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Anti-tumor engineering exosome, preparation method and application

An exosome, anti-tumor technology, used in biochemical equipment and methods, anti-tumor drugs, genetic engineering, etc., can solve the problems of limiting the application and transformation of siRNA, and low gene silencing efficiency.

Active Publication Date: 2021-09-07
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, both liposomes and polymers have the problem of low gene silencing efficiency
This is because, after the carrier carries siRNA into the cytoplasm, it enters the endosome / lysosome, and less than 1% of the siRNA can escape from the endosome / lysosome and enter the cytoplasm to perform biological functions. This greatly limits the application and transformation of siRNA

Method used

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  • Anti-tumor engineering exosome, preparation method and application
  • Anti-tumor engineering exosome, preparation method and application
  • Anti-tumor engineering exosome, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The extraction and identification of embodiment 1 Nex

[0034] Culture NK92MI cells (purchased from the Cell Bank of the Chinese Academy of Sciences) with exosome-free medium or serum-free medium. When the cells are in the logarithmic growth phase, centrifuge at 300g for 10 minutes to collect the supernatant (the supernatant can be stored at -80°C for one week) ).

[0035]The above supernatant (thawed in cold water if taken out from -80°C) was performed as follows: centrifuge at 2000g for 15 minutes to remove dead cells, collect the supernatant and discard the precipitate; centrifuge at 10000g for 30 minutes to remove cell debris, collect the supernatant and discard Precipitation; filter the supernatant with a 0.22 μm filter membrane to remove microvesicles with larger particle sizes. The obtained filtrate was transferred to a 100kD ultrafiltration tube, centrifuged at 3000g for 20 minutes, and the lower layer filtrate was discarded to obtain a culture supernatant conc...

Embodiment 2

[0037] Example 2 Anti-tumor activity of Nex at the cellular level

[0038] HepG2-luc and CT26 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were trypsinized and resuspended in DMEM medium and RPMI-1640 medium (Gibco) respectively, so that the cell density was about 1×10 5 individual / mL. Add 100 μL of cell suspension to each well of the 96-well plate, and continue to store at 37°C, 5% CO 2 cultured in a cell culture incubator for 24 hours. Add different doses of Nex to the cells, and after 48 hours of treatment, add an equal amount of MTT solution (Sigma, also known as tetrazolium salt) to the 96-well plate, incubate at 37°C for 4 hours, remove the medium containing MTT, and wash with PBS 3 times. Add 100 μL DMSO solution (dimethyl sulfoxide solution) to each well and let stand at room temperature for 5-10 minutes. The absorbance values ​​at 540nm and 650nm were recorded respectively with a microplate detector, and the cell survival rate was calcul...

Embodiment 3

[0044] Example 3 Preparation of Nex@siRNA / Ce6 and analysis of cell entry effect

[0045] The specific steps are:

[0046] (1) Take 100 μL of the purified Nex suspension (1 mg / mL) obtained in Example 1 into a 1.5 mL EP tube and place it on ice.

[0047] (2) PLK1 (Polo-like Kinase 1, Polo-like Kinase 1) siRNA dry powder (siPLK1, Suzhou Ruibo Biotechnology Co., Ltd.) was dissolved in DEPC (diethylpyrocarbonate) water to make the concentration 1 mg / mL.

[0048] (3) Take 100 μL of siPLK1 solution and add it to Nex, pipette gently to evenly transfer 200 μL of the mixture to a pre-cooled 0.4 cm electroporation cup.

[0049] (4) Add 200 μL of PBS to the electroporation cup, and gently pipette evenly to make the total volume of the system 400 μL.

[0050] (5) Use the Bio-RAD electroporator for electroporation, and set the electroporation parameters as: 200V, 125μF, 200Ω.

[0051] (6) Aspirate the mixed solution from the electroporation cup and transfer it to a new EP tube, and incub...

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Abstract

The invention relates to an anti-tumor engineering exosome, a preparation method and application. The anti-tumor engineering exosome comprises an exosome body, wherein an anti-tumor nucleic acid medicine and a photosensitizer are coated in the exosome body. Compared with a natural exosome Nex, the engineering exosome Nex @ siRNA / Ce6 has the following characteristics that 1, tumor cells are killed in a targeted way, and no killing effect is achieved on normal cells; 2, the tumor cell apoptosis is promoted through silencing PLK1 genes; the mouse immune system is activated through inhibiting the PD-L1 expression; and 3, the photosensitizer Ce6 in the engineering exosome generates active oxygen under the illumination; the targeted killing of the tumor cells is promoted through the photodynamic effect generated under the illumination; the active oxygen has the effect of damaging the endosome membrane; the siRNA is promoted to escape into the cytoplasm from the endosome; the gene inhibition effect of the cancer cells is improved; the active oxygen can also promote the M2 type macrophage to turn to the M1 type macrophage; and the anti-tumor effect is synergistically enhanced.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an anti-tumor engineered exosome, a preparation method and an application. Background technique [0002] Exosomes are small vesicles with a diameter of about 30-150 nm secreted by living cells, which have a typical lipid bilayer structure. Exosomes exist in cell culture supernatant, serum, plasma, saliva, urine, amniotic fluid, and other biological fluids, and carry important information such as a variety of proteins, lipids, and RNA. In recent years, with the deepening of exosome research, its application has involved in the fields of tumor treatment, medical foundation and immunotherapy. NK cell-derived exosomes retain the immune function of mother cells, and can play an immune role through the Fas / FasL pathway to kill tumor cells. It shows good anti-tumor properties in vivo and in vitro, but due to the cumbersome extraction process and extremely low yield of exosomes...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/113A61K35/17A61K31/7088A61K41/00A61K47/46A61P35/00
CPCC12N5/0646C12N5/0636C12N5/0635C12N5/0639C12N15/113C12N15/1138A61K35/17A61K31/7088A61K41/0071A61K47/46A61P35/00C12N2510/00C12N2310/14C12N2310/141A61K2300/00
Inventor 黄渊余张萌洁
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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