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Method for preparing fluorescent probe with tumor targeting effect by taking ferritin as carrier

A technology of ferritin and carrier, which is applied in the field of preparation of fluorescent probes and fluorescent probes, can solve the problems of poor solubility of coumarin-6, etc., and achieve the effect of uniform particle size, high utilization rate and small particle size

Inactive Publication Date: 2020-11-17
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is a fat-soluble fluorescent dye with high quantum yield, good cell penetration and relatively stable performance. However, due to the extremely poor solubility of coumarin-6 and almost insoluble in water, its application is limited to a certain extent.

Method used

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  • Method for preparing fluorescent probe with tumor targeting effect by taking ferritin as carrier
  • Method for preparing fluorescent probe with tumor targeting effect by taking ferritin as carrier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] E. coli expressing the protein of interest was inoculated into LB medium containing ampicillin, 37 [deg.] C temperature, 210r / min shaking cultured to an OD600 of between 0.6 to 0.8, IPTG was added to the cultures to a final concentration of 0.5 mM, the culture was 30 ℃, 180r / min continued induced culture 6-8h. The bacteria 8000r / min 5min to collect the cells after centrifugation, the cells were dissolved in lysis buffer (50mM NaH 2 PO 4 , 200mM NaCl, 10mM imidazole, pH = 7.9) for hanging weight. Suspension was broken at intervals of 5s after 15min, 8000r / min centrifugal supernatant was collected 5min, 10min the supernatant was heat-treated in water bath 60 ℃. The treated crude protein centrifuged at 8000r / min 30min the supernatant was collected, and the supernatant bound 6-12h nickel column, after elution through imidazol 5mM, 10mM, 250mM ferritin obtained crude sample, using an FPLC to give more pure HFtn .

Embodiment 2

[0031] 1:200 proportion (molar ratio) was weighed ultrasonic coumarin-6 was dissolved in absolute ethanol. The coumarin-6 was dissolved ferritin mixture was stirred at 4 ℃ 5min, mixed thoroughly. Using a mixed solution of 6M HCl solution the pH of the protein solution was adjusted to 2.5, were mixed and stirred at 4 ℃ 30min, depolymerization -6 sufficient contact with coumarin ferritin subunits. The mixing is complete the solution of 6M NaOH solution adjusted to pH 7.2, mixed and stirred at 4 ℃ 2h, depolymerization ferritin reassembled into the cage structure.

[0032] The protein solution using pH = PBS buffer 7.4 8-12h dialysis at 4 ℃, to remove non-entrapped coumarin-6. The dialyzed solution was 5000r / min centrifugal 10min, to remove the denatured proteins, it will give COU-HFtn nanoprobe solution.

[0033] HFtn-COU nanoprobes obtained in this embodiment, wherein the content of the coumarin-6 ferritin 1:200 ratio (molar mass ratio)

Embodiment 3

[0035] Fluorescence intensity HFtn-COU nanoparticles Analysis

[0036] (1) Take 1 × 10 4 The concentration of cells per well is inoculated with 24-well plate, 100 μL per well; at 37 ° C, 5% CO 2 The culture tank was cultured 24h to make cellular wall.

[0037] (2) 100 μl of the medium containing 0.5 μg / ml of COU and HFTN-COU is added to each well, each need to be repeated, and all solutions are added to 37 ° C, 5% CO. 2 Culture in the incubator for 24 h.

[0038] (3) After 24 hours of incubation, carefully suck all the supernatings in the well, rinse three times using the PBS buffer, wash away the residual medium. The cells were digested using trypsin and centrifuged and dissolved in 200 μl of DMSO solution after centrifugation.

[0039] (4) Determination of fluorescence intensity, recording data is detected using the enzymatic wavelength of 456 nm, and the emission wavelength is 504 nm, and the data is detected, and the drug name is the abscissa, the fluorescence intensity is a l...

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Abstract

The invention discloses a method for preparing a fluorescent probe with a tumor targeting effect by taking ferritin as a carrier. The ferritin carrier comprises human heavy chain ferritin. The methodcomprises the following steps: depolymerizing the human heavy chain ferritin with a cage-shaped structure into a monomer state; adding coumarin-6 with fluorescent properties into the depolymerized human heavy chain ferritin and combining the coumarin-6 with the ferritin; and re-polymerizing the depolymerized human heavy chain ferritin combined with the coumarin-6 into nanoparticles. The nano probeprepared by the invention has good biocompatibility and can be specifically enriched on the surface of tumor cells of an overexpression transferrin receptor 1(TfR1); and an efficient detection methodis provided for in-vivo tracing of a drug delivery system by utilizing the fluorescent property of the coumarin-6.

Description

Technical field [0001] The present invention belongs to the field of biotechnology, in particular to a method for preparing target tumor cells with a fluorescent probe, by loading the fluorescent dye coumarin inside ferritin -6 preparing a fluorescent probe targeting a tumor, for tumor tumor cells or in vivo targeted fluorescence imaging detection. Background technique [0002] In order to achieve efficient use and a tracer drug in vivo, and to reduce the side effects of each tissue damage to the human body, to absorb fluorescence labeling operation and the tracking of drugs it has become an important method. The researchers hope to find a suitable carrier and release the appropriate way to make it to be released at a specific time, specific location. It requires not only good targeting vector, which also requires good biocompatibility, and larger amount of loading. Inorganic non-metallic nano probe, probe metal nanoparticles, polymers, liposomes, and the like as a carrier a biof...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K49/00
CPCA61K49/0039A61K49/0056A61K49/0093
Inventor 张瑜马原蒙李瑞珂戚岚岚王飞李迅
Owner NANJING FORESTRY UNIV
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