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siRNA conveying carrier and application thereof

A carrier, superparamagnetic technology, applied in the field of biomedicine, which can solve the problems of not easy to scale up, poor repeatability, etc.

Inactive Publication Date: 2012-01-25
黄开红 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the carrier systems currently used are cationic liposomes or water-soluble cationic polymers. These carrier molecules are mixed with siRNA solutions to obtain a complex of the two, so as to achieve the purpose of delivering siRNA. The disadvantage of this method is that it does not Easy to scale up and less reproducible

Method used

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  • siRNA conveying carrier and application thereof
  • siRNA conveying carrier and application thereof
  • siRNA conveying carrier and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] The preparation of embodiment 1, PPS nanoparticles

[0054] 1. Synthesis of polyethylene glycol-grafted polyethyleneimine (mPEG-g-PEI).

[0055] Weigh 4.0g of monomethyl ether polyethylene glycol (mPEG-OH, 2kD) in a dry two-neck flask at 60°C for 8 hours in vacuum, after cooling, add 30mL of dried tetrahydrofuran (THF) under the protection of Ar to fully dissolve. Weigh 3.2 g of N, N'-carboxydiimidazole (CDI) and place it in another dry two-necked bottle, slowly add the THF solution dissolved in mPEG-OH into the two-necked bottle under the protection of Ar, and continue to stir and react at room temperature for 12 hours . 200 μL of water was added to inactivate excess CDI, and the reaction was carried out for 0.5 h, precipitated twice with a large amount of anhydrous ether, filtered and dried to obtain a white powdery solid.

[0056] Then weigh branched polyethyleneimine (hy-PEI, 25kD) 1.1g and above-mentioned white powder 0.8g and dissolve in 20mL CHCl 3 , Continue ...

Embodiment 2

[0060] Embodiment 2, the preparation of PPS / siRNA complex and the mensuration of its physicochemical property

[0061] 1. PPS is dissolved in sterilized deionized water. According to different N / P ratios, a certain amount of siRNA solution and PPS solution are gently mixed in deionized water or PBS, and incubated at room temperature for 10-15 minutes. That is, PPS / siRNA complexes with different N / P values ​​were obtained.

[0062] Among them, the calculation formula of PPS / siRNA N / P ratio

[0063]

[0064] 2. Determination of particle size and potential

[0065] According to the N / P ratio of 5, 10, 15, 20 to form PPS / siRNA complexes, the amount of siRNA contained in each complex was set to 10 μg, diluted to 500 μl with deionized water, and the Zeta-Plus particle size analyzer was used to measure the complex. particle size and potential. Each sample was measured 5 times, and the obtained values ​​were expressed as mean ± standard deviation.

[0066] The measurement resul...

Embodiment 3

[0073] Embodiment 3, cell culture and cytotoxicity test

[0074] 1. Cell culture

[0075] Human gastric adenocarcinoma cell line SGC-7901, human gastric normal epithelial cell line GES-1, and human malignant melanoma cell line A375 were placed in high-glucose DMEM medium containing 10% fetal bovine serum at a constant temperature of 37°C and 5% CO 2 cultured in an incubator.

[0076] 2. Cytotoxicity test

[0077] To evaluate the cytotoxicity of PPS / siRNA complexes to SGC-7901, MTT assay was performed. Three replicate wells were set up in each group, and the experiment was repeated three times independently, and the obtained values ​​were expressed as mean ± standard deviation.

[0078] (1) SGC-7901 cells were seeded in 96-well plates, 5×10 3 / well, adherent growth for 24 hours.

[0079] (2) The old medium was removed, and 200 μl of PPS / siRNA complexes with N / P ratios of 1.25, 5, 10, and 20 and fresh medium were added, and the amount of siRNA contained in each complex was ...

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Abstract

The invention discloses a siRNA conveying carrier. The active ingredient of the conveying carrier is nanaparticles formed by coupling superparamagnetic iron oxide nanoparticles and polyethylene glycol-polyethyleneimine. In addition, the invention also discloses a eukaryotic cell transfection liquid which comprises a composite made from siRNA and the siRNA conveying carrier. The siRNA conveying carrier provided by the invention has the physical and chemical properties of small particle size and suitable surface potential, has good siRNA compounding ability, low cell toxicity and high transfection rate, is a safe and efficient nano carrier with siRNA conveying function and has broad application prospect in siRNA conveying and disease treatment based on RNA interference.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to an siRNA delivery carrier and its application in the preparation of gene therapy drugs for cancer. Background technique [0002] RNA interference (RNA interference, RNAi) is an ancient biological phenomenon discovered in recent years and widely exists in the biological world, and it is the product of biological evolution. Specifically, it refers to the phenomenon of silencing specific target genes at the post-transcriptional level under the guidance of double-stranded RNA (double strand RNA, dsRNA), with the participation of specific enzymes, with the degradation target of exogenous or endogenous mRNA, that is, without Express. Current research shows that RNAi widely exists in most eukaryotes from fungi to higher plants, from invertebrates to mammals, and prokaryotes have not been found yet. RNAi not only participates in the regulation of normal cell growth, development, senescence a...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/113C12N15/79A61K48/00A61P35/00
Inventor 黄开红陈茵婷
Owner 黄开红
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