Gene transfection vector based on low-algebraic polyamidoamine and preparation method and application thereof

A technology of gene transfection carrier and dendrimer, which is applied in the field of polymer chemistry and biomaterials, can solve the problems of high cytotoxicity and high cost, and achieve low synthesis cost, low cost and high transfection efficiency effect

Inactive Publication Date: 2013-01-16
EAST CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, researchers have chemically modified polyamide-amine dendrimers by grafting amino acids, fatty chains and targeting molecules on the surface of high-algebra polyamide-amine dendrimers. The gene transfection efficiency of dendrimer may reduce its cytotoxicity, but the high cost of dendrimer gene transfection vectors and the high cytotoxicity associated with high gene transfection efficiency have not been improved

Method used

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  • Gene transfection vector based on low-algebraic polyamidoamine and preparation method and application thereof
  • Gene transfection vector based on low-algebraic polyamidoamine and preparation method and application thereof
  • Gene transfection vector based on low-algebraic polyamidoamine and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Preparation of novel gene transfection vectors based on low-generation polyamide-amine dendrimer

[0039] Taking the low-generation polyamide-amine dendrimer gene transfection vector G2DSP1-x as an example, such as figure 1 As shown, G2 is the second-generation polyamide-amine dendrimer, the central core is ethylenediamine, the terminal group is a primary amine group, n is 3, m is 2; DSP is 3,3'- Dithiodipropionic acid two (N-hydroxysuccinimide ester), 1-x is 1 molar equivalent G Corresponding to the crosslinking agent DSP of x molar equivalent, x=0.25-4.0, its structural formula is:

[0040]

[0041] In formula (I), R is the second generation polyamide-amine dendrimer G2; Its structure is as shown in formula (II):

[0042]

[0043] In formula (II), M is the central core of the second-generation polyamide-amine dendrimer G2: ethylenediamine.

[0044] In the specific implementation of the present invention, M can also be ammonia, ethylenediamine, butan...

Embodiment 2

[0048] Embodiment 2: the stability of gene transfection carrier G2DSP1-1 and DNA complex

[0049] The gene transfection reagent G2DSP1-1 prepared in Example 1 and the green fluorescent protein particle DNA (pEGFP-1) form a complex at room temperature, and the complex DNA of the gene transfection reagent G2DSP1-1 is detected by agarose gel electrophoresis. ability.

[0050] The specific method is: the gene transfection reagent G2DSP1-1 and the green fluorescent protein particle DNA (pEGFP-1) are mixed according to different nitrogen and phosphorus ratios (0:1, 0.5:1, 1:1, 2:1, 4:1, respectively). 1 and 8:1) were mixed at room temperature, incubated for 30 minutes, and then diluted with DNA loading buffer, and finally the sample was subjected to agarose gel electrophoresis at 90 volts for 50 minutes. The concentration is 1%. In the experiment, the second-generation polyamidoamine dendrimer G2 and the fifth-generation polyamidoamine dendrimer G5 were used as controls.

[0051]...

Embodiment 3

[0055] Embodiment 3: the gene transfection efficiency of gene transfection carrier G2DSP1-1

[0056] The gene transfection reagent G2DSP1-1 prepared in Example 1 and the luciferase plasmid or green fluorescent protein plasmid form a complex at room temperature, and then transfect in HEK293 cells and HeLa cells, by detecting luciferase or The expression level of green fluorescent protein was used to evaluate the gene transfection efficiency of the vector. The specific method is: culture HEK293 or HeLa cells in a 24-well plate and incubate for 48 hours, mix 1.6 micrograms of luciferase or green fluorescent protein particles with G2DSP1-1 at different nitrogen-to-phosphorus ratios, and then add them to the medium and mix well; After the cells were incubated together for 6 hours, 500 microliters of medium containing 10% serum was added. After 24 hours, the cells were processed, digested with trypsin, and the luciferase expression level was detected according to the instructions p...

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Abstract

The invention provides a novel gene transfection vector based on low-algebraic polyamidoamine. The gene transfection vector comprises low-algebraic polyamidoamine and a difunctional cross-linking agent which contains a disulfide bond and of which the reaction points to amino, wherein a primary amine group of the low-algebraic polyamidoamine is reacted and connected with active groups at two ends of the difunctional cross-linking agent. The invention also provides a method for preparing the gene transfection vector and application of the gene transfection vector which is used as a nucleic acid molecule delivery vector in vivo or in vitro. The gene transfection vector is low in synthetic cost and small in cytotoxicity, can deliver gene molecules to cells effectively and safely and can be used as the gene transfection vector which has the advantages of high efficiency, low toxicity, low cost and the like.

Description

technical field [0001] The invention relates to the technical fields of polymer chemistry and biomaterials, in particular to a novel gene transfection carrier based on low-generation polyamide-amine dendrimers and its preparation method and application. Background technique [0002] Gene transfection refers to the process of introducing foreign genes into cells to obtain new genetic traits. Gene transfection vector is the core of gene transfection technology. An ideal gene transfection vector should have the following characteristics: high transfection efficiency, low cytotoxicity, good biological safety, and low price. Currently used gene transfection vectors mainly include viral vectors and non-viral vectors. The main application research still uses viral vectors with high transfection efficiency, but viral vectors have problems such as limited ability to carry genes and potential safety hazards. [0003] Polyamidoamine (PAMAM) was first reported by Tomalia D.A. in 1985,...

Claims

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Application Information

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IPC IPC(8): C12N15/87C08G83/00C08J3/24
Inventor 程义云王辉刘红梅
Owner EAST CHINA NORMAL UNIVERSITY
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