Glutathione-modified chitosan copolymer serving as non-viral gene carrier material and preparation and application thereof

A technology of chitosan copolymer and gene carrier, which is applied in the field of preparation of non-viral gene carrier, can solve the problems of low transfection rate of active gene, unfavorable biological distribution, inefficient intracellular operation, etc., and achieves decreased cytotoxicity, good Protective effect, low immune rejection effect

Active Publication Date: 2013-07-10
NANKAI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Gene vectors are usually divided into viral vectors and non-viral vectors. In most cases, viral vectors have high transfection efficiency, but there are problems such as toxicity, immunogenicity and gene transfection targeting, which limit their application in clinical treatment. Application: Although non-viral vectors have no toxicity problem, their unfavorable biodistribution and inefficient intracellular operation lead to low transfection rate of active genes. Therefore, improving the transfection rate of non-viral gene vectors has become a hot spot of current research

Method used

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  • Glutathione-modified chitosan copolymer serving as non-viral gene carrier material and preparation and application thereof
  • Glutathione-modified chitosan copolymer serving as non-viral gene carrier material and preparation and application thereof
  • Glutathione-modified chitosan copolymer serving as non-viral gene carrier material and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] 1) Weigh 10.0g chitosan and dissolve it in deionized water, then add appropriate amount of sodium hydroxide and sodium borohydride, dissolve evenly, then add 10ml allyl bromide at 40°C, heat up to 60°C and stir for 3 After that, it was neutralized with acetic acid, and the product was repeatedly precipitated and extracted in ethanol, then vacuum-dried to constant weight to obtain allyl chitosan.

[0041] 2) Weigh 2.104g of polyethylene glycol methacrylate macromonomer (MPEG), dissolve 5.6mg of ACVA and 27.9mg of CPADB in methanol, and then, after three times of liquid nitrogen freezing-evacuation-thawing cycle degassing, at 60°C Under reaction for 24 hours. Then, the reaction was terminated with ice water, and viscous active polymer PMPEG was obtained through repeated precipitation in cold ether and vacuum drying.

[0042] 3) Weigh 0.2g of allyl chitosan obtained in step 1), and 0.25g of PMPEG and initiator ACVA obtained in step 2) are dissolved in dimethylformamide (D...

Embodiment 2

[0045] The cell transfection steps of copolymer (CS-PMPEG-GSH) are:

[0046] 1) Preparation of CS-PMPEG-GSH / pDNA complex:

[0047] Dissolve the polymer in sodium acetate solution at pH 5.5 to prepare a CS-PMPEG-GSH solution, then add the prepared plasmid DNA solution labeled with green fluorescent protein at a concentration of 0.5 μg / μL to the polymer in equal volume Add 2.5 μg pDNA to each complex, mix, let stand at room temperature for 30 minutes, and then stand still to obtain CS-PMPEG-GSH / pDNA complex solution.

[0048] 2) In vitro cell transfection experiments:

[0049] NIH3T3 cells at 1×10 per well 5 Several kinds of cells were put into 24-well culture plate and cultivated for 24 hours, then replaced with new medium containing glutathione-grafted chitosan copolymer CS-PMPEG-GSH / pDNA complex, continued to cultivate for 48 hours, and flow cytometry The transfection efficiency was quantified by cytometer. The result is as figure 2 shown.

[0050] figure 2It was sho...

Embodiment 3

[0051] Embodiment 3: gel retardation experiment:

[0052] First, prepare electrophoresis gel after dissolving agarose with a concentration of 1%, then CS-PMPEG-GSH / pDNA complex solutions with different N / P ratios, in which each sample contains 0.066 μg of pDNA, and finally electrophoresis at a constant voltage of 100V After electrophoresis, place the gel plate in EB solution for staining, soak for 30 minutes, and observe the pDNA band under ultraviolet light. The result is as image 3 shown.

[0053] image 3 Agar gel electrophoresis images of different N / P ratios, (a) chitosan / pDNA; (b) copolymer CS-PMPEG / pDNA and (c) graft copolymer CS-PMPEG-GSH / pDNA. It can be seen that the glutathione-grafted chitosan copolymer can wrap pDNA well when the N / P ratio is above 4.

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Abstract

The invention discloses a glutathione-modified chitosan copolymer serving as a non-viral gene carrier material and preparation and application thereof, which belong to the fields of gene therapy and novel materials. A preparation method of the copolymer comprises the following steps of: (1) synthesizing an allyl-modified chitosan derivative; (2) synthesizing brush-like PEG (Polyethylene Glycol) polymer chains with different molecular weights through RAFT (Reversible Addition-Fragmentation Chain Transfer); (3) grafting the brush-like PEG onto a chitosan framework by adopting a free radical coupling method; and (4) linking glutathione to the chain end of the brush-like PEG by adopting an EDC (1-Ethyl-3-(3-Dimethyllaminopropyl) Carbodiimide hydrochloride) / NHS (N-hydroxysuccinimide) activation method to obtain a glutathione-modified chitosan copolymer carrier material. The glutathione-modified chitosan copolymer obtained by adopting the technology serves as the non-viral gene carrier material. By adopting the copolymer, the endocytosis function of a composite nanoparticle formed from the copolymer and DNA (Deoxyribonucleic Acid) can be remarkably enhanced, the releasing mechanism of DNA from a composite particle after cell entrance is improved, and the non-viral gene carrier material with a high transfection efficiency is further obtained.

Description

technical field [0001] The invention belongs to the field of gene therapy and new materials, in particular to a preparation method of a non-viral gene carrier based on glutathione modified chitosan. Background technique [0002] Gene therapy has attracted increasing attention in the treatment of various human diseases. Gene therapy is a technology that uses molecular biology methods to introduce a target gene into a patient to express the target gene product, so that the disease can be treated. The carrier problem has always been one of the core technologies in the field of gene therapy research, and the key technology of gene therapy is the selection of gene delivery carrier. Gene vectors are usually divided into viral vectors and non-viral vectors. In most cases, viral vectors have high transfection efficiency, but there are problems such as toxicity, immunogenicity and gene transfection targeting, which limit their application in clinical treatment. Application: Althoug...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08G81/02C08F120/28C08B37/08C12N15/63
Inventor 郭天瑛李丛欣周德重胡玉玲
Owner NANKAI UNIV
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