modified HEK293T cell, preparation method thereof, drug-loaded exosome, and preparation of drug-loaded exosome

A technology of exosomes and cells, which is applied in the direction of genetically modified cells, biochemical equipment and methods, cells modified by introducing foreign genetic materials, etc. Problems such as small amount of exosome collection, low cost, realization of therapeutic potential, and easy implementation

Active Publication Date: 2020-07-17
承启医学(深圳)科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, exosomes derived from immature DC cells have weak ability to cause the body's immune response and low toxicity, but the amount of exosomes collected is relatively small, and it is difficult to prepare a large amount of exosomes to meet the needs of clinical trials.

Method used

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  • modified HEK293T cell, preparation method thereof, drug-loaded exosome, and preparation of drug-loaded exosome
  • modified HEK293T cell, preparation method thereof, drug-loaded exosome, and preparation of drug-loaded exosome
  • modified HEK293T cell, preparation method thereof, drug-loaded exosome, and preparation of drug-loaded exosome

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Experimental program
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preparation example Construction

[0034] After this modification, HEK293T cells can greatly increase the secretion of exosomes, thereby loading more drugs, including but not limited to small molecule chemical drugs, proteins and peptides, and nucleic acid drugs. In one embodiment, the modified HEK293T cells are used to load anticancer drugs, and the preparation method thereof comprises the following steps:

[0035] S3: Plasmid construction: using the peptide iRGD (CRGDK\GPDC) that can specifically bind to the tumor surface αv integrin to be fused and expressed on the exosome membrane protein (lamp2b) as a targeted genetically engineered exosome that can target αv integration Protein-positive cancer cells; wherein the amino acid sequence of iRGD is: CRGDKGPDC;

[0036] S4: express plasmid iRGD-lamp2b in HEK293T cells obtained in S2, and screen stable cell lines;

[0037] S5: The HEK293T cells obtained in S4 were cultured in an exosome-free medium, and the cell supernatant was collected and subjected to ultrace...

Embodiment 1

[0040] Embodiment 1 Transformation of HEK293T cells

[0041] plasmid backbone Plasmid containing gene pcDNA3.1 STEAP3 Species: Homo sapiens, Gene ID: 55240 pcDNA3.1 SDC4 Species: Homo sapiens, Gene ID: 6385 pcDNA3.1 NadBfragment Species: Homo sapiens, Gene ID: 8027

[0042] The above three plasmids were transfected into HEK293T cells at a ratio of 1:1:1, and stable cell lines were screened to obtain the transformed HEK293T cell lines;

Embodiment 2

[0043] Example 2 Preparation of targeted exosomes

[0044] Plasmid construction: using the peptide iRGD (CRGDK\GPDC) that can specifically bind to the tumor surface αv integrin to be fused and expressed on the exosome membrane protein (lamp2b) as a targeted genetically engineered exosome that can target αv integrin positive Cancer cells; express the plasmid iRGD-lamp2b in the HEK293T cells obtained in the first step, and select stable cell lines;

[0045] The modified HEK293T cells carrying the iRGD-lamp2b expression plasmid were cultured in an exosome-free medium, the cell supernatant was collected, and the HEK293T cell exosomes were separated; the extraction steps were as follows:

[0046] (1) Collect about 150mL of samples and store them at 4 degrees (not more than one week);

[0047] (2) Centrifuge at 300×g for 10 minutes at 4 degrees, and take the supernatant;

[0048] (3) Centrifuge at 16500×g for 20 minutes at 4 degrees, and take the supernatant;

[0049] (4) Use an ul...

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Abstract

The invention relates to a method for preparing and modifying an HEK293T cell, a modified HEK293T cell, a method for preparing drug-loaded exosome and the drug-loaded exosome. The method for preparingand modifying the HEK293T cell comprises the following steps: S1, constructing plasmids: separately constructing three plasmids, namely pcDNA3.1-STEAP3, pcDNA3.1-SDC4 and pcDNA3.1-NadB fragments by using a pcDNA3.1 expression vector as a plasmid skeleton; and S2, transfecting the three plasmids in S1 into HEK293T cells according to ratios of (1-3):(1-3):(1-3), and screening a stable cell strain to obtain a modified HEK293T cell strain. By adopting the technical scheme, due to the synergistic effect of the three plasmids, the HEK293T cells with larger exocrine secretion can be reformed, so that the exocrine secretion is further improved, and more choices are provided for clinical tests.

Description

technical field [0001] The invention relates to the field of drug carrier preparation, in particular to transformed HEK293T cells and a preparation method, drug-loaded exosomes and a preparation method. Background technique [0002] Targeted delivery of drug molecules to specific types of cells is a major challenge in precision medicine. Natural exosomes have biofunctionalized nanovesicles that can display a variety of targeting proteins through genetic engineering. Or the ligand of the polypeptide and directly package the drug to exercise the ability of drug targeted delivery. [0003] Exosomes are a kind of membranous vesicles actively secreted by living cells under physiological or pathological conditions, which carry proteins and transport RNA, and their contents include proteins, short chain peptides, DNA fragments, lncRNA, phospholipids and miRNA . Studies have found that mast cells, dendritic cells, lymphocytes, fibroblasts, mesenchymal stem cells, and tumor cells c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10A61K47/46A61K45/00A61P35/00
CPCA61K45/00A61K47/46A61P35/00C12N5/0636C12N15/85C12N2510/00
Inventor 王程董鸣何慧琼
Owner 承启医学(深圳)科技有限公司
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