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Cationic polymer nanomaterial gene carrier and its preparation method and application

A technology of copolymers and complexes, applied in the field of gene therapy, can solve the problem of inability to effectively deliver pDNA and siRNA at the same time

Active Publication Date: 2019-09-20
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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  • Cationic polymer nanomaterial gene carrier and its preparation method and application
  • Cationic polymer nanomaterial gene carrier and its preparation method and application
  • Cationic polymer nanomaterial gene carrier and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] mPEG-SS-Lys n -r-His m Construction and Characterization of

[0122] The preparation process is illustrated as follows:

[0123]

[0124] At room temperature, 9 g of cystamine dihydrochloride (40 mmol) was weighed and dissolved in 80 ml of double-distilled water, 80 μl of sodium hydroxide (1 M) was added dropwise, and the product was subjected to rotary evaporation at 60 °C after magnetic stirring for 1 h to remove all water. 60 ml of anhydrous dichloromethane was added dropwise to the spin-dried product, stirred magnetically for 30 min, then filtered with filter paper to remove salt, and the filtrate was spin-dried at 30° C. to remove dichloromethane. Finally, the product was placed in a vacuum oven to dry overnight.

[0125] Under nitrogen protection, mPEG-COOH (methoxy-polyethylene glycol-carboxyl group, MW2000g / mol, Shanghai Yanyi Biotechnology Co., Ltd.) was activated, and mPEG-COOH (2g, 1mmol), N-hydroxysuccinimide Amine (0.14 g, 1.2 mmol) and N,N'-dicycloh...

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 compounding ability for 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 siRN...

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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 cancer treatment. Gene therapy generally includes three parts: a functional gene (therapeutic gene), a vector carrying the therapeutic gene (gene delivery system), and the site of action of the therapeutic gene (target cell). [0003] At present, gene delivery systems are mainly divided into two categories: viral vectors and non-viral vectors. Common viral vectors are: lentivirus, retrovirus, herpes simplex virus, adenovirus and adeno-associated virus. Non-viral gene carriers mainly include three categories: liposomes, polymers and liposome-polymers. Compared with viral gene vectors, non-viral vect...

Claims

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Application Information

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Patent Type & Authority Patents(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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