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Compound of tumor targeted nonviral gene vector and plasmid DNA and preparation method thereof

A tumor-targeting, gene carrier technology, applied in the field of complex preparation, can solve problems such as macrocytotoxicity, and achieve the effects of low cytotoxicity, excellent transfection ability, and high sensitivity

Inactive Publication Date: 2010-06-09
常熟紫金知识产权服务有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the high positive charge density makes PEI exhibit greater cytotoxicity at the same time.

Method used

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  • Compound of tumor targeted nonviral gene vector and plasmid DNA and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0027] (1) Mix cystamine hydrochloride, acryloyl chloride and sodium hydroxide at a molar ratio of 1:5:10 at 0°C, add deionized water to dissolve, react at room temperature for 12 hours, and filter to obtain a white crude product; wash with water for 3 After several times, it was crystallized and purified with ethyl acetate, and dried in vacuum to obtain bisacrylcystamine.

[0028] A methanol solution of diacrylcystamine with a concentration of 5 mg / ml was prepared, and an aqueous solution of polyethyleneimine with a molecular weight of 800 Da at a concentration of 20 mg / ml was prepared. Under the protection of nitrogen, at 25°C, mix the methanol solution of diacrylcystamine and the aqueous solution of polyethyleneimine, the molar ratio of diacrylcystamine and polyethyleneimine is 1:10, and react in the dark for 48h, Add hydrochloric acid to adjust the pH to 2, dialyze with water for 4 days, and obtain disulfide bond cross-linked polyethyleneimine after freeze-drying.

[0029...

Embodiment 2

[0035] (1) Mix cystamine hydrochloride, acryloyl chloride and sodium hydroxide at a molar ratio of 1:3:15 at 10°C, add deionized water to dissolve, react at room temperature for 20 hours, and filter to obtain a white crude product; wash with water for 3 After several times, it was crystallized and purified with ethyl acetate, and dried in vacuum to obtain bisacrylcystamine.

[0036] A methanol solution of diacrylcystamine with a concentration of 10 mg / ml was prepared, and an aqueous solution of polyethyleneimine with a molecular weight of 800 Da at a concentration of 15 mg / ml was prepared. Under the protection of nitrogen, at 25°C, mix the methanol solution of diacryloylcystamine and the aqueous solution of polyethyleneimine, the molar ratio of diacryloylcystamine and polyethyleneimine is 1:5, and react in the dark for 24h. Add hydrochloric acid to adjust the pH to 4, dialyze with water for 2 days, and obtain disulfide bond cross-linked polyethyleneimine after freeze-drying. ...

Embodiment 3

[0043] The complexes of the tumor-targeting non-viral gene carrier and plasmid DNA prepared in Example 1 and Example 2 were observed with a scanning electron microscope, and the results showed that the complex prepared in Example 1 had a spherical shape, and its diameter between 200-300nm, while the complexes prepared in Example 2 have very irregular shapes, and the particle diameters are all greater than 500nm.

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Abstract

The invention relates to a compound of a tumor targeted nonviral gene vector and plasmid DNA and a preparation method thereof. The method comprises the following steps:(1) synthesizing biotinylated disulfide polyethyleneimine, and then taking avidin as a bridge, coupling the biotinylated disulfide polyethyleneimine with biotinylated human transferrin to obtain the tumor targeted nonviral gene vector; (2) preparing a water solution of the tumor targeted nonviral gene vector, of which the concentration is 0.1-2mg / ml; (3) preparing a plasmid DNA solution with the concentration of 50-800mu m g / ml; and (4) adding the solution of step (2) to an equal amount of solution of step (3), swirling for 1-30s, and standing for 1-120min to obtain the compound of the tumor targeted nonviral gene vector and the plasmid DNA. The compound prepared by the invention has low cytotoxicity, good biocompatibility, obvious tumor targeting effect, simple synthesis steps, fast reaction speed and easy control of conditions, and can obviously improve the transfection efficiency of the plasmid DNA in tumor cells.

Description

technical field [0001] The invention relates to the technical field of biomedical polymer materials, in particular to a method for preparing a compound of a tumor-targeting non-viral gene carrier and plasmid DNA. Background technique [0002] Gene therapy was originally proposed for genetic diseases with single gene defects. The purpose is to replace the defective gene with a normal gene or a therapeutic gene or to remedy the pathogenic factors of the defective gene, so as to achieve the purpose of treating the disease. Gene therapy is a new method for the treatment of human diseases. The use of genes to treat diseases is to introduce functional genes into the patient's body to express them, and to perform functions through protein expression so that diseases can be cured. Gene therapy is currently mainly used to treat diseases that seriously threaten human health, including: genetic diseases, malignant tumors, cardiovascular diseases and infectious diseases. Successful gen...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K47/42A61K47/34A61P35/00
Inventor 张先正曾旋孙云霞瞿玮卓仁禧
Owner 常熟紫金知识产权服务有限公司
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