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Intracellular polymerization method for hypoxia response type alkyne-amine click polymerization and application of intracellular polymerization method

A technology of click polymerization, polymerization method, applied in the field of biochemistry, can solve the problem of difficult to distinguish between normal cells and tumor cells

Pending Publication Date: 2022-07-12
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, when the alkyne-amine click polymerization reaction is constructed in tissue cells, the carbonyl-activated alkyne monomer and the diamine of tetraphenylethylene (TPE) can be taken up by normal cells and tumor cells simultaneously, and undergo intracellular polymerization spontaneously to achieve "light-up" imaging, which makes it difficult to distinguish normal cells from tumor cells

Method used

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  • Intracellular polymerization method for hypoxia response type alkyne-amine click polymerization and application of intracellular polymerization method
  • Intracellular polymerization method for hypoxia response type alkyne-amine click polymerization and application of intracellular polymerization method
  • Intracellular polymerization method for hypoxia response type alkyne-amine click polymerization and application of intracellular polymerization method

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Embodiment 1

[0071] HeLa cells grown to 80% cell density were digested, resuspended in fresh DMEM (10% fetal bovine serum) medium, and diluted to 1.5 x 10 5 cells / mL, inoculate 1 mL in each laser confocal culture dish, and culture until the cells adhere to the wall, add 150 μM aromatic binary nitro compound 1 solution (solvent is DMEM medium containing 10% fetal bovine serum), Placed at 37°C, 5% O 2 After the hypoxia culture, the culture medium was discarded at room temperature (20 °C), and the cells were carefully washed 3 times with 1 ml of PBS phosphate buffered saline solution, and then the washed cells were added to the concentration of 10 μM bismuth The solution of the alkynyl compound M2 (the solvent is PBS) was placed at 37°C in normoxia (21% O 2 ) cultured for 10 min; after the end of normoxia culture, the culture medium was discarded at room temperature (20°C), and the cells were carefully washed 3 times with 1 ml of PBS phosphate buffered saline solution, and then fresh 10% fet...

Embodiment 2

[0079] Detect the luminescence of the aromatic dibasic nitro compound 1 and the aromatic dibasic amine compound M1 in Example 1, and the results are as follows figure 1 shown. from figure 1 It can be seen that compound M1 has strong fluorescence, while compound 1 hardly emits light. It shows that the "lighting" of the fluorescent signal cannot occur before the binary nitro compound is converted into the binary amine compound.

Embodiment 3

[0081] Set the concentration to 1.5 x 10 5 1 mL of HeLa cells / mL were added to a laser confocal culture dish, and after the cells adhered to the wall, 150 μM of aromatic binary nitro compound 1 solution (the solvent was DMEM containing 10% fetal bovine serum) was added. base) at 37 °C in normoxia (21% O 2 ), 5%O 2 , 1%O 2 and 5% O 2 + Dicoumarin (nitroreductase inhibitor) for incubation, after incubation, the culture medium was discarded at room temperature (20°C), and the cells were carefully washed 3 times with 1 ml of PBS phosphate buffered saline, and then the The washed cells were added with 10 μM dibasic alkynyl compound M2 solution (the solvent was PBS), and then all were cultured in normoxia at 37°C. After the normoxia culture, the culture medium was discarded at room temperature (20°C). , and carefully washed the cells 3 times with 1 ml of PBS phosphate buffered saline solution, and then added fresh DMEM medium containing 10% fetal bovine serum to the washed cells...

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Abstract

The invention provides an intracellular polymerization method for hypoxia response type alkyne-amine click polymerization and application of the intracellular polymerization method, and belongs to the technical field of biochemistry. The intracellular polymerization method for hypoxia response type alkyne-amine click polymerization, provided by the invention, comprises the following steps: (1) performing hypoxia culture on cells in a binary nitro compound solution; (2) transferring the cells subjected to hypoxic culture in the step (1) into a binary alkynyl compound solution for normal oxygen culture; and (3) transferring the cells subjected to the normal oxygen culture in the step (2) into a culture medium for normal oxygen culture, so as to realize intracellular polymerization of hypoxia response type alkyne-amine click polymerization. The binary nitro compound is reduced into a diamine compound through nitroreductase in a cell under the hypoxic condition, and the reduced diamine compound and a binary alkynyl compound are polymerized in the cell, so that anchoring of fluorescent small molecules is realized, a fluorescent signal is lightened, and fluorescence imaging of the cell is realized.

Description

technical field [0001] The invention relates to the technical field of biochemistry, in particular to an intracellular polymerization method of hypoxia-responsive alkyne-amine click polymerization and application thereof. Background technique [0002] In 2014, in the process of expanding the click polymerization process based on alkyne monomers, the project of Academician Tang Benzhong established a spontaneous thiol-alkyne click polymerization reaction without catalyst. The reaction can be carried out under very mild conditions without the use of external catalysts. High molecular weight polymers were obtained in as little as 2 hours with yields as high as 97% (Macromalecules, 2014, 47: 1325-1333). However, when the alkyne-amine click polymerization is constructed in tissue cells, the carbonyl-activated alkyne monomer and the diamine of tetraphenylethylene (TPE) can be taken up by normal cells and tumor cells at the same time, and spontaneously undergo intracellular polyme...

Claims

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Application Information

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IPC IPC(8): C12P13/00G01N15/10G01N21/64
CPCC12P13/001G01N15/10G01N21/6458G01N2015/1006
Inventor 唐本忠邱燕萍秦安军胡蓉
Owner SOUTH CHINA UNIV OF TECH
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