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Novel double-targeting gene conveying system based on magnetic nanoparticles and preparing method thereof

A magnetic nanoparticle and gene delivery technology, which can be used in powder delivery, medical preparations with non-active ingredients, medical preparations containing active ingredients, etc., can solve the problems of poor stability and targeting, easy degradation, and gene transfection efficiency Low-level problems, to achieve high targeting selectivity, good biocompatibility, and targeting-specific effects

Active Publication Date: 2016-05-11
HENAN UNIVERSITY OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to construct a nano-gene delivery system with specific dual-targeting functions of antibodies and cationic transfection agents for the urgent problems of low gene transfection efficiency, easy degradation, poor stability and targeting.

Method used

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  • Novel double-targeting gene conveying system based on magnetic nanoparticles and preparing method thereof
  • Novel double-targeting gene conveying system based on magnetic nanoparticles and preparing method thereof
  • Novel double-targeting gene conveying system based on magnetic nanoparticles and preparing method thereof

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Embodiment 1

[0042] Schematic diagram of the construction of a novel dual-targeted gene delivery system based on magnetic nanoparticles, see figure 1 , its specific preparation steps are as follows:

[0043] (1) Preparation of the magnetic core, that is, superparamagnetic iron oxide nanoparticles (SPIONPs): draw 10.33 mL of deionized water into a three-necked flask, add 3 mL of 2mol / L FeCl 3 The solution was fixed on a magnetic stand for stirring. Then add 2mL of 1mol / L Na at a constant speed 2 SO 3 The solution was added dropwise within one minute. When the color changed from reddish brown to yellow, slowly add 80mL of 0.85mol / L NH 3 ·H 2 O, and vigorously stirred, there will be a black precipitate, continue to stir for 40min. The precipitate was washed with anaerobic water to pH less than 7.5, and the precursor material was diluted to 3 mg / mL with anaerobic water. Then use 0.1mol / L HCl to adjust the pH to 3.0, and maintain this state for 5 minutes. Raise the temperature to 90°C wi...

Embodiment 2

[0058] Cell Culture Experiment

[0059] Glioma U251 cells in 5% CO 2RPMI-1640 cell culture medium containing 10% newborn bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin was used for routine culture at 37 °C. When the cells reached 90% confluence, routine subculture was carried out at a ratio of 1:3 to ensure that the cells were in the logarithmic growth phase.

[0060] The tumor stem cells in the glioblastoma multiforme U251 cell line were enriched and isolated by serum-free suspension culture combined with the cell cycle-specific drug vincristine (VCR). Before the cells were used, the cells were digested with 0.25% trypsin, centrifuged at 1000rpm for 5min to collect the cells and reintroduced with VCR (8ng / mL) or VCR-free serum-free DMEM / F12 medium supplemented with growth factors (rhEGF, bFGF, LIF, B27). Suspended, counted, ready to use. Cells were seeded to a suitable density in a 24-well plate, 2x10 4 Cells / well, after culturing for 24 hours, discard one-t...

Embodiment 3

[0062] Cell uptake experiment of dual-targeted nanocarriers: In order to verify whether the prepared dual-targeted nanocarriers could successfully enter cells, we labeled the prepared carrier material with fluorescent dye RBITC and conducted cell uptake experiments. The results showed that CD133-PEI-CTS-TPPSPIONPs or PEI-CTS-TPPSPIONPs could successfully enter the cells, and CD133-PEI-CTS-TPPSPIONPs could enter the cells more easily because of the active targeting of CD133 , showing a more intense red fluorescence ( Figure 9 ).

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Abstract

The invention discloses a novel double-targeting gene conveying system based on magnetic nanoparticles and a preparing method thereof. The superparamagnetic nanoparticles and carboxymethyl chitosan serve as a magnetic nucleus and a coating material of the gene conveying system respectively, and the surface of carboxymethyl chitosan is modified with a targeted molecule CD133 antibody and a cationic transfection agent polyethyleneimine. The double-targeting gene conveying system has the advantages of being high in biocompatibility, stability and targeting specificity, so that the system has high targeting selectivity to tumor cells, can directionally release and convey the target genes to a tumor position to conduct targeted inhibition and kill tumor cells, and provides a new strategy for targeted tumor treatment. The preparing method for a nano carrier and the gene conveying system is easy to operate, reaction conditions are mild, a prepared product can serve as a wide-range carrier for genes, and a new strategy is provided for research on efficient targeted gene conveying carriers.

Description

technical field [0001] The invention relates to the fields of biological nanometer drug-carrying materials and targeted gene delivery, in particular to a novel dual-targeted gene delivery system based on magnetic nanoparticles and a preparation method thereof. Background technique [0002] Malignant tumors (cancers) have the characteristics of high morbidity, high mortality, difficult treatment and easy recurrence. According to the latest "World Cancer Report" by the World Health Organization, there were 14 million new cancer cases and 8.2 million cancer deaths worldwide in 2012. There are 3.07 million newly diagnosed cancer cases in my country, accounting for 21.8% of the global total, and about 2.2 million cancer deaths, accounting for 26.9% of the global cancer deaths. And according to the current trend of cancer incidence, the incidence of cancer worldwide will increase by 50% by 2020. Conventional tumor treatment methods are mainly surgical resection, radiotherapy and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K9/14A61K47/42A61K47/36A61K47/34A61K47/02A61K31/7105A61P35/00A61K47/58A61K47/68
CPCA61K9/143A61K9/146A61K31/7105
Inventor 王雪琴伊艳杰李瑞芳张慧茹景红娟李翠香
Owner HENAN UNIVERSITY OF TECHNOLOGY
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