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Magnetic nanocomposite for specifically capturing and effectively releasing circulating tumor cells and preparation method thereof

A tumor cell and magnetic nanotechnology, applied in cell dissociation methods, tumor/cancer cells, biochemical equipment and methods, etc., can solve problems such as invasiveness, interference with cell microenvironment, damage to cell integrity structure, etc., to achieve convenient The effect of detection analysis, fast capture, and narrow particle size distribution

Pending Publication Date: 2020-05-05
HUBEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, most of these release methods are invasive and may damage the integrity of the cell structure and disturb the cellular microenvironment

Method used

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  • Magnetic nanocomposite for specifically capturing and effectively releasing circulating tumor cells and preparation method thereof
  • Magnetic nanocomposite for specifically capturing and effectively releasing circulating tumor cells and preparation method thereof
  • Magnetic nanocomposite for specifically capturing and effectively releasing circulating tumor cells and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 2

[0043] Embodiment 1 disulfide dipyridyl triiron tetroxide (Fe 3 o 4 @SiO 2 -SSPy) preparation

[0044] Take 2ml Fe 3 o 4 Nanoparticle (30mg / ml) solution, after magnetic separation, add 60ml absolute ethanol and 15ml ultrapure water, stir magnetically to make it evenly mixed, then add 1ml concentrated ammonia water, and then add 20ul tetraethyl orthosilicate every half hour Ester (TEOS), add 3 times in total, react at 40°C for 12h, then add 200ul concentrated ammonia water and 200ulmercaptopropyltrimethoxysilane (MPTMS), react at 60°C for 6h, magnetically separate the resulting product and use anhydrous Wash with ethanol for 3-4 times, and finally disperse in absolute ethanol to obtain Fe 3 o 4 @SiO 2 - SH dispersion (10 mg / ml).

[0045] Take 1ml Fe 3 o 4 @SiO 2 -SH solution was magnetically separated and dispersed in 10ml methanol, adjusted its pH to 4-5 with acetic acid, then added 2ml, 5mg / ml 2,2'-dithiobispyridylmethanol solution, stirred at room temperature for ...

Embodiment 2

[0046] Example 2 Preparation of Cysteine ​​(Cys) Hyaluronic Acid (HA-Cys).

[0047] Dissolve hyaluronic acid HA (Mw=31000D, 310mg, 0.8mmol-COOH) in 20ml ultrapure water, then add 1-(3-methylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC (766.8mg, 4mmol) stirred for 0.5h, then added N-hydroxysuccinimide NHS (460.36mg, 4mmol), stirred for 1.5h, then added cysteine ​​ethyl ester hydrochloride (29.70mg, 0.16mmol) After 24 hours of reaction at room temperature, the reaction solution was transferred to a dialysis bag (1000D), dialyzed with ultrapure water for 3 days, and freeze-dried to obtain the HA-Cys complex.

[0048]Dissolve RhB (95.8mg, 0.2mmol) in 10ml ultrapure water, add EDC (38.34mg, 0.2mmol), stir for half an hour, add HA (155mg, 0.4mmol) aqueous solution, then add DMAP (25mg, 0.2mmol ), react overnight at room temperature and dialyze the resulting reaction solution to remove unreacted reactants, dialyze with ultrapure water for 3 days, freeze-dry and store in the da...

Embodiment 3

[0049] Embodiment 3 magnetic (fluorescent) nanocomposite Fe 3 o 4 @SiO 2 - Preparation of SS-HA-(RhB).

[0050] Take the above-prepared HA-Cys (RhB-HA-Cys) and dissolve it in 20ml of ultrapure water. After it is completely dissolved, add 3ml (10mg / ml) of Fe under magnetic stirring 3 o 4 @SiO 2 -SSPy solution, react at room temperature for 48h, magnetically separate and wash the product several times with ultrapure water, freeze-dry to obtain Fe 3 o 4 @SiO 2 -SS-HA-(RhB) nanoparticles.

[0051] TEM images show that Fe 3 o 4 , Fe 3 o 4 @SiO 2 -SH,Fe 3 o 4 @SiO 2 -SSPy,Fe 3 o 4 @SiO 2 -SS-HA nanoparticles are spherical and uniform in size, and MPTMS(b), SSPy(c), HA(d) have been successfully modified to Fe 3 o 4 The surface of magnetic nanoparticles ( figure 2 a-d), the resulting Fe 3 o 4 @SiO 2 -SS-HA particle size distribution between 100-300nm. DLS results showed that Fe 3 o 4 , Fe 3 o 4 @SiO 2 -SH, Fe 3 o 4 @SiO 2 -SSPy, Fe 3 o 4 @SiO 2 -SS...

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Abstract

The invention discloses a magnetic nanocomposite for specifically capturing and effectively releasing circulating tumor cells and a preparation method thereof. The method comprises the following steps: mixing ferroferric oxide, tetraethyl orthosilicate and mercaptopropyltrimethoxysilane, and performing reacting to obtain Fe3O4@SiO2-SH; reacting dithiodipyridine with Fe3O4@SiO2-SH to obtain Fe3O4@SiO2-SSPy; mixing hyaluronic acid, 1-(3-methylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and cysteine ethyl ester hydrochloride and performing reacting to prepare HA-Cys; grafting rhodamine on HA-Cys to obtain RhB-HA-Cys; and reacting magnetic nanoparticles Fe3O4@SiO2-SSPy with HA-Cys or RhB-HA-Cys to obtain a targeted functional magnetic nanocomposite Fe3O4@SiO2-SS-HA-(RhB). The magnetic nanocomposite is uniform in particle size, has good thermal stability, magnetic responsiveness and biocompatibility, can realize efficient capture and nondestructive release of circulating tumor cells, and thus develops application prospects for detection and analysis of the circulating tumor cells.

Description

technical field [0001] The invention belongs to the technical field of biomaterials, and specifically refers to a magnetic nanocomposite for specifically capturing and effectively releasing circulating tumor cells and a preparation method thereof. Background technique [0002] Cancer metastasis is the main cause of cancer death, and circulating tumor cells (Circulating TumorCells, CTCs) are the key factors of cancer metastasis. The detection and analysis of CTCs has very important clinical significance for early diagnosis, prognosis judgment, recurrence detection and individualized treatment of cancer. However, the number of CTCs in peripheral blood is very small (about 1-10 CTCs in 1 mL of peripheral blood), so it is difficult to capture and separate CTCs by conventional means. At present, methods for enriching CTCs include density gradient centrifugation, membrane filtration, and immunomagnetic bead enrichment. Among them, immunomagnetic bead enrichment technology is one...

Claims

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Application Information

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IPC IPC(8): C12N5/09
CPCC12N5/0693C12N2509/00
Inventor 郭惠玲王文静张溢刘明星祝红达孙红梅
Owner HUBEI UNIV OF TECH
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