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Preparation method of light-responsive pegized gene delivery system based on host-guest assembly

A technology of gene delivery and photoresponse, applied in other methods of inserting foreign genetic materials, gene therapy, pharmaceutical formulations, etc., can solve the problems of complex preparation and affecting the ability of gene carriers to associate DNA molecules, etc., to achieve simple preparation and improve gene expression. Simple effects of transfection efficiency, responsive regulation

Inactive Publication Date: 2011-12-07
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the existing research is to introduce PEG segments containing sensitive groups into the gene delivery system through chemical bonding, but this method is complicated to prepare and will affect the ability of the gene carrier to associate with DNA molecules

Method used

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  • Preparation method of light-responsive pegized gene delivery system based on host-guest assembly
  • Preparation method of light-responsive pegized gene delivery system based on host-guest assembly
  • Preparation method of light-responsive pegized gene delivery system based on host-guest assembly

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Experimental program
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Effect test

Embodiment 1

[0019] Prepare a N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes) buffer solution with a NaCl concentration of 20 mM, a pH of 7.4, and a concentration of 20 mM. Using this buffer solution, a solution of polyethyleneimine grafted cyclodextrin (PEI-CD, PEI molecular weight 25,000) at a concentration of 1 mg / mL and azobenzene-modified polyethylene glycol (Azo -PEG, PEG molecular weight 5000) solution. The green fluorescent protein reporter gene pEGFP (4733bp) was selected as the target gene, and a pEGFP solution with a concentration of 500 μg / mL was prepared. Take 162 μl of the above PEI-CD solution and 88 μl of the above Azo-PEG solution, the molar ratio of CD and Azo-PEG in PEI-CD is 1:1, sonicate for 15 min and then let it stand for 1 h. Then it was mixed with 250 μl DNA solution in an equal volume and left to stand to prepare a light-responsive PEGylated gene delivery system based on host-guest assembly.

Embodiment 2

[0021] Use N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes) buffer solution with a NaCl concentration of 20 mM, pH=7.4, and a concentration of 20 mM, and prepare polyethyleneimine with a concentration of 0.1 mg / mL Grafted cyclodextrin (PEI-CD, PEI molecular weight 25000) solution and azobenzene-modified polyethylene glycol (Azo-PEG, PEG molecular weight 5000) solution at a concentration of 1.5 mg / mL. Prepare a pEGFP solution with a concentration of 50 μg / mL. Take 162 μl of the above PEI-CD solution and 88 μl of the above Azo-PEG solution, the molar ratio of CD and Azo-PEG in PEI-CD is 1:4, sonicate for 15 min and then let it stand for 1 h. Then it was mixed with 250 μl DNA solution in an equal volume and left to stand to prepare a light-responsive PEGylated gene delivery system based on host-guest assembly.

Embodiment 3

[0023] Use N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes) buffer solution with a concentration of 20 mM NaCl, pH=7.4, and a concentration of 20 mM, and prepare polyethylene oxide with a concentration of 1 mg / mL. Amine-grafted cyclodextrin (PEI-CD, PEI molecular weight 25000) solution and azobenzene-modified polyethylene glycol (Azo-PEG, PEG molecular weight 5000) solution at a concentration of 0.4 mg / mL. Prepare a pEGFP solution with a concentration of 50 μg / mL. Take 22 μl of the above PEI-CD solution and 228 μl of the above Azo-PEG solution, the molar ratio of CD and Azo-PEG in PEI-CD is 1:2, sonicate for 15 min and then let it stand for 1 h. Then it was mixed with 250 μl DNA solution in an equal volume and left to stand to prepare a light-responsive PEGylated gene delivery system based on host-guest assembly.

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Abstract

The invention discloses a preparation method of a light-responsive PEGylated gene delivery system based on host-guest assembly. Its steps are as follows: 1) Using N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid buffer solution, prepare polyethyleneimine grafted cyclodextrin solution with a concentration of 0.1-1 mg / mL and a concentration of 0.4-2 mg / mL azobenzene-modified polyethylene glycol solution; 2) Use the buffer solution in step 1) to prepare a DNA solution with a concentration of 50-500 μg / mL; 3) Mix the two solutions in step 1) and polymerize The molar ratio of cyclodextrin and azobenzene-modified polyethylene glycol contained in ethyleneimine-grafted cyclodextrin is 1:1-1:4, and the supramolecular compound solution is obtained after ultrasonication for 15 minutes and standing for 1 hour; 4) Add the solution prepared in step 3) to an equal volume of the solution in step 2), vortex to mix and let stand. The invention can improve its stability in physiological saline solution. Through the photoisomerization of azobenzene, the detachment of the PEG shell of the intracellular gene delivery system can be achieved, which can effectively improve the efficiency of gene transfection.

Description

technical field [0001] The invention relates to a preparation method of a light-responsive PEGylated gene transfer system based on host-guest assembly, which belongs to the preparation method and technology of a novel high-efficiency non-viral gene transfer system in the field of gene therapy. Background technique [0002] Gene therapy is a biomedical new technology that introduces human normal genes or genes with therapeutic effects into human target cells in a certain way to correct gene defects or exert therapeutic effects, so as to achieve the purpose of treating diseases. Since naked nucleic acid molecules are difficult to stably exist in body fluids, only low-level expression can be achieved in vivo. Although viral vectors have high transfection efficiency, potential toxicity, immunogenicity and targeting limit their application in clinical treatment. It is of great significance to develop a highly biocompatible non-viral gene delivery system with high efficiency, saf...

Claims

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Application Information

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IPC IPC(8): C12N15/87A61K48/00
Inventor 王幽香李文宇陈丽娜唐三胡巧玲
Owner ZHEJIANG UNIV
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