of the human body to make its function disorder. The main cause of disease is gene defect and gene deletion. The appropriate foreign gene is introduced into human cells to make up for the defect or missing gene, express the corresponding prote...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K5/037C12N15/85
Inventor 顾忠伟聂宇徐翔晖程刚姜倩岳冬罗奎
Owner SICHUAN UNIV
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