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PEI (Polyetherimide)-chitosan triply compound gene vector with low molecular weight and preparation method and application thereof

A technology of gene carrier and chitosan, which is applied in the fields of low molecular weight PEI-chitosan triple composite gene carrier and its preparation and application, can solve the problems of unstable transfection efficiency and low efficiency of composite particles, and prolong the circulation time in vivo. , mild conditions, reduce the effect of adsorption

Inactive Publication Date: 2010-09-29
TIANJIN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The instability of complex particles in the body fluid circulation is the single most important factor for the low transfection efficiency of non-viral vectors in vivo

Method used

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  • PEI (Polyetherimide)-chitosan triply compound gene vector with low molecular weight and preparation method and application thereof
  • PEI (Polyetherimide)-chitosan triply compound gene vector with low molecular weight and preparation method and application thereof
  • PEI (Polyetherimide)-chitosan triply compound gene vector with low molecular weight and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Add 32 ml of 40% NaOH solution into the beaker, add 3.2 g of chitosan (Mw=50 kDa, 0.02 mol) and soak in the beaker overnight. Pour off the lye in the upper layer, add 13ml of isopropanol, and then add chloroacetic acid (12.8g, 0.13mol), leave it at room temperature for 2h, then continue to react at 65°C for 3h, and cool down. Add 100ml of water to dissolve the reaction product, centrifuge at 10000r / min for 5min, discard the insoluble matter, and take the upper layer solution. Add excess ethanol to precipitate a white flocculent solid, filter the precipitate, add 50ml of water to dissolve, and dissolve the precipitate several times until the pH is neutral. The precipitate was put into a 37 degree oven for drying to obtain carboxymethyl chitosan.

[0028] Get 0.22g carboxymethyl chitosan (about 1mmol) in the there-necked flask, add 15ml water to dissolve, add 4gPEI (Mw=800, 5mmol), adjust the pH value to 5.0 with 38% hydrochloric acid, then add NHS (0.117g , 1 mmol), stir...

Embodiment 2

[0032] Add 32 ml of 40% NaOH solution into the beaker, add 3.2 g of chitosan (Mw=50 kDa, 0.02 mol) and soak in the beaker overnight. Pour off the lye in the upper layer, add 13ml of isopropanol, and then add chloroacetic acid (12.8g, 0.13mol), leave it at room temperature for 2h, then continue to react at 65°C for 3h, and cool down. Add 100ml of water to dissolve the reaction product, centrifuge at 10000r / min for 5min, discard the insoluble matter, and take the upper layer solution. Add excess ethanol to precipitate a white flocculent solid, filter the precipitate, add 50ml of water to dissolve, and dissolve the precipitate several times until the pH is neutral. The precipitate was put into a 37 degree oven for drying to obtain carboxymethyl chitosan.

[0033] Get 0.22g carboxymethyl chitosan (about 1mmol) in the there-necked flask, add 15ml water to dissolve, add 4g PEI (Mw=800), adjust the pH value to 5.0 with 38% hydrochloric acid, then add NHS (0.117g, 1 mmol), stirred a...

Embodiment 3

[0036] Add 32 ml of 40% NaOH solution into the beaker, add 3.2 g of chitosan (Mw=50 kDa, 0.02 mol) and soak in the beaker overnight. Pour off the lye in the upper layer, add 13ml of isopropanol, and then add chloroacetic acid (12.8g, 0.13mol), leave it at room temperature for 2h, then continue to react at 65°C for 3h, and cool down. Add 100ml of water to dissolve the reaction product, centrifuge at 10000r / min for 5min, discard the insoluble matter, and take the upper layer solution. Add excess ethanol to precipitate a white flocculent solid, filter the precipitate, add 50ml of water to dissolve, and dissolve the precipitate several times until the pH is neutral. The precipitate was put into a 37 degree oven for drying to obtain carboxymethyl chitosan.

[0037] Get 0.22g carboxymethyl chitosan (about 1mmol) in the there-necked flask, add 15ml water to dissolve, add 4gPEI (Mw=800), adjust pH value to 5.0 with 38% hydrochloric acid, then add NHS (0.117g, 1mmol ), stirred at roo...

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Abstract

The invention relates to PEI (Polyetherimide) modified chitosan triply compound gene vector coated with RGD (Arginyl-Glycyl-Aspartic acid)-chondroitin sulfate and a preparation method and application thereof. The triply compound gene vector is formed by mixing RGD polypeptide modified chondroitin sulfate, pDNA and PEI grafted chitosan in the mass ratio of 15:1:4. The transfection efficiency of a chitosan vector is improved by using a PEI grafting method; by using a coating method of electronegative chondroitin sulfate, red cells in blood plasma and plasma protein can be prevented from being adsorbed and reduced and the stability of blood circulation can be enhanced; in addition, by using the special targeted RGD polypeptide to modify the chondroitin sulfate, the transmembrane capability of compound particles can be enhanced. A triply gene delivery system can be constructed by taking the PEI grafted chitosan and DNA compound particles as the core and the RGD-modified chondroitin sulfate as the casing. The preparation method is simple and convenient and has mild conditions; the modified chitosan is used as a gene vector, so that the transfection efficiency is high and the cytotoxicity is low; in addition, the invention can effectively prevent the adsorption to the red cells and electronegative macromolecules, has favorable body fluid circulating stability and is hopeful to be used for gene treatment clinical experiments.

Description

technical field [0001] The invention relates to an RGD polypeptide-modified chondroitin sulfate-coated low-molecular-weight PEI-chitosan triple composite gene carrier, a preparation method and application thereof, which can be used as an efficient targeting non-viral gene carrier in gene therapy. Background technique [0002] In recent years, gene therapy has received extensive attention. Although viral vectors have made some breakthroughs in gene therapy, their immunogenicity and potential carcinogenicity are still major hidden dangers that are difficult to overcome during clinical application. Non-viral vectors have been extensively studied as an alternative route of gene delivery. Currently, non-viral vectors mainly include two types: cationic liposomes and cationic polymers. [0003] Viral vectors have a great advantage in transfection rate due to the full use of the infectious and parasitic characteristics of highly evolved viruses. Since non-viral vectors lack this ...

Claims

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Application Information

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IPC IPC(8): C12N15/63A61K47/42A61K47/36A61K47/18
Inventor 刘文广刘源翟欣昀孙鹏徐军罗永峰
Owner TIANJIN UNIV
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