Application of vorinostat in oxidation responded cationic polymer gene vector mediated gene therapy

A cationic polymer, gene carrier technology, applied in the field of biomedicine, to enhance the effect of gene transfection, improve the therapeutic effect, and promote the effect of charge reversal

Inactive Publication Date: 2018-11-27
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Application of vorinostat in oxidation responded cationic polymer gene vector mediated gene therapy
  • Application of vorinostat in oxidation responded cationic polymer gene vector mediated gene therapy
  • Application of vorinostat in oxidation responded cationic polymer gene vector mediated gene therapy

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Embodiment 1

[0044] The cultivation of embodiment 1 cell and the preparation of nanocomposite

[0045] A549 cells and HeLa cells were cultured in RPMI-1640 medium containing 10% FBS in a constant temperature incubator, and the temperature of the incubator was set at 37°C, CO 2 The concentration is 5%.

[0046]B-PDEAEA / DNA complex configuration: Dilute DNA to 40 μg / mL with pH 7.4 HEPES buffer, weigh an appropriate amount of B-PDEAEA and dissolve it with HEPES to dilute to 507 μg / mL, mix the two in equal volume and vortex for 10 seconds , standing for 30min, that is to form nanoparticles (polyplex, B-PDEAEA / DNA complex).

Embodiment 2

[0047] Example 2 Intracellular reactive oxygen species (ROS) detection

[0048] By using 2,7-dichlorofluorescein diacetate to detect the level of ROS in the cell, 2,7-dichlorofluorescein diacetate can be converted into a fluorescent substance dichlorofluorescence under the action of ROS after entering the cell yellow.

[0049] HeLa and A549 cells were plated in 6-well plates, and the number of cells was 200,000 / well; after the cells adhered to the wall, different concentrations of vorinostat were added to the experimental group (the concentration of SAHA was 2 μM or 4 μM), and no vorinostat was added to the control group; Cultivate for 24 hours, discard the medium, add 1ml of 2,7-dichlorofluorescein diacetate at a concentration of 10μM and incubate at 37°C for 30 minutes, collect the cells and detect the fluorescence intensity of dichlorofluorescein by flow cytometry; excitation wavelength 488nm, emission wavelength 520nm.

[0050] The results showed that after A549 cells we...

Embodiment 3

[0051] Example 3 Cytotoxicity Detection

[0052] 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (MTT) method was used to detect the effect of transfection of TRAIL plasmid on the growth of PSCs.

[0053] Detection principle: Mitochondria of living cells can produce succinate dehydrogenase, under the action of this enzyme, MTT will be reduced to water-insoluble blue-purple crystalline formazan (Formazan) to form deposits, dead cells will not cause this reaction . Dimethyl sulfoxide (DMSO) can dissolve the generated formazan crystals, measure its maximum light absorption value at a wavelength of 562nm with a microplate reader, and subtract the background absorption value at a wavelength of 620nm, thereby indirectly reflecting the cell growth in the experiment.

[0054] 200 μL containing 4000 A549 or HeLa cell suspensions were added to each well of a 96-well plate, and then the cells were placed in a 37° C. constant temperature incubator (5% CO2 concentration, 95% hu...

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Abstract

The invention relates to a novel application of vorinostat in an oxidation responded cationic polymer gene vector mediated gene therapy. The treatment effect on tumor cells is improved obviously by being combined with a TRAIL gene. Experiments find that vorinostat can lead to up-regulation of ROS in cells at a concentration without generating cytotoxicity and can promote charge reversal of an ROSresponded cationic polymer so as to further promote release of DNA, so that the final gene transfection effect is enhanced.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the application of vorinostat in gene therapy mediated by an oxidation-responsive cationic polymer gene carrier. Background technique [0002] Gene delivery (gene therapy) is considered a promising modality for disease treatment, especially for cancer. Successful gene delivery largely depends on the research and development of safe and efficient delivery vehicles. Viral vectors have received extensive attention and research because of their extremely high transfection efficiency, but their possible safety issues, including immunogenicity and biocompatibility, limit the further development of viral vectors. This also makes gene delivery based on non-viral vectors an option for gene therapy. However, the key problem of non-viral vector gene delivery is the low transfection efficiency, which cannot meet the needs of clinical application. The main reason is that the ability of the non-v...

Claims

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Application Information

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IPC IPC(8): A61K31/16A61K48/00A61P35/00
CPCA61K31/16A61K48/0041A61K48/005A61P35/00
Inventor 刘祥瑞周雪飞周珠贤唐建斌申有青
Owner ZHEJIANG UNIV
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