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Process for preparing transgenic compound particles of alkylated chitosan

A technology of chitosan and compound, which is applied in the field of preparation of chitosan derivative non-viral transgene carrier, can solve the problems of reducing transfection rate and complex dissociation hindrance, so as to improve transfection rate and affinity Effects of compatibility and transmembrane capacity

Inactive Publication Date: 2002-12-25
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

[0012] Take 5 grams of chitosan with a molecular weight of 50,000 and a degree of deacetylation of 90%, add 20ml of 10% sodium hydroxide and 100ml of isopropanol, stir for 20 minutes at 40°C, add 1.5g of bromododecane, and react at 60°C For 4 hours, adjust the pH to neutral with hydrochloric acid, filter, wash with ethanol three times, filter with suction, and dry in an oven at 50°C for 24 hours. The product was dissolved in dilute acetic acid, dialyzed in deionized water for 5 days, and freeze-dried at -50°C to obtain the final carrier.

[0013] Dissolve the dialyzed 10mg dodecyl chitosan in 0.02M acetic acid / 0.02 sodium acetate solution, the concentration is 1mg / ml, and the plasmid DNA containing the CAT reporter gene is dissolved in 0.02M acetic acid / 0.02 sodium acetate solution, the concentration is 10mg / ml, filter the bacteria after standing for 1 hour, take 20μl of dodecyl chitosan solution and 20μl of plasmid DNA solution and mix, vortex and shake, and stand for 40 minutes...

Embodiment 2

[0015] Take 5 grams of chitosan with a molecular weight of 40,000 and a degree of deacetylation of 90%, add 10ml of 10% sodium hydroxide and 50ml of isopropanol, stir for 20 minutes at 40°C, add 1.2g of bromododecane, and react at 60°C For 4 hours, adjust the pH to neutral with hydrochloric acid, filter, wash with ethanol three times, filter with suction, and dry in an oven at 50°C for 24 hours. The product was dissolved in dilute acetic acid, dialyzed in deionized water for 5 days, and freeze-dried at -50°C to obtain the final carrier.

[0016] Dissolve the dialyzed 10mg dodecyl chitosan in 0.02M acetic acid / 0.02 sodium acetate solution, the concentration is 1mg / ml, and the plasmid DNA containing the CAT reporter gene is dissolved in 0.02M acetic acid / 0.02 sodium acetate solution, the concentration is 10mg / ml, filter the bacteria after standing for 1 hour, take 20μl of dodecyl chitosan solution and 20μl of plasmid DNA solution and mix, vortex and shake, and stand for 40 minutes....

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PUM

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Abstract

A process for preparing the transgenic compound particles of alkylated chitosan includes dispersing the chitosan with 40000-50000 of molecular weight and more than 90% of deactylated level in the solvent of sodium hydroxide-ispropanol mixture, adding the alkylating reagent, reaction, washing to obtain alkylated chitosan with 20-30% of substituted level, respectively dissolving said alkylated chatosan and the plasmid DNA containing CAT report gene in the solution of acetic acid / sodium acetate, still standing, mixing with phosphate of DNA, still standing and filtering to obtain 80-100 nm particles. It has high transfection efficiency.

Description

Technical field [0001] The invention relates to a preparation method of a chitosan derivative non-viral transgenic vector. It belongs to the preparation method and technology of a novel high-efficiency non-viral vector in the field of gene therapy. 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. At present, gene transfection mainly uses methods such as microinjection, viral vector, non-viral vector transformation, etc., all of which have disadvantages such as easy DNA damage, low transfection efficiency, expensive equipment, and superb operating skills, which restrict the improvement of this technology. Wide range of applications. The core issue and key step of gene transfection is how to introduce foreign ge...

Claims

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Application Information

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IPC IPC(8): A61K47/26A61K48/00C08B37/08C12N15/89
Inventor 姚康德刘文广孙光洁张镜宇梁东春郭刚
Owner TIANJIN UNIV