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Novel cationic-polymer nanometer-material genetic carrier, preparing method and application

A technology of copolymers and complexes, applied in the field of gene therapy, can solve the problem of not being able to effectively deliver pDNA and siRNA at the same time

Active Publication Date: 2016-03-02
上海市口腔病防治院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the constructed cationic polymer gene carriers cannot effectively deliver pDNA and siRNA at the same time.

Method used

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  • Novel cationic-polymer nanometer-material genetic carrier, preparing method and application
  • Novel cationic-polymer nanometer-material genetic carrier, preparing method and application
  • Novel cationic-polymer nanometer-material genetic carrier, preparing method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] mPEG-SS-Lys n -r-His m construction and characterization

[0122] The preparation process is shown as follows:

[0123]

[0124] At room temperature, weigh 9 g of cystamine dihydrochloride (40 mmol) and dissolve it in 80 ml of double-distilled water, add dropwise 80 μl of sodium hydroxide (1 M), and react with magnetic stirring for 1 hour. After reacting for 1 h, the product is rotary-evaporated at 60° C. to remove all water. Add 60 ml of anhydrous dichloromethane dropwise to the spin-dried product, stir it magnetically for 30 min, then filter it through filter paper to remove salt, and spin the filtrate to dry at 30°C to remove dichloromethane. Finally, the product was dried overnight in a vacuum oven.

[0125] Activate mPEG-COOH (methoxy-polyethylene glycol-carboxyl, MW2000g / mol, Shanghai Yanyi Biotechnology Co., Ltd.) under the protection of nitrogen, mix mPEG-COOH (2g, 1mmol), N-hydroxysuccinyl Amine (0.14g, 1.2mmol) and N,N'-dicyclohexylcarbodiimide (0.25g, ...

Embodiment 2

[0136] mPEG-SS-Lys n -r-His m Preparation and characterization of / pDNA complexes

[0137] mPEG-SS-Lys n -r-His 20 Dissolved in DMF and dialyzed to freeze-dry, take its fresh freeze-dried sample to prepare the complex. Take a certain amount of mPEG-SS-Lys n -r-His m Or PEI was dissolved in 150mM NaCl solution (220nm membrane filter), sterilized by 220nm filter membrane to prepare a 1mg / ml solution, and according to different mass ratios (0, 0.1, 0.25, 0.5, 0.75, 1 , 1.5 and 2), mixed with pDNA, vortexed slightly for 10s, and placed in a 37°C incubator for 30min to form a stable copolymer / gene complex.

[0138] The ability of the gene delivery system to compound genes is a prerequisite for its function as a gene carrier. In this experiment, the mPEG-SS-Lys was observed by gel electrophoresis n -r-His m Complexity to pDNA. Samples of the complex were taken, wherein the pDNA was 0.5 μg / sample. After 30 minutes, add loading buffer solution to each sample, then add the c...

Embodiment 3

[0143] mPEG-SS-Lys n -r-His m Preparation and characterization of / siRNA complex

[0144] mPEG-SS-Lys n -r-His 20 Dissolved in DMF and dialyzed to freeze-dry, take its fresh freeze-dried sample to prepare the complex. Take a certain amount of mPEG-SS-Lys n -r-His m Or PEI was dissolved in 150mM NaCl solution (220nm membrane filter), sterilized by 220nm filter membrane to prepare a 1mg / ml solution, and according to different mass ratios (0, 0.1, 0.25, 0.5, 0.75, 1 , 1.5 and 2), mixed with siRNA, vortexed slightly for 10s, and placed in a 37°C incubator for 30min to form a stable copolymer / gene complex.

[0145] Adopt the similar method of embodiment 2 to detect the complex ability of compound, the result is as follows Figure 12 shown, indicating that mPEG-SS-Lys n -r-His 20 It has better compound ability to siRNA, such as mPEG-SS-Lys when the mass ratio is 0.75:1 and 0.5:1, respectively 55 -r-His 20 and mPEG-SS-Lys 95 -r-His 20 Can effectively complex with siRNA. ...

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Abstract

The invention discloses a novel cationic-polymer nanometer-material genetic carrier, a preparing method and an application. The carrier is a copolymer and comprises a polyethylene glycol core, a polylysine block (a), a polylysine block (b) and a poly-histidine block, or a random or alternating polymer zone (c), wherein the polylysine block (a), the polylysine block (b) and the poly-histidine block are covalently connected with the polyethylene glycol core, and the random or alternating polymer zone (c) is formed by a lysine monomer, a histidine monomer and an optional amino acid monomer in a polymerization mode. The invention further discloses a compound formed by the copolymer and nucleic acid and is used for preparing medicine for treating a tumor. By means of the gene transmitting carrier, DNA can be effectively transmitted, and RNA can also be effectively transmitted.

Description

technical field [0001] The invention relates to the field of gene therapy, in particular to a novel cationic polymer nanomaterial gene carrier capable of efficiently transfecting stem cells and its construction and application. Background technique [0002] With the rapid development of modern medicine and molecular biology, gene therapy has gradually matured as a new method of treating cancer. Gene therapy generally includes three parts: a functional gene (therapeutic gene), a carrier carrying the therapeutic gene (gene delivery system), and the site of action of the therapeutic gene (target cells). [0003] At present, gene delivery systems are mainly divided into two categories: viral vectors and non-viral vectors. Common viral vectors mainly include: lentivirus, retrovirus, herpes simplex virus, adenovirus and adeno-associated virus. Non-viral gene vectors mainly include three categories: liposomes, polymers and liposome-polymers. Compared with viral gene vectors, non...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08G65/48C12N15/87A61K47/34A61K48/00A61P35/00
Inventor 刘上峰李永勇
Owner 上海市口腔病防治院
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