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Multi-capture-ligand-modified multi-layer nanoparticle flexible stent of target cell and application of multi-layer nanoparticle flexible stent

A technology of target cells and nanoparticles, which is applied in the field of multi-layered nanoparticle flexible scaffolds, can solve problems such as the inability to capture CTCs, and achieve the effects of high specific capture efficiency, high roughness, and low non-specific adsorption.

Active Publication Date: 2016-07-06
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Antibodies are mainly used for specific ligands. However, whether it is positive enrichment (such as anti-EpCAM antibody used in the most representative CellSearch method) or negative sorting (such as anti-CD45 antibody, or anti-CD61 antibody), both Unable to achieve specific CTC capture

Method used

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  • Multi-capture-ligand-modified multi-layer nanoparticle flexible stent of target cell and application of multi-layer nanoparticle flexible stent
  • Multi-capture-ligand-modified multi-layer nanoparticle flexible stent of target cell and application of multi-layer nanoparticle flexible stent
  • Multi-capture-ligand-modified multi-layer nanoparticle flexible stent of target cell and application of multi-layer nanoparticle flexible stent

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specific Embodiment 1

[0031] Specific embodiment 1: SiO 2 Preparation of nanoparticles and their silanization

[0032] Add 25 mL of absolute ethanol at constant temperature to 40°C, 1.5 mL of ammonia water, and 0.5 mL of water into the two-neck flask in turn, and quickly add 0.75 mLTEOS (tetraethyl orthosilicate) in a constant temperature water bath at 40°C under stirring at 500 rpm / min. 7h, then add 0.5mLTEOS, continue to react for 15h, stop the reaction. Centrifuge at high speed, discard the supernatant, wash the precipitate with absolute ethanol and three-distilled water in turn, and finally redissolve in 25mL three-distilled water for later use to obtain SiO 2 Nanoparticle solution (11 mg / mL).

[0033] A silanized hydrolyzate was prepared by stirring with 19 mL of absolute ethanol, 0.3 mL of APTES (3-aminopropyltriethoxysilane) and 0.3 mL of triple-distilled deionized water for 5 min. Take 11mg / mLSiO 2 2 mL of nanoparticle solution, centrifuged for 20 min, redissolved in 1 mL of absolute et...

specific Embodiment 2

[0034] Specific Example 2: Preparation of flexible nanoparticle scaffold and connection of its aptamer

[0035] Preparation of silanized carrier matrix: 30% hydrogen peroxide and 98% sulfuric acid were mixed at a volume ratio of 3:7 to prepare a lotion. Cut the slide into 25.4×19mm 2 sized rectangular glass slides as carrier substrates. Wash the carrier matrix with water and absolute ethanol respectively, blow dry, put into the lotion, heat at 80-100°C for 1 hour, cool naturally, wash the carrier matrix with triple-distilled deionized water, and blow dry with cold wind. The carrier matrix was reacted in 2% APTES absolute ethanol solution for 18 hours, rinsed with triple distilled deionized water, dried, and baked at 120°C for 6 hours to obtain a silanized carrier matrix with surface-coupled amino groups.

[0036] Preparation of activated PAA solution: Take 100 μL of polyacrylic acid (PAA, 25wt% aqueous solution, Mw=240000) in a 10mL single-necked bottle, add 5mL of three-dis...

specific Embodiment 3

[0039] Specific Example 3: Cell Culture and Cell Capture

[0040] Cell culture and staining: For Ramos and HL-60 cells, in the prepared culture solution (the culture solution was prepared with RPMI-1640 medium, in which FBS contained 15%, and philin-streptomycin was 100 U / mL), 37 5% CO at °C 2 Cultivate in saturated humidity. Ramos cells were labeled with orange-red DiI dye, while HL-60 cells were labeled with green DiO dye. Suspend the cells in serum-free medium, the corresponding cell density is 1.0×10 6 cells / mL, add 5 μL of cell labeling solution to every 1 mL of cell suspension, mix well, incubate at 37°C for 10-20 minutes, centrifuge at 1500 rpm for 5 minutes, and gently resuspend the cells with 37°C medium. Repeat 2 washes with 5 mL of Wash Buffer WB, and finally suspend it in 6 mL of BB to obtain 1.0×10 6 cells / mL of stained cell suspension. Wherein DiI and DiO solutions adopt DMSO as the solvent, respectively prepare solutions containing DiI and DiO dyes at a con...

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Abstract

The invention relates to a multi-capture-ligand-modified multi-layer nanoparticle flexible stent of a target cell and application of the multi-layer nanoparticle flexible stent.Active macromolecules serve as a cross-linking agent, and rough surfaces of multi-layer nanoparticles are established on a carrier matrix; active macromolecules are chemically connected to the rough surfaces, and a multi-layer nanoparticle flexible stent is obtained; then, multi-capture ligands are fixed to the multi-layer nanoparticle flexible stent, and the multi-layer nanoparticle flexible stent connected with the multi-capture ligands is obtained.The orderly multi-layer nanoparticle flexible stent is easily, quickly and controllably assembled based on the macromolecule cross-linking method, and the stent is provided with both the nanoparticle structure and the flexible structure, and shows the high roughness degree advantage; for the target cell, the stent shows extremely-low nonspecific adsorption, the multivalence-aptamer-modified stent has extremely-high specific capture efficiency and can be prepared into an in-vitro circulation capture system to be applied to the aspect of in-vivo target cell capture in an in-vitro circulation mode.

Description

technical field [0001] The present invention relates to a scaffold for sequentially capturing target cells in vivo, an in vitro flow-through capture system containing the scaffold and its application, in particular to a flexible multi-layered nanoparticle modified by multiple capture ligands for cyclically capturing target cells in vivo The scaffold and its application belong to single cell capture technology. Background technique [0002] The main cause of cancer death is that some tumor cells acquire the ability to invade and cause distant metastasis. The shedding, invasion and entry of tumor cells into the blood circulation is the initial stage of tumor metastasis and provides the possibility for the final formation of tumor metastases. In theory, circulating tumor cells (circulating tumor cells, CTCs) in peripheral blood are directly related to hematogenous metastasis of tumors, so CTCs in peripheral blood of cancer patients are regarded as ideal biomarkers for tumor di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078
CPCC12N5/0634C12N2537/10
Inventor 谢洪平刘玲蔡雪萍
Owner SUZHOU UNIV
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