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Chitosan derivative used as gene vector, and preparation method and application thereof

A technology of chitosan derivatives and chitosan, which is applied in the direction of introducing foreign genetic material, gene therapy, and pharmaceutical formulations by using a carrier, can solve the problems of unfavorable transfection efficiency, poor water solubility of chitosan, and low transfection efficiency. , to achieve high transfection efficiency, high biocompatibility, and improved water solubility.

Inactive Publication Date: 2013-04-10
SHANGHAI INST OF MATERIA MEDICA CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, with chitosan without any modification, the transfection efficiency is very low, and high molecular weight chitosan has poor water solubility, which is not conducive to improving the transfection efficiency, so it must be chemically modified

Method used

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  • Chitosan derivative used as gene vector, and preparation method and application thereof
  • Chitosan derivative used as gene vector, and preparation method and application thereof
  • Chitosan derivative used as gene vector, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The synthetic method of embodiment 1 chitosan derivative

[0044] The raw material of chitosan (Cs) was purchased from Shanghai Boao Biotechnology Co., Ltd., with a molecular weight of 60 kilodaltons and a deacetylation degree of 90%. The specific synthesis method is:

[0045] Step (a): Weigh 2.5 g of Cs and swell overnight in 250 ml of dimethylformamide (DMF). After filtration, the filter cake was redispersed in 100 ml of DMF, 7 g of phthalic anhydride was added, and reacted at 120° C. for 10 hours until the reaction liquid was completely clear. After the reaction was completed, the reaction solution was poured into ice water to separate out a light yellow precipitate, filtered, and the filter cake was rinsed with ether and dried to obtain the product ( 13 C NMR: δ 22.5, 57.8, 61.5, 71.3, 75.9, 83.0, 101.0, 131.4, 168.08 ppm.).

[0046] Step (b): Weigh 1 gram of the product obtained in step (a), dissolve it in 100 milliliters of N-methylpyrrolidone (NMP), add 6.5 gr...

Embodiment 2

[0057] The preparation of embodiment 2 chitosan derivatives / nucleic acid nanocomposite

[0058] The chitosan derivative (prepared by Example 1 (substitution degree 12.5%)) is dissolved with deionized water, and is made into a solution of 0.1~10 mg / ml; plasmid DNA (for the plasmid DNA containing the green fluorescent protein gene, The plasmid number is pEGFP-N1 vector, GenBank Accession #U55762) was dissolved in deionized water to obtain a DNA solution with a concentration of 0.2 mg / ml; Mix for 30 seconds and stand at room temperature for 0.5-1 hour. The chitosan derivative and plasmid DNA self-assemble into a nanocomposite through electrostatic self-assembly.

Embodiment 3

[0059] Embodiment 3 gel retardation experiment

[0060] According to the method of Example 2, a series of chitosan derivatives (substitution degree 12.5%) and plasmid DNA nanocomposites with different N / P ratios were prepared. Take 20 microliters of samples of each ratio respectively, use 10 microliters of pure plasmid as a reference, perform 1% agarose gel electrophoresis, ethidium bromide staining, and photograph through a gel imaging system (UVP Bioimaging Systems). The result is as figure 1 shown.

[0061] figure 1 Among them, the first electrophoresis lane is the pure plasmid control, and the second to seventh lanes are the N / P ratios of chitosan derivatives and plasmid DNA at 0.5, 0.8, 1.2, 5, 10, and 20, respectively. from figure 1 It can be seen that chitosan derivatives can compress DNA very well, and can completely block the migration of DNA when N / P is greater than 1.

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Abstract

The invention discloses a novel chitosan derivative used as a non-viral gene vector, and a preparation method and application thereof. The chitosan derivative is synthesized by introducing N,N,N-trimethyl-(1,2,3-triazol-4-)methyl ammonium bromide groups into the 6-site of chitosan, and the structural formula of the chitosan derivative is shown in the specification, wherein n is the number of the repeating units of the chitosan derivative. The chitosan derivative disclosed by the invention not only retains the efficacy of 2-amino in the gene delivery process and the characteristics of no toxicity and high biocompatibility of chitosan, but also greatly improves the water solubility and gene transfection efficiency of chitosan; and the non-viral gene vector prepared by using the chitosan derivative has the characteristics of high biocompatibility, low toxicity and high transfection efficiency and the like.

Description

technical field [0001] The present invention relates to a chitosan derivative and its preparation method and application, in particular to a novel chitosan derivative used as a non-viral gene carrier and its preparation method and application, and also relates to a chitosan derivative derived from the chitosan Nanocomplex composed of substance and nucleic acid and its preparation method and application. Background technique [0002] With the development of bioengineering technology, more and more biomacromolecular drugs (such as proteins, polypeptides and nucleic acids, etc.) have been used clinically in recent years. This type of drug has strong biological activity, small dose and high therapeutic index, and has become a very important class of drugs in clinical practice. In particular, nucleic acid drugs used in gene therapy not only have the characteristics of high efficiency, long-term treatment, and few side effects, but also enable the therapeutic gene to stably expre...

Claims

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Application Information

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IPC IPC(8): C08B37/08C08L5/08C12N15/63A61K48/00
Inventor 李亚平高瑜张志文顾王文
Owner SHANGHAI INST OF MATERIA MEDICA CHINESE ACAD OF SCI
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