Hyperbranched polyethyleneimine-grafted poly L-glycine copolymer and preparation method thereof

A technology of glycine copolymer and polyethyleneimine, applied in the biological field, can solve the problems of troublesome preparation and not much improvement of transfection efficiency, etc.

Inactive Publication Date: 2018-07-06
CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the preparation of such polymers is relatively troublesome, and the transfection efficiency is not greatly improved.

Method used

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  • Hyperbranched polyethyleneimine-grafted poly L-glycine copolymer and preparation method thereof
  • Hyperbranched polyethyleneimine-grafted poly L-glycine copolymer and preparation method thereof
  • Hyperbranched polyethyleneimine-grafted poly L-glycine copolymer and preparation method thereof

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preparation example Construction

[0030] The present invention provides a kind of preparation method of the poly-L-glycine copolymer of the hyperbranched polyethyleneimine graft of formula (I) structure, comprising:

[0031] L-glycine N-carboxylic acid anhydride and hyperbranched polyethyleneimine are reacted in solvent, obtain the poly-L-glycine copolymer of the hyperbranched polyethyleneimine graft of formula (I) structure;

[0032]

[0033] Wherein, n1, n2, n3, x, y are degrees of polymerization, 600≥x+y≥40; preferably, 550≥x+y≥50; more preferably, 520≥x+y≥60;

[0034] 3≤n1+n2+n3≤180; preferably 10≤n1+n2+n3≤170; more preferably, 20≤n1+n2+n3≤150.

[0035] Wherein, the polyethyleneimine molecular weight is preferably 1800-25000; more preferably 1900-24000;

[0036] The molecular weight of the poly-L-glycine copolymer grafted with hyperbranched polyethyleneimine of the product formula (I) structure is preferably 2,370 to 35,260.

[0037] In the present invention, at first under anhydrous and oxygen-free c...

Embodiment 1

[0081] The preparation of embodiment 1 polyethyleneimine-polyglycine

[0082] (1) Under anhydrous and anaerobic conditions, according to the mass g of glycine N-carboxylic acid anhydride (Gly-NCA) monomer, mix with anhydrous chloroform, anhydrous dichloromethane or anhydrous N'N'-dimethylformaldehyde respectively The ratio of the volume mL of the amide is 1-100, 1-10, feed the two into a dry reactor, fill with nitrogen protection, stir and dissolve; then dissolve the branched polyethyleneimine as a polyamine initiator in Anhydrous chloroform, anhydrous dichloromethane or anhydrous N'N'-dimethylformamide, and added to the reactor, wherein polyethyleneimine mass g, and anhydrous chloroform, anhydrous dichloromethane or anhydrous The volume of water N'N'-dimethylformamide is mL, and the ratio is 1 to 100, 1 to 10; the mass ratio of polyethyleneimine to Gly-NCA is 1:10-1:120; temperature control is 0 to Stir and react at 50°C for at least 12 hours. After the reaction is completed...

Embodiment 2

[0087] In vitro transfection of pGL3 (luciferase plasmid), pEGFPN1 (green fluorescent protein plasmid) and pCMV-lac Z (-galactosidase plasmid) mediated by polyethyleneimine-polyglycine

[0088] Culture of HeLa cells

[0089] (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).

[0090] (2) In vitro transfection

[0091] 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 inoculated in a 96-well culture plate at a density of 37°C in an incubator with a volume fraction of 5% carbon dioxide and continued to culture until the confluence reached 80-90%. For transfection, discard the culture medium in the cell culture plate that was added the day before, wash twice with phosphate buffered saline, and remove the complex particles of genome ...

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Abstract

The invention provides hyperbranched polyethyleneimine-grafted poly L-glycine copolymer with a structure shown in formula (I). Hyperbranched polyethyleneimine is taken as a macroinitiator and is dissolved in an organic solvent, and L-glycine N-carboxylic acid anhydride ring opening polymerization is initiated in a waterless and oxygen-free condition, so that the hyperbranched polyethyleneimine-grafted poly L-glycine copolymer is obtained. The polymer belongs to a polycation gene carrier, and is high in transfection efficiency, the maximum transfection efficiency of polymer to MCF7 and CHO cell-mediated luciferase plasmid is 10 times and 8 times that of the commercial PE125K, and the cell survival rate is 80% or higher within a transfection proportional range; when the polymer is applied tosiRNA transfection, the maximum gene silencing efficiency of the polymer to Huh7 and CT26 cells for constantly expressing luciferase can reach 80% or higher, and the polymer has high gene silencing efficiency and small cytotoxicity for a commercial transfection reagent PE125K.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a poly-L-glycine copolymer grafted with hyperbranched polyethyleneimine and a preparation method thereof. Background technique [0002] Gene therapy has become an important treatment for human genetic diseases and cancer [see Olefsky JM.Diabetes–gene therapy for rats and mice.Nature.2000 408: 420-421.Chen YC, Huang L.Tumor- Targeted delivery of siRNA by nonviral vector: safe and effective cancer therapy. Expert. Opin. Drug. Deliv. 2008 5: 1301-1311.]. Gene therapy refers to the introduction of 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. How to efficiently introduce genes into cells to play a role has become an important factor restricting the development of gene therapy. The vectors currently used in gene therapy can be divided i...

Claims

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Application Information

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IPC IPC(8): C08G69/10C08G69/04C12N15/63C12N15/87
CPCC12N15/63C08G69/04C08G69/10C12N15/87
Inventor 田华雨郭兆培陈学思陈杰林琳孙平杰
Owner CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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