Preparation method and application of liposome-chitosan compound gene carrier

A technology of gene carrier and chitosan, which is applied in the field of preparation of liposome-chitosan composite gene carrier, can solve problems such as unsatisfactory, increase absorption and bioavailability, solve excessive cytotoxicity, reduce cell toxic effect

Inactive Publication Date: 2011-07-27
DALIAN NATIONALITIES UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There are domestic research patents on targeting chitosan gene carriers, and the transfection efficiency has been improved to a certain extent, but it is still not ideal (public number: CN1884551)

Method used

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  • Preparation method and application of liposome-chitosan compound gene carrier
  • Preparation method and application of liposome-chitosan compound gene carrier
  • Preparation method and application of liposome-chitosan compound gene carrier

Examples

Experimental program
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Effect test

Embodiment 1

[0036] The preparation process adopts a 55 μl complex system and a high-throughput transfection procedure in a 96-well plate. The mass ratio of chitosan and DNA is 1 / 1, the preparation method of 0.1-1 μ g DNA calculation per hole of 96-well plate: 0.1-1 μ g DNA is diluted to 25 μ l with DMEM, 0.3-8 μ g DOTAP (according to DOTAP / DNA=3- The mass ratio of 8 / 1) was diluted to 25 μl with DMEM, and the two were gently mixed (after the liposome dilution was added to the plasmid dilution, it was incubated at room temperature for 5-10min, and 0.1-1μg (according to chitosan / DNA =1 / 1) chitosan, diluted to 5 μ l with DMEM, mixed gently, and incubated at room temperature for 20-30min. That is, the liposome-chitosan composite gene carrier was obtained.

Embodiment 2

[0038] The mass ratio of chitosan and DNA is 2 / 1, the preparation method of 0.1-1 μ g DNA calculation per well of 96-orifice plate: 0.1-1 μ g DNA is diluted to 25 μ l with DMEM, 0.3-8 μ g DOTAP (according to DOTAP / DNA=3- 8 / 1 mass ratio) was diluted to 25 μl with DMEM, after the two were gently mixed (liposome dilution was added to plasmid dilution), room temperature was incubated for 5-10min, and 0.2-2 μg (according to chitosan / DNA=2 / 1) chitosan, diluted to 5 μl with DMEM, mixed gently, and incubated at room temperature for 20-30min. That is, the liposome-chitosan composite gene carrier is prepared.

Embodiment 3

[0040] The mass ratio of chitosan and DNA is 3 / 1, the preparation method of 0.1-1 μ g DNA calculation per well of 96-orifice plate: 0.1-1 μ g DNA is diluted to 25 μ l with DMEM, 0.3-8 μ g DOTAP (according to DOTAP / DNA=3- 8 / 1 mass ratio) was diluted to 25 μ l with DMEM, after the two were gently mixed (liposome dilution was added to the plasmid dilution), room temperature was incubated for 5-10 min, and 0.3-3 μg (according to chitosan / DNA=3 / 1) chitosan, diluted to 5 μl with DMEM, mixed gently, and incubated at room temperature for 20-30min. That is, the liposome-chitosan composite gene carrier is prepared.

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Abstract

The invention relates to a preparation method and application of a liposome-chitosan compound gene carrier, wherein the preparation method comprises the steps of: slowly dipping DOTAP dilution in DNA dilution with the same volume and preserving the heat at room temperature for 5-10min; and adding chitosan with molecular weight range of 50-190KDa and deacetylation degree of 75-85% and preserving the heat at room temperature for 20-30min so as to prepare the compound gene carrier. Through the carrier, the problem that the transfection efficiency of cation liposome DOTAP is low is overcome, higher transfection efficiency is provided, the cytotoxicity is reduced and the problem that the cytotoxicity of the cation liposome is higher is solved. The compound gene carrier provided by the invention can slowly release genetic medicines and increase the absorption and the bioavailability of the genes, has the advantages of efficiency, safety, good cell compatibility and convenience for use, and is a novel efficient gene carrier and transfection reagent.

Description

technical field [0001] The invention belongs to a preparation method and application of a gene carrier, in particular to a preparation method and application of a liposome-chitosan composite gene carrier. Background technique [0002] Gene therapy is a biomedical treatment method that uses vectors to deliver target genes into target cells for moderate expression in order to treat diseases. As a new treatment method, gene therapy can treat a variety of diseases, including cancer, genetic diseases, infectious diseases, cardiovascular diseases and autoimmune diseases. Among them, cancer gene therapy is the main application field of gene therapy. Over the past few years, global gene therapy clinical trials have made great progress. But at the same time, gene therapy also encountered many difficulties. In the future, one of the main goals of gene therapy is to develop safe and efficient gene delivery systems. The vectors that can be used for gene transfer mainly include viral ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/36A61K48/00C12N15/63
Inventor 王冰张树彪崔韶晖赵轶男陈会英
Owner DALIAN NATIONALITIES UNIVERSITY
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