Multifunctional graphene gene vector and gene transfection reagent based on gene vector and preparation method thereof

A gene carrier, graphene technology, applied in chemical instruments and methods, introduction of foreign genetic material using carriers, inorganic chemistry, etc., can solve the problems of low transfection efficiency of non-tumor cells, limited transfection efficiency, and unstable graphene. , to achieve the effect of small impact on dosage, convenient operation and low toxicity

Inactive Publication Date: 2013-06-05
刘遵峰 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, polyethyleneimine-functionalized graphene obtained by physical adsorption method is unstable and precipitates in brine
Although it is stable when linked to graphene oxide through covalent bonds, its stability is still seriously affected by the charge properties of the solution after being mixed with DNA plasmids. The transfection process is greatly affected by the operation method and reagent dosage, and there is still a certain toxicity
In addition, the gene transfection process is only controlled by the cationic polymer, and its transfection efficiency is still limited, especially for non-tumor cells.

Method used

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  • Multifunctional graphene gene vector and gene transfection reagent based on gene vector and preparation method thereof
  • Multifunctional graphene gene vector and gene transfection reagent based on gene vector and preparation method thereof
  • Multifunctional graphene gene vector and gene transfection reagent based on gene vector and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1. Polyethyleneimine and 2-arm polyethylene glycol functionalized graphene gene carrier and gene transfection reagent

[0032] (1) Preparation of polyethyleneimine and 2-arm polyethylene glycol functionalized graphene gene carrier and gene transfection reagent

[0033] Disperse 1 g of graphene oxide in water, add 0.5 g of polyethyleneimine (Mw=2,500), 0.5 g of 2-arm polyethylene glycol (Mw=2000) with amino groups at the end, 0.2 g of 1 g (3-dimethylamino Propyl)-3-ethylcarbodiimide, ultrasonic and stirring, the reaction time is 5-24h. Filtrating and washing to obtain graphene oxide functionalized with cationic polymer and surfactant. Disperse it in water to make gene transfection reagent. Atomic force results show that the average size is less than 30nm, such as figure 1 shown. The zeta potential is +30mV.

[0034] (2) Evaluation of gene transfection efficiency

[0035] The cell species required for the experiment were placed in a 24-well plate to achieve...

Embodiment 2

[0038] Embodiment 2. Polyethylenimine and 6-arm polyethylene glycol functionalized graphene gene carrier and gene transfection reagent

[0039] (1) Preparation of gene carrier and gene transfection reagent: except that 6-arm polyethylene glycol was used instead of 2-arm polyethylene glycol as the reaction raw material, other experimental steps were the same as in Example 1. The atomic force results show that the average size is less than 30nm and the zeta potential is +33mV.

[0040] (2) Evaluation of gene transfection efficiency: the procedure was the same as that in Example 1 except that 1 μl, 2 μl, 5 μl, 10 μl, 15 μl, and 20 μl of transfection reagent were added. The mixture of the gene transfection reagent and the GFP plasmid was centrifuged at 10000 g for 30 min in the cell culture medium without aggregation. 48h transfection efficiencies were 50%, 75%, 77%, 75%, 70%, 65%.

[0041] (3) Cytotoxicity evaluation of gene transfection reagent: the steps are the same as in Ex...

Embodiment 3

[0042] Example 3. Polyethyleneimine and 4-arm polyethylene glycol functionalized graphene gene carrier and gene transfection reagent

[0043] (1) Preparation of gene carrier and gene transfection reagent: except that 4-arm polyethylene glycol was used as the reaction material instead of 2-arm polyethylene glycol, other experimental steps were the same as in Example 1. The atomic force results show that the average size is less than 30nm and the zeta potential is +28mV.

[0044] (2) Evaluation of gene transfection efficiency: the steps are the same as in Example 1. The mixture of the gene transfection reagent and the GFP plasmid was centrifuged at 10000 g for 30 min in the cell culture medium without aggregation. 48h transfection efficiency was 65%.

[0045] (3) Cytotoxicity evaluation of gene transfection reagent: the steps are the same as in Example 1. The cell viability was measured to be 92%.

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Abstract

The invention belongs to the technical field of biological medicine, and designs a multifunctional graphene gene vector, a gene transfection reagent based on the gene vector and a preparation method thereof. The multifunctional graphene comprises the following three parts of graphene, cationic polymer, and an active reagent, wherein the active reagent comprises one or more than one of a surfactant, or cell-penetrating peptide, or nuclear localization signal peptide; the cationic polymer and the active reagent are connected to the graphene. According to the invention, through multi-functionalization, the stability, dispersibility, biocompatibility of the gene vector in the gene transfection process are improved; the toxicity is reduced; and a plurality of gene transfection promotion mechanisms are adopted to improve transfection efficiency. Gene transfection experiment results show that high transfection efficiency and low toxicity are realized in a plurality of cell strains. The main purpose of the invention is gene transfection, and is applicable to cytobiological research, gene product production, and gene therapy.

Description

[0001] Technical field: the present invention relates to a multifunctional nanomaterial and its preparation method, especially a multifunctional graphene gene transfection reagent and its preparation method Background technique: [0002] The gene transfection process is a process in which eukaryotic cells obtain new genetic markers due to the incorporation of foreign DNA. Gene transfection technology is not only an important tool for studying transgene and gene expression, but also a key step in gene therapy. At present, the main gene transfection reagents are viral transfection reagents and synthetic transfection reagents. Viral transfection reagents have limited gene carrying capacity, lack of targeting, and are dangerous to operate (Putnam, D. Nat Mater 5, 439-451, 2006), so people tend to use synthetic gene transfection reagents. At present, synthetic gene transfection reagents are mainly cationic liposomes, cationic peptides and cationic polymers, or complexes of the abo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C01B31/04C01B32/194
Inventor 刘遵峰
Owner 刘遵峰
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