Method for preparing glutathione response shell disulfide bond crosslinking non-virogene vector

A technology of glutathione and gene carrier, which is applied in the field of preparation of non-viral gene carrier, can solve the problems of reducing the circulation time of gene carrier, unstable PEI/DNA assembly, affecting gene transfection efficiency, etc., and achieves a simple method. Effect

Inactive Publication Date: 2008-09-17
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the PEI / DNA assembly is unstable in physiological saline solution, prone to aggregation, and easily cleared by the reticuloendocytic system in the body, which reduces the circulation time of the gene carrier in the body and affects the gene transfection efficiency

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] (1) Under nitrogen protection, dissolve 3mmol mercaptoacetic acid in 15mL triple-distilled water, add 15mmol 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 15mmol N-hydroxy Succinimide (NHS), reacted at 0°C for 6h. Weigh 0.12mmol branched polyethyleneimine (PEI 25k ), dissolved in 10 mL triple distilled water, added to the above mixed reaction solution, and reacted overnight at room temperature. Under nitrogen protection, the above solution was dialyzed for one week and lyophilized to obtain a viscous white solid. Comparing the proton magnetic spectrum of polyethyleneimine, thiolated polyethyleneimine has been successfully prepared.

[0019] The mercapto-polyethylenimine prepared above was used to measure the content of mercapto groups by the Ellman method. Using cysteine ​​as a standard curve, due to the reaction of 1 molecule of cysteine ​​with 1 molecule of 5,5'-dithio-bis-(2-nitrobenzoic acid (DTNB), 1 molecule of nitrate There is a strong ...

Embodiment 2

[0024] (1) Under the protection of nitrogen, dissolve 1mmol of thioglycolic acid in 10mL of three-distilled water, add 3mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 3mmol of N-hydroxy Succinimide (NHS), reacted at 0°C for 4h. Weigh 0.06 mmol of branched polyethyleneimine and dissolve it in 10 mL of triple distilled water, add it to the above mixed reaction solution, and react overnight at room temperature. Under nitrogen protection, the above solution was dialyzed for one week and lyophilized to obtain a viscous white solid. Comparing the proton magnetic spectrum of polyethyleneimine, thiolated polyethyleneimine has been successfully prepared.

[0025] The mercapto-polyethylenimine prepared above was used to measure the content of mercapto groups by the Ellman method. Using cysteine ​​as a standard curve, due to the reaction of 1 molecule of cysteine ​​with 1 molecule of 5,5'-dithio-bis-(2-nitrobenzoic acid (DTNB), 1 molecule of nitrate The re...

Embodiment 3

[0030] (1) Under nitrogen protection, dissolve 2mmol mercaptoacetic acid in 15mL triple-distilled water, add 12mmol 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 12mmol N- Hydroxysuccinimide (NHS), reacted at 0°C for 4h. Weigh 0.2 mmol of branched polyethyleneimine and dissolve it in 10 mL of triple distilled water, add it to the above mixed reaction solution, and react overnight at room temperature. Under nitrogen protection, the above solution was dialyzed for one week and lyophilized to obtain a viscous white solid. Comparing the proton magnetic spectrum of polyethyleneimine, thiolated polyethyleneimine has been successfully prepared.

[0031] The mercapto-polyethylenimine prepared above was used to measure the content of mercapto groups by the Ellman method. Using cysteine ​​as a standard curve, due to the reaction of 1 molecule of cysteine ​​with 1 molecule of 5,5'-dithio-bis-(2-nitrobenzoic acid (DTNB), 1 molecule of nitrate The release of benzoi...

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Abstract

The invention discloses a method for preparing a glutathione-response shell disulfide bond crosslinking non-virus gene carrier. The method comprises the following steps: (1) synthesizing sulfhydrylation polyethyleneimine; (2) preparing sulfhydrylation polyethyleneimine aqueous solution with concentration of 0.1-1mg / ml; (3) preparing plasmid DNA solution with concentration of 50-250Mu g / ml; and (4) adding the solution prepared in step (2) to the solution of the same volume prepared in step (3), whirling to mix, standing to obtain sulfhydrylation gene carrier solution, stirring in air, and obtaining the glutathione-response shell disulfide bond crosslinking non-virus gene carrier. Through the preparation of the shell disulfide bond bionic crosslinking non-virus gene carrier, the stability of the gene carrier in physiological salt solution can be improved. The degradation of high concentration reduced glutathione in cells on disulfide bond can release associated DNA molecule so as to realize effective gene transfection.

Description

technical field [0001] The invention relates to a preparation method of a non-viral gene carrier, especially a method for preparing a glutathione-responsive shell disulfide bond cross-linked non-viral gene carrier. Background technique [0002] The completion of modern gene technology and the human genome engineering map provides broad prospects for the use of gene molecular biology methods to treat various diseases and improve the quality of human life. Gene therapy has become one of the most active biological high-tech fields. Since naked nucleic acid molecules are difficult to exist stably in body fluids and can only achieve low-level expression in major organs, seeking gene carriers that can achieve widespread gene expression has become a key issue in the large-scale clinical application of gene therapy. Although viral vectors have high transfection efficiency, potential toxicity, immunogenicity and targeting limit their application in clinical treatment. It is of grea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/64
Inventor 王幽香胡巧玲沈家骢
Owner ZHEJIANG UNIV
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