Modified polyethyleneimine and application thereof in the preparation of gene transfection vector reagent

Abstract

The invention relates to the field of polymer modification, and in particular to a modified polyethyleneimine and application thereof in the preparation of a gene transfection vector reagent. According to the present invention, hydrophobic rosin acid or dehydroabietic acid is grafted onto polyethyleneimine to obtain the modified polyethyleneimine. The hydrophobic group of the modified polyethyleneimine forms a dense hydrophobic core with strong hydrophobicity due to the intermolecular forces in water, hydrophilic polyethyleneimine forms hydrophilic shell, thereby forming a nano micelles with shell-core structure, and nanoparticles with small particle size formed after DNA binding. The composite nanoparticles in smaller size gains tighter structure, so that composite nanoparticles are easier to pass through the cell membrane structure of a lipid bilayer, and the efficiency of transfection is improved. The modified polyethyleneimine can be applied to the preparation of gene transfection vectors and has the characteristics of high biological safety, high transfection efficiency, and low cost.

Description

technical field

[0001] The invention relates to the field of polymer modification, in particular to a modified polyethyleneimine and its application in the preparation of gene transfection carrier reagents. Background technique

[0002] Gene transfection technology refers to the transfer of exogenous nucleic acid with certain functions into target cells and expression in the cells to achieve the desired purpose. Therefore, as a key technology, it is widely used in gene therapy, agricultural biotechnology, and biopharmaceuticals Engineering and other fields. The introduction of exogenous genes into biological cells requires the use of gene transfection vectors. Currently, commonly used vectors are divided into two categories: viral vectors and non-viral vectors. Although the viral vector has a high transfection rate, it has a small capacity for exogenous genes (about 4.5-30kbp) and poor stability, and the viral DNA may replicate in the human body, which can induce the body t...

Examples

Embodiment 1

[0045] (1) 0.24g of N-hydroxysuccinimide and 0.60g of dehydroabietic acid were mixed and fully dissolved in 50mL of tetrahydrofuran, and reacted at 25°C for 6h;

[0046] (2) After fully dissolving 10g polyethyleneimine (molecular weight: 10000) in 200mL of tetrahydrofuran, drop the solution prepared in step (1) into the polyethyleneimine solution, add 0.84g O-benzotriazole -N,N,N',N'-tetramethylurea tetrafluoroboric acid and 0.28g N,N-diisopropylethylamine, and nitrogen protection, reacted at 25°C for 16h; after the reaction was completed, lyophilized , dialyzed and then freeze-dried to obtain modified polyethyleneimine (the degree of substitution of the substituent R is 0.1%, and the molecular weight is 6000);

[0047] The particle size of the modified polyethyleneimine detected by laser scattering in the aqueous solution is 46nm.

Embodiment 2

[0049] (1) 0.36g of N-hydroxysuccinimide and 0.90g of dehydroabietic acid were mixed and fully dissolved in 50mL of tetrahydrofuran, and reacted at 15°C for 12h;

[0050] (2) After fully dissolving 10g polyethyleneimine (molecular weight: 6000) in 200mL of tetrahydrofuran, drop the solution prepared in step (1) into the polyethyleneimine solution, add 1.26g O-benzotriazole -N,N,N',N'-tetramethylurea tetrafluoroboric acid and 0.42g N,N-diisopropylethylamine, and nitrogen protection, reacted at 25°C for 8h; after the reaction was completed, lyophilized , dialyzed and then freeze-dried to obtain modified polyethyleneimine (the degree of substitution of the substituent R is 3%, and the molecular weight is 11000);

[0051] The particle size of the modified polyethyleneimine detected by laser scattering in aqueous solution is 58nm.

Embodiment 3

[0053] (1) 0.48g of N-hydroxysuccinimide and 1.2g of dehydroabietic acid were mixed and fully dissolved in 50mL of tetrahydrofuran, and reacted at 0°C for 24h;

[0054] (2) After fully dissolving 10g of polyethyleneimine (molecular weight: 40,000) in 200mL of tetrahydrofuran, drop the solution prepared in step (1) into the polyethyleneimine solution, and add 1.68g of O-benzotriazepam Azole-N,N,N',N'-tetramethyluronium tetrafluoroboric acid and 0.56g N,N-diisopropylethylamine, under nitrogen protection, reacted at 25°C for 24h; after the reaction was completed, freeze Dry, dialyze and then freeze-dry to obtain modified polyethyleneimine (the degree of substitution of the substituent R is 12%, and the molecular weight is 19000);

[0055] The particle size of the modified polyethyleneimine detected by laser scattering in the aqueous solution is 76nm.

technical field

[0001] The invention relates to the field of polymer modification, in particular to a modified polyethyleneimine and its application in the preparation of gene transfection carrier reagents. Background technique

[0002] Gene transfection technology refers to the transfer of exogenous nucleic acid with certain functions into target cells and expression in the cells to achieve the desired purpose. Therefore, as a key technology, it is widely used in gene therapy, agricultural biotechnology, and biopharmaceuticals Engineering and other fields. The introduction of exogenous genes into biological cells requires the use of gene transfection vectors. Currently, commonly used vectors are divided into two categories: viral vectors and non-viral vectors. Although the viral vector has a high transfection rate, it has a small capacity for exogenous genes (about 4.5-30kbp) and poor stability, and the viral DNA may replicate in the human body, which can induce the body t...

Examples

Embodiment 1

[0045] (1) 0.24g of N-hydroxysuccinimide and 0.60g of dehydroabietic acid were mixed and fully dissolved in 50mL of tetrahydrofuran, and reacted at 25°C for 6h;

[0046] (2) After fully dissolving 10g polyethyleneimine (molecular weight: 10000) in 200mL of tetrahydrofuran, drop the solution prepared in step (1) into the polyethyleneimine solution, add 0.84g O-benzotriazole -N,N,N',N'-tetramethylurea tetrafluoroboric acid and 0.28g N,N-diisopropylethylamine, and nitrogen protection, reacted at 25°C for 16h; after the reaction was completed, lyophilized , dialyzed and then freeze-dried to obtain modified polyethyleneimine (the degree of substitution of the substituent R is 0.1%, and the molecular weight is 6000);

[0047] The particle size of the modified polyethyleneimine detected by laser scattering in the aqueous solution is 46nm.

Embodiment 2

[0049] (1) 0.36g of N-hydroxysuccinimide and 0.90g of dehydroabietic acid were mixed and fully dissolved in 50mL of tetrahydrofuran, and reacted at 15°C for 12h;

[0050] (2) After fully dissolving 10g polyethyleneimine (molecular weight: 6000) in 200mL of tetrahydrofuran, drop the solution prepared in step (1) into the polyethyleneimine solution, add 1.26g O-benzotriazole -N,N,N',N'-tetramethylurea tetrafluoroboric acid and 0.42g N,N-diisopropylethylamine, and nitrogen protection, reacted at 25°C for 8h; after the reaction was completed, lyophilized , dialyzed and then freeze-dried to obtain modified polyethyleneimine (the degree of substitution of the substituent R is 3%, and the molecular weight is 11000);

[0051] The particle size of the modified polyethyleneimine detected by laser scattering in aqueous solution is 58nm.

Embodiment 3

[0053] (1) 0.48g of N-hydroxysuccinimide and 1.2g of dehydroabietic acid were mixed and fully dissolved in 50mL of tetrahydrofuran, and reacted at 0°C for 24h;

[0054] (2) After fully dissolving 10g of polyethyleneimine (molecular weight: 40,000) in 200mL of tetrahydrofuran, drop the solution prepared in step (1) into the polyethyleneimine solution, and add 1.68g of O-benzotriazepam Azole-N,N,N',N'-tetramethyluronium tetrafluoroboric acid and 0.56g N,N-diisopropylethylamine, under nitrogen protection, reacted at 25°C for 24h; after the reaction was completed, freeze Dry, dialyze and then freeze-dry to obtain modified polyethyleneimine (the degree of substitution of the substituent R is 12%, and the molecular weight is 19000);

[0055] The particle size of the modified polyethyleneimine detected by laser scattering in the aqueous solution is 76nm.

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