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Carbon nano-tube as siRNA carrier and transfection method thereof

A carbon nanotube, single-walled carbon nanotube technology, applied in the biological field

Inactive Publication Date: 2008-07-16
CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, carbon nanotubes have not been reported as siRNA carriers so far.

Method used

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  • Carbon nano-tube as siRNA carrier and transfection method thereof
  • Carbon nano-tube as siRNA carrier and transfection method thereof
  • Carbon nano-tube as siRNA carrier and transfection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1: carbon nanotube

[0024] The single-walled carbon nanotubes of the present invention are purchased from Adrich (St. Louis, MO, USA), with a diameter of 1.1 nm.

[0025] Carboxylated carbon nanotubes were prepared as follows: the single-walled carbon nanotubes were ultrasonically treated in a mixed acid of 3:1 (v / v) concentrated sulfuric acid (98%) and concentrated nitric acid (70%) for 24 hours, and then washed with water to a pH close to neutral.

[0026] The carbon nanotubes functionalized with hexamethylenediamine are prepared according to related patented technology (reference: Chinese patent application number: 200610016734.4).

Embodiment 2

[0027] Example 2: Preparation of siRNA: carbon nanotube complex

[0028] siRNA was prepared by conventional methods. The siRNA described in the embodiments of the present invention specifically inhibits human cyclin A 2 siRNA, the target sequence corresponding to the siRNA is human cyclin A 2 The mRNA of the gene, the siRNA is 16-30bp in length and contains the nucleic acid sequence as follows:

[0029] 5’-CCAUUG GUC CCU CUU GAU UTT-3’

[0030] 5'-AAU CAA GAG GGA CCAAUG GTT-3' (SEQ ID NO: 1)

[0031] Preferably, the 3' end of the siRNA contains an extended structure of 2-4 dT or 2-6 U;

[0032] The negative control siRNA is:

[0033] 5’-UUC UCC GAA CGU GUC ACG UTT-3’

[0034] 5’-ACG UGA CAC GUU CGG AGA ATT-3’

[0035] Mix the siRNA and the carbon nanotubes at a ratio of 1:40-1:200 (w / w), and place them at room temperature (15-30° C.) for 30 minutes to one hour.

[0036] Non-denaturing polyacrylamide gel electrophoresis experiments confirmed that when the ratio of siRNA...

Embodiment 3

[0037] Example 3: Transfection

[0038] Transfection mainly has the following steps:

[0039] (1) Preparation of cells to be transfected: K562 cells were inoculated in IMDM (Iscove's modified Dulbecco's medium) medium containing 10% fetal bovine serum and 2mmol / L glutamine, placed at 37°C, 5%CO 2 In the saturated humidity incubator with (v / v) concentration, no drug was cultivated two weeks before the experiment.

[0040] (2) The siRNA:carbon nanotube complex was prepared by the method in Example 2.

[0041] (3) Transfection: cells were seeded in 6-well or 24-well culture plates at a density of 0.5-1.2×10 5cells / mL, add the siRNA:carbon nanotube complex into the cells, the final concentration of siRNA is 40nmol / L, and then place it at 37℃, 5%CO 2 (v / v) concentration in a saturated humidity incubator.

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Abstract

The invention relates to a method for utilizing a carbon nano-tube as a siRNA vector and the transfection. The invention utilizes the carbon nano-tube as the siRNA transfection vector, the vector has very low cytotoxicity and can effectively transfect the siRNA into a cell for leading the siRNA to play biological function. The transfection proposal is that: (1) the siRNA is prepared by a conventional method; (2) the carbon nano-tube and the siRNA are mixed evenly and placed for a period of time at the room temperature; (3) the treated mixture of the carbon nano-tube and the siRNA is utilized for culturing cells. The method for utilizing a carbon nano-tube as the siRNA vector and the transfection of the invention has the advantages of low cytotoxicity, high transfection efficiency and so on. The siRNA takes the hexamethylene diamine-functionized carbon nano-tube as the vector, and the mRNA expression level of the human cyclin A2 of K562 cell is reduced by 60 to 95 percent via the transfection.

Description

technical field [0001] The invention belongs to the field of biotechnology. The invention relates to the use of carbon nanotubes as siRNA carrier and its transfection method. Background technique [0002] RNA interference (RNA interfering, RNAi) is a sequence-specific gene post-transcriptional silencing process triggered by double-stranded RNA homologous to the target gene sequence and widely present in organisms. RNAi was first discovered in Caenorhabditis elegans by Andrew Fire in 1998, and then widely found in most eukaryotes such as fungi, Arabidopsis, Hydra, planarians, trypanosoma, and zebrafish. Elbashir SM et al. used artificially synthesized small molecule double-stranded RNA to exert RNAi effect in mammalian cells in 2001. Since then, RNAi has become a powerful tool in molecular medicine. [0003] siRNA is unstable, easily degraded by enzymes, and has a low cellular uptake rate. Therefore, an efficient transfection carrier is crucial for its effectiveness. Commo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/87
Inventor 曲晓刚王晓辉
Owner CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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