Construction of bio-reducible efficient cation gene vectors taking polysaccharides as frameworks with ATRP (Atom Transfer Radical Polymerization) method

A gene carrier and cationic technology, applied in the field of bioreducible cationic polysaccharide gene carrier, can solve the problems of different protonation ability and carrier transfection efficiency, and achieve the effect of easy regulation, simple use method and good storage stability

Inactive Publication Date: 2013-07-24
BEIJING UNIV OF CHEM TECH
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  • Description
  • Claims
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Problems solved by technology

[0012] 2. When preparing high-performance polysaccharide gene vectors, different monomers can be grafted to obtain cationic polymers with different properties, but the protonation ability of different monomers is different, resulting in different transfection efficiencies of the vectors. How to screen out highly efficient polysaccharides? High-performance monomer is a problem that needs to be considered

Method used

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  • Construction of bio-reducible efficient cation gene vectors taking polysaccharides as frameworks with ATRP (Atom Transfer Radical Polymerization) method
  • Construction of bio-reducible efficient cation gene vectors taking polysaccharides as frameworks with ATRP (Atom Transfer Radical Polymerization) method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Continuous reaction under nitrogen protection conditions at 25°C, add 0.1g dextran macromolecular initiator (recorded as SS-Dextran-Br) into methanol to dissolve, then add water, N,N-dimethylaminoethyl methacrylate in sequence (DMEMA), 2,2-bipyridine, and finally add CuBr to initiate active controlled free radical polymerization, wherein water / SS-Dextran-Br=30 (mass ratio), methanol / SS-Dextran-Br=20 (mass ratio) , DMEMA / SS-Dextran-Br=10 (mass ratio), 2,2-bipyridine / SS-Dextran-Br=0.36 (mass ratio), CuBr / SS-Dextran-Br=0.18 (mass ratio); after 5min Open the bottle stopper to accelerate stirring for 10 minutes, fully contact with air to stop the reaction; the polymer product is repeatedly precipitated with ether until the shape becomes solid, and then put it in a vacuum drying oven to remove the ether; then dissolve the product in 40ml of water and put it in a Put it in a dialysis bag, then transfer it to a 5L beaker and fill it with deionized water to start dialysis for 6 ...

Embodiment 2

[0043] Continue the reaction under nitrogen protection at 25°C, dissolve 0.1g of dextran macroinitiator (referred to as SS-Dextran-Br) in methanol, then add water, N,N-dimethylaminoethyl methacrylate in sequence (DMEMA), 2,2-bipyridine and CuBr initiate active controllable radical polymerization, the reaction conditions and the post-treatment method of the polymerized product are the same as in Example 1, except that the reaction time is 30min, and the bioreducible cationic gene with polysaccharide as the skeleton is obtained. 0.3 g of the carrier is recorded as a polymer (SS-Dextran-PDMEMA), its Mn is 102000, and its Mw / Mn is 1.80.

[0044] The skeleton structure of SS-Dextran-Br is dextran with a molecular weight of 20,000-25,000, and the hydroxyl groups on it are replaced by groups of the following structural formula

[0045] The above-mentioned bioreducible cationic gene carrier with polysaccharide as the backbone is denoted as DPD2, and its transfection efficiency is de...

Embodiment 3

[0047] Continuous reaction in an oxygen-free environment at 25°C, dissolve 0.1g dextran macroinitiator (referred to as SS-Dextran-Br) in dimethyl sulfoxide (DMSO), and then add glycidyl methacrylate in sequence (GMA), CuBr, CuBr 2 , and finally add pentamethyldiethylenetriamine (PMDTETA) to initiate living controllable radical polymerization, wherein dimethyl sulfoxide (DMSO) / SS-Dextran-Br=100 (mass ratio), glycidyl methacrylate ( GMA) / SS-Dextran-Br=40 (mass ratio), CuBr / SS-Dextran-Br=0.33 (mass ratio), CuBr 2 / SS-Dextran-Br=0.06 (mass ratio), pentamethyldiethylenetriamine (PMDTETA) / SS-Dextran-Br=0.5 (mass ratio); after 15min, open the cork and pour the liquid in the bottle slowly into the Prepare a beaker with 200mL of methanol, stir at high speed to stop the reaction, then add 200mL of methanol to precipitate the product, and then put it in a vacuum oven to remove methanol; dissolve the product in 40ml of water and put it in a dialysis bag with a molecular weight cut-off of...

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Abstract

The invention discloses construction of a series of bio-reducible cation polysaccharide gene vectors taking polysaccharides including dextran, cyclodextrin and prolan as frameworks and having high gene transfer efficiency with an ATRP (Atom Transfer Radical Polymerization) method, which belongs to the technical field of non-viral gene vectors. In the ATRP method, a polymerization reaction is stable and is easy to adjust and control, a plurality of high-performance cation polysaccharide gene vectors which belong to different molecular weight series and have narrow weight distribution can be prepared as required, and the prepared cation polysaccharide gene vectors have high storage stability and can keep original performance after being stored for certain days or certain months. The polysaccharide cation gene vectors have higher transfer efficiency than a liposome lipfect2000 which is applied commercially internationally in cells such as Hepg2, Hela, C6, Cos7, HEK293 and the like, are easy to use, and have commercial potentials.

Description

technical field [0001] The invention belongs to the technical field of non-viral gene carriers, and specifically relates to the construction of a series of bioreducible cationic polysaccharide gene carriers with high gene transfection efficiency using ATRP method as the backbone, including dextran, cyclodextrin and pullulan. Background technique [0002] Gene therapy plays a pivotal role in overcoming major diseases such as human cancer, genetic diseases and cardiovascular diseases. Future medical treatment is to introduce normal or therapeutic genes into target cells in a certain way to interfere with the occurrence, development and process of diseases , so as to achieve the purpose of treatment. There are three important steps in gene therapy, namely, the search for the target gene, the development of the gene carrier, and the specific expression of the target gene in cells. Among them, the gene transfer system is the core technology of gene therapy, and the lack of safe ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08F251/00C08F120/34C08F120/32C08F120/54C08F8/00C08B37/02C08B37/16C08B37/00A61K48/00
Inventor 徐福建王增辉杨鑫超胡杨朱韵柴明英
Owner BEIJING UNIV OF CHEM TECH
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