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Functional red blood cell targeting circulating tumor cells (CTCs)

A technology for tumor cells and red blood cells, applied in the field of functional red blood cells, can solve the problems of low separation purity, difficulty in in-depth analysis, and difficulty in the purity of CTCs samples, and achieve the effect of avoiding interference and stable structure.

Inactive Publication Date: 2018-09-28
段莉
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the contact interface between this type of device and CTCs is a three-dimensional nanostructure, and there is non-specific physical adsorption for white blood cells, so it is still difficult to greatly improve the purity of CTCs samples.
[0005] To sum up, the current CTCs isolation technology is facing the technical bottleneck of low isolation purity caused by a large number of leukocytes mixed in the sample, which cannot meet the purity requirements of subsequent cell / molecular biology analysis for CTCs, which seriously restricts the extraction of CTCs. The in-depth study of the key role in the early detection of cancer and the mechanism of cancer metastasis has led to the current clinical application of CTCs limited to simple applications such as counting, and it is difficult to study the biological macromolecules (such as proteins, DNA, etc.) that regulate the metabolic activities of CTCs. etc.) level of analysis

Method used

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  • Functional red blood cell targeting circulating tumor cells (CTCs)
  • Functional red blood cell targeting circulating tumor cells (CTCs)
  • Functional red blood cell targeting circulating tumor cells (CTCs)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Erythrocyte modification of folic acid (FA) for specific recognition, capture and activity regulation of CTCs in vitro

[0037] (1) Erythrocyte modified folic acid (FA): first extract 100 μL erythrocytes from 1 mL of normal human blood, and then incubate with 50 μL (1 mg / mL) DSPE-PEG-FA (purchased from American Nanocs Company) (purchased from US nanocs company) for 30 minutes, and all samples were prepared at 1000 g After centrifugation for 10 minutes, they were washed three times with PBS before use to form FA-modified functional erythrocytes.

[0038] (2) In order to confirm that DSPE was effectively embedded in the red blood cell membrane, DSPE-PEG-Cy5 was used instead of DSPE-PEG-FA. First extract 100 μL red blood cells from 1 mL of normal human blood, then incubate with 50 μL (1 mg / mL) DSPE-PEG-Cy5 for 30 minutes, centrifuge all samples at 1000 g for 10 minutes, wash with PBS three times before use, and form Cy5-modified red blood cells . DSPE-PEG-Cy5 m...

Embodiment 2

[0044] Example 2 Red blood cell modified epithelial cell adhesion molecule antibody (RBC-antiEpCAM)

[0045] (1) Modification of biotin (Biotin) on the surface of red blood cells (RBC): 500 μL of red blood cells were suspended in 4 ml of PBS (pH7.4), and then 0.5 mg of DSPE-PEG-Biotin (purchased from American nanocs company) (3700D, 100 μg / mL) was added ), incubated at 37°C for 30 minutes with continuous stirring, DSPE was embedded in the phospholipid bilayer of RBC to obtain RBC-Biotin. The obtained RBC-Biotin was washed twice (PBS, pH 7.4, 400g, 5min),

[0046] (2) Modification of avidin (Avidin) on RBC-Biotin: under constant stirring, add 1 mg of Avidin solution (6.30×104 molecules / red blood cells) to the RBC-Biotin solution, and react at 4°C for 60 minutes . Formation of red cell RBC-Biotin-Avidin. The obtained RBC-Biotin-Avidin solution was washed twice (PBS, pH 7.4, 400 g, 5 minutes) to remove unbound avidin.

[0047] (3) Modification of epithelial cell adhesion mole...

Embodiment 3

[0053] Example 3 Red blood cell modified sialylated Lewis oligosaccharide-X antibody (RBC-anti-Sialyl Lewis X)

[0054] (1) Modification of biotin on the surface of red blood cells (RBC): 500 μL of red blood cells were suspended in 4 ml of PBS (pH 7.4), and then 0.5 mg of DSPE-PEG-Biotin (purchased from American nanocs company) (3700D, 100 μg / mL) was added ), incubated at 37°C for 30 minutes with continuous stirring, DSPE was embedded in the phospholipid bilayer of RBC to obtain RBC-Biotin. The obtained RBC-Biotin was washed twice (PBS, pH 7.4, 400g, 5min),

[0055] (2) Modification of avidin (Avidin) on RBC-Biotin: under constant stirring, add 1 mg of Avidin solution (6.30×104 molecules / red blood cells) to the RBC-Biotin solution, and react at 4°C for 60 minutes . Formation of red cell RBC-Biotin-Avidin. The obtained RBC-Biotin-Avidin solution was washed twice (PBS, pH 7.4, 400 g, 5 minutes) to remove unbound avidin.

[0056] (3) Modification of epithelial cell adhesion m...

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Abstract

The invention provides a functional red blood cell targeting circulating tumor cells (CTCs) and belongs to the technical field of red blood cell modifying and circulating tumor cell catching. A functional molecule is embedded in a cytomembrane of the functional red blood cell; one end of the functional molecule is used as an embedded end and used for being embedded in the red cell membrane; the other end of the functional molecule is used as a targeting end, the targeting end is used as a CTCs targeting antibody or folic acid and can specifically recognize and be combined with CTCs surface biomarkers; and the embedded end of the functional molecule is connected with the targeting end of the functional molecule through polyethylene glycol (PEG) to form the functional molecule with an "embedded end-PEG-targeting end" structure. The functional red blood cell can recognize, catch or inhibit the CTCs, is used for specific recognition and catching research for microscale CTCs of blood samples of clinic patients in vitro and adjusting and controlling activity of caught CTCs, and therefore the application prospect is developed on forecast and precaution of tumor metastasis.

Description

technical field [0001] The invention relates to a functional red blood cell capable of identifying, capturing, enriching or inhibiting circulating tumor cell CTCs. Background technique [0002] Circulating tumor cells are a key factor in the metastasis of malignant tumors, and high-purity samples have great research value. In my country, the incidence of malignant tumors (cancers) continues to increase, and the mortality rate ranks first among all death diseases all the year round, seriously threatening the lives and health of Chinese people. Cancer metastasis is the main cause of death, accounting for up to 90% of cancer deaths. The key carrier of cancer metastasis is the diseased cells called circulating tumor cells (Circulating Tumor Cells, CTCs). On the tissue, a new malignant tumor will eventually form. Therefore, the separation and analysis technology of peripheral blood CTCs is expected to become a highly feasible non-invasive means of cancer diagnosis, and can hel...

Claims

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Application Information

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IPC IPC(8): C12N5/078C12Q1/02G01N33/569
CPCC12N5/0641G01N33/5005G01N33/56966
Inventor 段莉
Owner 段莉
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