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Gene vector system containing targeted shading system, preparation and use thereof

A gene carrier and targeting ligand technology, applied in the biological field, can solve the problems of reduced transfection efficiency, cumbersome reactions, and difficult access to raw materials, and achieve high transfection efficiency, improved performance, and low cytotoxicity

Inactive Publication Date: 2012-01-25
CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the shielding of positive charges by PEG molecules will also reduce the phagocytosis of complex particles by target tissue cells, thereby reducing transfection efficiency [5] , the amount of grafted PEG molecules needs to be carefully controlled to get better results. Our previous work also confirmed the above results
In addition, using the method of linking targeting ligands with double-hetero-PEG, the raw materials are not easy to obtain, and the reaction is relatively cumbersome.
The present invention develops a more convenient and easy-to-obtain shielding system, introduces the shielding system carrying the targeting ligand into the gene carrier system by means of electrostatic compounding, adjusts the shielding ratio, controls the reduction of transfection efficiency caused by excessive shielding, and utilizes the targeting ligand Ligands improve the specific recognition and delivery of gene carriers to target tissues is a technical problem that the industry urgently needs to solve

Method used

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  • Gene vector system containing targeted shading system, preparation and use thereof
  • Gene vector system containing targeted shading system, preparation and use thereof
  • Gene vector system containing targeted shading system, preparation and use thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1: Preparation of a shielding system with a targeting ligand

[0031] According to the feeding ratio described in Table 1, the molecular weight was 10 mg of hyaluronic acid of 1.3M, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and RGD The aqueous solution was mixed, and the pH of the reaction solution was adjusted to 4.75 with hydrochloric acid solution. The reaction was carried out at room temperature for 5 hours. After the reaction, the product was dialyzed against 3 liters of distilled water for 3 days and then freeze-dried to obtain a white flocculent product. The content of RGD in the product was calculated by NMR, and the results are shown in Table 1.

[0032] Table 1 Preparation table of hyaluronic acid masking system with RGD short peptide

[0033] product number

[0034] complex unit contains an RGD)

Embodiment 2

[0035] Embodiment 2: the preparation of the gene carrier system containing targeting shielding system

[0036]Take HA-RGD, add secondary water to dissolve, configure an aqueous solution with a concentration of 0.02-2 mg / mL, filter and sterilize with a microporous membrane with a pore size of 0.45 μm, and set aside. Take cationic polymer polyethyleneimine (PEI) and polyethyleneimine-polybenzyl glutamate (PEI-PBLG), add secondary water to dissolve, prepare an aqueous solution with a concentration of 0.02-2 mg / mL, and use a pore size of 0.45 μm microporous membrane filter to sterilize and set aside. Take the plasmid and dissolve it with secondary water to prepare an aqueous solution with a concentration of 0.02 mg / mL.

[0037] The cationic polymer aqueous solution and the plasmid aqueous solution of different concentrations are mixed in equal volumes to ensure that the mass ratio of the cationic polymer to the plasmid is 1:1. After the mixed aqueous solution of complex particle...

Embodiment 3

[0041] Example 3: Using gel electrophoresis to identify the stability of complex particles

[0042] Take 4 μL of 0.1 μg / μL DNA aqueous solution and 4 μL of 1 μg / μL polyethyleneimine-polybenzyl glutamate aqueous solution to complex, let it stand at room temperature for 10 minutes, then add 4 μL of different concentrations of HA aqueous solution, and detect the complex particle The stability of the masking system after addition. figure 1 The results of electrophoresis showed that adding 40 times of DNA to the hyaluronic acid shielding system would not destroy the complex formed by the cationic polymer and DNA. Example 4: In vitro transfection of HeLa cells mediated by green fluorescent protein particles using a gene carrier system with targeting shielding

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Abstract

The invention relates to a gene delivery system containing a target shielding system and a preparation method and application thereof. The gene delivery system consists of a shielding system with a target ligand, a cation polymer material and plasmid DNA, wherein, the cation polymer material and the plasmid DNA are compounded to form compound particles, and the shielding system with the target ligand is shielded on the surface of the compound through electrostatic effect. The gene delivery system containing the target shielding system can transfer a loaded genic material into cells to realizethe expression of the genic material and finish a transfection process, and the targeting of gene transfection can be improved. The preparation method has the advantages that: the target ligand is grafted onto the shielding system; on the one hand, the targeting strategy can not affect the compound ability of a cationic polymer to the DNA, on the other hand, the targeting strategy can also guarantee that the target ligand is extended to the periphery of the gene delivery system so as to improve the combination efficiency of the target ligand and a cell surface receptor. The highest transfection efficiency of the gene delivery system to a HeLa cell is 73 percent, and the toxicity of the cell is small.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a gene carrier system containing a targeting masking system, its preparation method and application Background technique [0002] With the completion of the Human Genome Project and the advancement of cell biology technology, gene therapy will play an important role in the process of overcoming the stubborn disease of human beings, and will gradually become a common method [1] . Gene therapy refers to a new biomedical 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. [0003] Introducing gene material into cells for expression is an essential step for gene therapy, and successful gene therapy depends on effective gene carriers. The use of viral vector-mediated gene transfer is the most widely used metho...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/64C12N15/85
Inventor 田华雨陈学思林琳夏加亮陈磊郭兆培景遐斌
Owner CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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