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Application of cationic imidazole Gemini surfactant [Cn-s-Cnim]Br2 in gene transfection

A cn-s-cnim, surfactant technology, applied in the field of biomedicine, can solve the problems of different carrier/DNA structures, difficulty in release research, etc.

Inactive Publication Date: 2013-02-27
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the research on the interaction between non-viral gene carriers and DNA has received attention, a major limitation of the commonly used cationic polymers as gene carriers lies in the heterogeneity of the size and valence state of the polymer molecules. The uniformity directly leads to the difference in the prepared vector / DNA structure, which brings great difficulties to the release research in vivo

Method used

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  • Application of cationic imidazole Gemini surfactant [Cn-s-Cnim]Br2 in gene transfection
  • Application of cationic imidazole Gemini surfactant [Cn-s-Cnim]Br2 in gene transfection
  • Application of cationic imidazole Gemini surfactant [Cn-s-Cnim]Br2 in gene transfection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1. [C 12 -4-C 12 im]Br 2 Preparation and Characterization of DNA / DNA Nanocomplexes

[0056] 1.1. The used [C 12 -4-C 12 im]Br 2 with DNA

[0057] Imidazole type Gemini surfactant ([C 12 -4-C 12 im]Br 2 ), which incorporates the characteristics of traditional Gemini surfactants and long-chain imidazole ionic liquids, and has more outstanding advantages. [C 12 -4-C 12 im]Br 2 Can be prepared according to relevant literature (referring to people such as Xu, G.Y., Synthesis and Properties of Ionic Liquid-Type Gemini Imidazolim Surfactants, J.ColloidInterface Sci.2008,326490-495; Aggregation and Thermodynamic Properties of Ionic Liquid-Type Gemini Imidazolium Surfactants with Different Spacer Length, Colloid Polym Sci. 2009, 287, 395-402).

[0058] DNA is plasmid DNA, pBR322DNA (4363bp) and pEGFP-N1DNA (4733bp) were purchased from Sigma-Aldrich Company. Among them, pBR322DNA (4363bp) was used for [C 12 -4-C 12 im]Br 2 Characterization of the basic ph...

Embodiment 2

[0067] Example 2. [C 12 -4-C 12 im]Br 2 Cytotoxicity assay

[0068] Using MTT (thiazolium blue) analysis method to investigate [C 12 -4-C 12 im]Br 2 The selected cells include human embryonic kidney cells HEK293 cells, human cervical cancer cells HeLa cells, human non-small cell lung cancer cells A549Y-10 cells and human oral epidermoid cancer cells KB cells. Will 1×10 4 Cells per well were inoculated in 96-well culture plate, 100 μL of medium (containing 10% bovine serum albumin, 1% penicillin and streptomycin) was added to each well, and placed at 37°C containing 5% CO 2 Incubate for 24 hours in an incubator (humid atmosphere). Replace with fresh medium, add 100 μL of [C 12 -4-C 12 im]Br 2 (The final concentrations were 1.2 × 10 -3 mol / L, 6×10 -4 mol / L, 3×10 -4 mol / L, 1.5×10 -4 mol / L, 7.5×10 -5 mol / L, 3.75×10 -5 mol / L, 1.9×10 -5 mol / L, 9.5×10 -6 mol / L, 4.7×10 -6 mol / L, 2.4×10 -6 mol / L, 1.2×10 -6 mol / L, 6×10 -7 mol / L, 3×10 -7 mol / L), after the cells wer...

Embodiment 3

[0073] Example 3. [C 12 -4-C 12 im]Br 2 Gene transfection experiment of DNA / DNA nanocomplex

[0074] The use of non-viral gene vectors [C 12 -4-C 12 im]Br 2 The DNA is introduced into the cells to achieve gene expression. The test cells are human embryonic kidney cells HEK293 cells and human cervical cancer cells HeLa cells. The day before transfection, inoculate 4 × 10 -4 6×10 -4 For cells, add 2mL of culture medium (containing 10% bovine serum albumin, without antibiotics), which can make the cells confluent at 50%-60% at the time of transfection. Cells were placed at 37°C with 5% CO 2 cultured in an incubator (humid atmosphere). After incubation, the old medium was replaced with serum- and antibiotic-free medium. Prepare nanocomplexes as follows: Prepare 50 μL of pEGFP-N1DNA solution (2×10 -5mol / L), 50μL of [C 12 -4-C 12 im]Br 2 solution (1 x 10 -5 mol / L), after standing at room temperature for 5 minutes, the DNA solution and [C 12 -4-C 12 im]Br 2 The solu...

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Abstract

The invention relates to an application of a cationic imidazole Gemini surfactant [Cn-s-Cnim]Br2 in gene transfection. A non-viral gene vector is the cationic imidazole Gemini surfactant ([C12-4-C12im]Br2); and a [C12-4-C12im]Br2 solution prepared by a buffering solution can be directly applied in the gene transfection. The invention also provides a method of a [C12-4-C12im]Br2 / DNA nanocomposite formed by effectively inducing DNA with [C12-4-C12im]Br2. High-efficiency transfection of the genes in cells by adding a buffering solution of the [C12-4-C12im]Br2 / DNA nanocomposite to receptor cells. The [Cn-s-Cnim]Br2 and the [C12-4-C12im]Br2 / DNA nanocomposite have relatively high gene transfection efficiency for various cells, and have relatively low cytotoxicity for transfection cells.

Description

technical field [0001] The present invention relates to cationic imidazole Gemini surfactant [C n -s-C n im]Br 2 The application in gene transfection belongs to the technical field of biomedicine. Background technique [0002] The development of molecular biology has made gene therapy of diseases at the molecular level possible. The whole process of gene therapy is that the target gene enters the cell under the carrier and realizes gene correction and gene replacement, etc., to correct or compensate for diseases caused by gene defects and abnormalities, or to achieve the purpose of treatment by interfering with the expression of specific genes, etc. ( See Qasba, P.K. et al., DNA and Gene Therapy: Transfer of Mouse DNA to Human and Mouse Embryonic Cells by Polyoma Pseudovirions, Proc. Natl. Acad. Sci. USA 1971, 68, 2345-2349; and Xia, H.B. et al., siRNA -mediated gene silencing in vitro and in vivo, Nat.Biotechnol.2002, 20, 1006-1010), one of the key issues of gene therap...

Claims

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Application Information

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IPC IPC(8): C12N15/63
Inventor 徐桂英周亭杨延莲敖明祺李平王琛
Owner SHANDONG UNIV
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