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Polyethyleneimine-polylysine copolymer and preparation method thereof

A technology of polyethylenimine and polylysine, applied in other methods of inserting foreign genetic materials, recombinant DNA technology, etc., can solve the problem of low vector transfection rate, reduce cytotoxicity, promote transfection efficiency, Increase the effect of endocytosis

Active Publication Date: 2014-02-19
CHANGZHOU INST OF ENERGY STORAGE MATERIALS &DEVICES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the transfection efficiency of this vector is lower than that of PEI homopolymer with a molecular weight of 25,000

Method used

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  • Polyethyleneimine-polylysine copolymer and preparation method thereof
  • Polyethyleneimine-polylysine copolymer and preparation method thereof
  • Polyethyleneimine-polylysine copolymer and preparation method thereof

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preparation example Construction

[0034] Regarding the polyethyleneimine-polylysine having the structure of formula (I), the present invention also provides its preparation method, which specifically includes the following steps:

[0035] Copolymerizing polyethyleneimine and ε-benzyloxycarbonyl-L-lysine N-carboxylic acid anhydride in the first organic solvent to obtain polyethyleneimine polyε-benzyloxycarbonyl-L-lysine copolymer;

[0036] The hydrogen chloride solution, the second organic solvent and the polyethyleneimine polyε-benzyloxycarbonyl-L-lysine copolymer are mixed, and the polyethyleneimine-polylysine copolymer is obtained after reaction.

[0037] According to the present invention, the ε-benzyloxycarbonyl-L-lysine N-carboxylic acid anhydride (lysine-NCA) is extremely sensitive to oxygen and is easily oxidized in an aerobic environment. in an anhydrous environment. The present invention has no limitation on the order of adding the polyethyleneimine, lysine-NCA and organic solvent, and the polyethyle...

Embodiment 1

[0043] Embodiment 1: the preparation of polyethylenimine-polylysine

[0044] 1) Under anhydrous and oxygen-free conditions, put ε-benzyloxycarbonyl-L-lysine N-carboxylic acid anhydride (Lys(z)-NCA) and chloroform in a dry reactor according to the ratio of 1g:50mL , filled with nitrogen protection, stirred to dissolve; then branched polyethyleneimine was dissolved in chloroform as a polyamine initiator, and added to the reactor, wherein the ratio of polyethyleneimine to chloroform was 1:40; temperature control Stir and react at 0~50°C for at least 12 hours. After the reaction is completed, settle in ether, filter, and dry the filter cake to obtain polyethyleneimine polyε-benzyloxycarbonyl-L-lysine copolymer.

[0045] 2) Dissolve the obtained polyethylenimine polyε-benzyloxycarbonyl-L-lysine copolymer in an ice bath with trifluoroacetic acid, add hydrogen bromide in acetic acid solution for deprotection, and carry out the reaction in an ice bath for 2 hours Above, settle with e...

Embodiment 2

[0049] In vitro transfection of HeLa cells with pGL3 (luciferase plasmid), pEGFPN1 (green fluorescent protein plasmid) and pCMV-lac Z (β-galactosidase plasmid) mediated by polyethyleneimine-polylysine

[0050] (1) Human cervical cancer cells (HeLa cells) were taken and cultured continuously in culture medium containing 10% calf serum (5% CO 2 , 37°C incubator).

[0051] (2) In vitro transfection

[0052] Within 24 hours before transfection, cells in the logarithmic growth phase were collected, digested with trypsin and diluted with Dulbecco’s modified Eagle’s medium at 1×10 per well. 4 Cells were seeded in 96-well culture plates at a density of 37°C in an incubator containing 5% carbon dioxide by volume and continued to culture until the confluence reached 80-90%. During transfection, the culture medium in the cell culture plate added the day before was discarded, and after washing twice with phosphate buffer, the complex particles of genome transfection and Dulbecco contain...

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Abstract

The invention provides polyethyleneimine-polylysine copolymer which has the structure of a formula (I). The invention also provides a preparation method of the polyethyleneimine-polylysine copolymer. The method comprises the following steps of: carrying out polymerization reaction to the polyethyleneimine and epsilon-carbobenzoxy-L-lysineN-carboxylic acid anhydride in a first organic solvent so as to obtain polyethyleneimine poly-epsilon-carbobenzoxy-L-lysine copolymer; and mixing the halogen hydride solution, a second organic solvent and the polyethyleneimine poly-epsilon-carbobenzoxy-L-lysine copolymer, and reacting so as to obtain the polyethyleneimine-polylysine copolymer with the structure of the formula (I). The polyethyleneimine-polylysine copolymer provided by the invention is a novel polycation gene carrier, the properties of the polyethyleneimine and the polyamino acid are integrated, and thus the copolymer has relatively high transfection efficiency.

Description

technical field [0001] The invention relates to the technical field of polymer compounds, in particular to a polyethyleneimine-polylysine copolymer and a preparation method thereof. Background technique [0002] With the continuous advancement of biological science and technology, gene therapy will occupy an important position in the process of overcoming stubborn diseases such as cancer and genetic diseases, and will gradually become a common and effective method. Gene therapy refers to a new biomedical technology that introduces human normal genes or genes with therapeutic effects into human target cells in a certain way to correct gene defects or exert therapeutic effects, so as to achieve the purpose of treating diseases. Successful gene therapy depends on effective gene carriers, and common carriers are divided into viral vectors and non-viral vectors. [0003] Viral vectors include retrovirus, adenovirus (AV), adeno-associated virus (AAV), herpes simplex virus (HSV), ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08G69/48C08G69/10C12N15/87
Inventor 田华雨郭兆培陈学思焦自学林琳陈杰
Owner CHANGZHOU INST OF ENERGY STORAGE MATERIALS &DEVICES
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