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Preparation of temperature-sensitive chitosan-graft-NIPAAm/VL copolymer transgenic carrier

A temperature-sensitive chitosan and transgenic vector technology, applied in the field of gene therapy, can solve the problems of reducing the transfection rate, complex dissociation obstacles, etc. Effect

Inactive Publication Date: 2006-12-27
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Strong electrostatic interactions favor complex formation on the one hand, but hinder complex dissociation on the other hand, thereby reducing transfection efficiency

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Dissolve 2 g of isopropylacrylamide in 25 ml of tetrahydrofuran, add 0.25 g of vinyl laurate, stir magnetically for 20 minutes, add 0.01 g of azobisisobutyronitrile and 20 μl of mercaptoacetic acid, and store at 45 °C under nitrogen protection. After reacting for 5 hours, the product was precipitated and washed three times with ether, and the sample was dried in a vacuum oven.

[0019]Get 2.0 gram of last step reaction gained samples and be dissolved in 20ml deionized water, get 3.5 grams of molecular weight 1000, the chitosan that deacetylation degree is 95% is dissolved in 20ml deionized water, add 0.15 gram carbodiimide, with hydrochloric acid / N, N, N', N'tetramethylethylenediamine adjusted the pH value to 4.5, reacted with magnetic stirring at 5°C for 6 hours, dialyzed the product in deionized water for 5 days, and freeze-dried to obtain the final carrier.

[0020] Dissolve 5 mg of the dialyzed thermosensitive carrier in 0.02M acetic acid / 0.02M sodium acetate solut...

Embodiment 2

[0022] Dissolve 2 g of isopropylacrylamide in 25 ml of tetrahydrofuran, add 0.98 g of vinyl laurate, stir magnetically for 20 minutes, add 0.02 g of azobisisobutyronitrile and 50 μl of mercaptoacetic acid, and store at 60 °C under nitrogen protection. After reacting for 6 hours, the product was precipitated and washed three times with ether, and the sample was dried in a vacuum oven.

[0023] Get 2.0 gram of last step reaction gained samples and be dissolved in 20ml deionized water, get 3.5 gram molecular weight 2000, the chitosan that deacetylation degree is 90% is dissolved in 20ml deionized water, add 0.35 gram carbodiimide, with hydrochloric acid / N, N, N', N'tetramethylethylenediamine adjusted the pH value to 5.0, reacted with magnetic stirring at 10°C for 8 hours, dialyzed the product in deionized water for 5 days, and freeze-dried to obtain the final carrier.

[0024] Dissolve the dialyzed 5mg temperature-sensitive carrier in 0.02M acetic acid / 0.02M sodium acetate solut...

Embodiment 3

[0026] Dissolve 2 g of isopropylacrylamide in 25 ml of tetrahydrofuran, add 1.96 g of vinyl laurate, stir magnetically for 20 minutes, add 0.03 g of azobisisobutyronitrile and 70 μl of mercaptoacetic acid, and store at 60 °C under nitrogen protection. After reacting for 7 hours, the product was precipitated and washed three times with ether, and the sample was dried in a vacuum oven.

[0027] Get 2.0 gram of last step reaction gained samples and be dissolved in 20ml deionized water, get 3.5 grams of molecular weight 5000, the chitosan that deacetylation degree is 80% is dissolved in 20ml deionized water, add 0.50 gram carbodiimide, with hydrochloric acid / N, N, N', N'tetramethylethylenediamine adjusted the pH value to 5.0, reacted with magnetic stirring at 15°C for 8 hours, dialyzed the product in deionized water for 5 days, and freeze-dried to obtain the final carrier.

[0028] The dialyzed 10mg thermosensitive chitosan was dissolved in 0.02M sodium acetate / 0.02M acetic acid ...

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Abstract

The invention discloses the preparation of temperature-sensitive chitosan-grafting-isopropylacrylamide (NIPAA) / vinyl levulinate (VL) copolymer transgenic carrier, which comprises, using NIPAA and VL for polymerization synthesis into temperature sensing copolymer, coupling chitosan and copolymers, forming chitosan-grafting-isopropylacrylamide / vinyl levulinate copolymer, refining to obtain the carrier, dissolving the carrier and plasmid DNA which contains beta-galactosidase reporting gene into solution of acetic acid / sodium acetate, stewing and forming compound particles with 50-120 nm of grain size from the solution and carrier DNA according to different electric charge ratio.

Description

technical field [0001] The invention relates to a method for preparing a temperature-sensitive chitosan-graft-isopropylacrylamide (NIPAAm) / vinyl laurate (VL) copolymer non-viral transgene carrier, which belongs to the field of gene therapy and is a novel high-efficiency non-viral carrier preparation methods and techniques. Background technique [0002] Transgenic technology is playing an increasingly important role in the fields of biology, medicine, and environmental protection. Batches of transgenic animals and plants have been produced one after another, which has become a new bright spot in scientific research. For successful gene therapy, one of the important factors is the need for highly efficient carriers, which can carry DNA carrying genetic information into the nucleus, transcribe and translate into target proteins to achieve curative effect. At present, many beneficial attempts have been made in the development of natural and synthetic ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08F299/00C12N15/66
Inventor 刘文广孙书军程男姚康德梁东春左爱军郭刚张镜宇
Owner TIANJIN UNIV