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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  • 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 i

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

Example Embodiment

[0043] Example 1 Disulfide dipyridine 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 of absolute ethanol and 15ml of ultrapure water, magnetically stir to make it evenly mixed, then add 1ml of concentrated ammonia, and then add 20ul of tetraethyl orthosilicate every half an hour Ester (TEOS), add 3 times in total, react at 40℃ for 12h, then add 200ul concentrated ammonia and 200ul mercaptopropyltrimethoxysilane (MPTMS), react at 60℃ for 6h, magnetically separate the resulting product and use anhydrous Wash 3-4 times with ethanol, and finally disperse in absolute ethanol to obtain Fe 3 O 4 @SiO 2 -SH dispersion (10mg / 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'-dithiodipyridine methanol solution, stirred at room temperature for 10h, magnetic Separa...

Example Embodiment

[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) Stir for 0.5h, then add N-hydroxysuccinimide NHS (460.36mg, 4mmol), stir for 1.5h, then add cysteine ​​ethyl ester hydrochloride (29.70mg, 0.16mmol) After reacting at room temperature for 24 hours, 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, and then add DMAP (25mg, 0.2mmol) ), react overnight at room temperature, dialyze the resulting reaction solution to remove unreacted reactants, dialyzed with ultrapure water for 3 days, lyophilize and store at low temperature ...

Example Embodiment

[0049] Example 3 Magnetic (fluorescent) nanocomposite Fe 3 O 4 @SiO 2 -Preparation of SS-HA-(RhB).

[0050] Take the prepared HA-Cys (RhB-HA-Cys) and dissolve it in 20ml of ultrapure water. After it is completely dissolved, add 3ml (10mg / ml) Fe under magnetic stirring. 3 O 4 @SiO 2 -SSPy solution, after 48h reaction at room temperature, 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 image shows 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 all spherical, uniform in size, and MPTMS(b), SSPy(c), HA(d) have been successfully modified to Fe 3 O 4 Magnetic nanoparticle surface ( figure 2 a-d), the resulting Fe 3 O 4 @SiO 2 -SS-HA particle size distribution is between 100-300nm. DLS results 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 -The average particle size of SS-HA is 242.9±4.2nm, 390.2±7...

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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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