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Non-viral gene transfection carrier and its preparation method and use

A gene transfection carrier and gene transfection technology, applied in the field of gene transfection ability, can solve the problems of cytotoxicity, low transfection efficiency, and low transfection efficiency, and achieve low toxicity, improved hydrophobicity, and transfection efficiency exact effect

Inactive Publication Date: 2006-10-11
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of these polymers are chemically synthesized polyamino structures, including polyamino acids and polyacetimides. The problems in these studies are low transfection efficiency at low concentrations, and obvious cytotoxicity at high concentrations.
Studies have shown that this material has its unique advantages in terms of cytotoxicity and particle size control of dense DNA products; although key technologies including transferrin ligand modification and polyethylene glycol (PEG) grafting are used, but The problem of low transfection efficiency still exists

Method used

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  • Non-viral gene transfection carrier and its preparation method and use
  • Non-viral gene transfection carrier and its preparation method and use
  • Non-viral gene transfection carrier and its preparation method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: a kind of synthetic method of chitosan-fatty acid graft:

[0032] Take 0.4 g of chitosan oligosaccharides with average molecular weights of 1.5, 10, 19, and 51 kD, weigh them accurately, add 30 mL of distilled water and stir to dissolve, then add a proportioning amount of carbodiimide (EDC) (Table 1), and stir to dissolve. According to chitosan: stearic acid (molar ratio) 1: 100, take stearic acid and dissolve in 20mL ethanol solution. The above two solutions were mixed, at 400r·min -1 Under the condition of magnetic stirring, the reaction was carried out at a constant temperature of 80°C for 5h, and after 5h, the room temperature (25°C) was maintained, and the stirring was continued for 6h until the reaction solution was clear. The final reaction solution was placed in a dialysis bag and dialyzed with distilled water for 48 hours. After the dialysate was freeze-dried, the residual stearic acid was washed with absolute ethanol to obtain chitooligosacchar...

Embodiment 2

[0037] Embodiment 2: the synthetic method of the 2nd kind chitosan-fatty acid graft:

[0038] Take 0.4 g of chitosan oligosaccharides with an average molecular weight of 19 kD, accurately weigh, add 30 mL of distilled water and stir to dissolve, add 1.2 g of carbodiimide, and stir to dissolve. According to chitosan: fatty acid (molar ratio) 1: 20, weigh fatty acid (myristic acid, palmitic acid, stearic acid, behenic acid any one of them) respectively, dissolve in 20mL ethanol solution. The above two solutions were mixed, at 400r·min -1 Under the condition of magnetic stirring, the reaction was carried out at a constant temperature of 80°C for 5h, and after 5h, the room temperature (25°C) was maintained, and the stirring was continued for 6h until the reaction solution was clear. The final reaction solution was placed in a dialysis bag and dialyzed with distilled water for 48 hours. After the dialysate was freeze-dried, the residual fatty acid was washed with absolute ethanol...

Embodiment 3

[0042] Embodiment 3: the synthesis of the 3rd kind of chitosan-fatty acid graft:

[0043] Take 0.4 g of chitosan oligosaccharides with an average molecular weight of 19 kD, weigh them accurately, add 30 mL of distilled water and stir to dissolve, then add a proportioning amount of carbodiimide (Table 3), and stir to dissolve. According to chitosan: fatty acid (molar ratio) 1:1, 1:10, 1:20, 1:50, 1:100, stearic acid was weighed and dissolved in 20mL ethanol solution. The above two solutions were mixed, at 400r·min -1 Under the condition of magnetic stirring, the reaction was carried out at a constant temperature of 80°C for 5h, and after 5h, the room temperature (25°C) was maintained, and the stirring was continued for 6h until the reaction solution was clear. The final reaction solution was placed in a dialysis bag and dialyzed with distilled water for 48 hours. After the dialysate was freeze-dried, the residual stearic acid was washed with absolute ethanol to obtain oligoch...

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Abstract

The invention provide a kind of non-virogene transfection vector, by grafting chitose that average molecular weight is 1.5kD~51kD and C10~C22 fatty acid to forming the chitose-fatty acid grafting colloidal cluster, critical colloidal cluster concentration< 0.1mg / ml, particle size between 20 and 200 nm, colloidal cluster surface charge on positive charge, amidol degree of substitution of the chitose is 1%~50%. By synthesizing the chitose-fatty acid grafting substance to preparing the chitose-fatty acid grafting colloidal cluster of non-virogene transfection vector. The invention's synthetic colloidal cluster is low-toxic cationic polymer colloidal cluster, the colloidal cluster has characteristic of penetrate cell membrane fast, destroyed by intracellular lysosome rarely and the function of target nucleus, and by the cationic feature of itself joint seal the negative charge biomacromolecule to forming stable colloidal cluster / DNA complex administer drug system, it can apply in the field of medicine as highly effective and safety non-virogene transfection vector.

Description

technical field [0001] The invention belongs to the preparation and application of a non-viral gene carrier, and relates to the preparation conditions and gene transfection ability of chitosan-fatty acid graft micelles loaded with genes. Background technique [0002] Gene therapy is a treatment method based on gene transfer, which introduces foreign genes into the human body to achieve therapeutic purposes. At present, the primary problem of gene therapy is how to choose a suitable gene transfer system. Generally, gene transfer systems can be classified into viral vector (viral vector) systems and non-viral vector (non-viral vector) systems. Viral vectors have been widely and effectively used by making full use of the highly evolved infectious and parasitic characteristics of viruses. However, the viral vectors currently used in research still have many shortcomings, such as high immunogenicity, high toxicity, small capacity of the target gene, complicated preparation and ...

Claims

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Application Information

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IPC IPC(8): C12N15/63
Inventor 胡富强杜永忠袁弘游剑应晓英赵梦丹
Owner ZHEJIANG UNIV
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