Apoferritin-benzothiazole compound complex as well as preparation method and application thereof
A kind of technology of apoferritin and benzothiazole, applied in the field of medicine
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Embodiment 1
[0038] Embodiment 1: Preparation of apoferritin (Apoferritin, AFt)
[0039] Apoferritin is the iron-free form of ferritin, which is composed of 24 protein subunits and is a natural iron storage protein; in order to obtain apoferritin, we need to dialyze ferritin.
[0040] (1) Prepare dialysis buffer: dissolve sodium acetate in water at room temperature, adjust the pH to 5.5 with hydrochloric acid, and make a sodium acetate dialysis buffer with a concentration of 0.1mol / L;
[0041] (2) Prepare the dialysis reaction: put 1.5L of dialysis buffer into a 2L beaker, add 1.95mL of thioglycolic acid drop by drop in a dark environment, and then slowly fill with nitrogen for 2 hours before use;
[0042] (3) Prepare ferritin: Dilute ferritin (SIGMA, CAS: 9007-73-2) 10 times with 0.1mol / L sodium acetate dialysis buffer, and put it into a dialysis bag wetted with sodium acetate dialysis buffer in advance (MWCO 12000-14000), immerse the dialysis bag in the beaker containing the sodium acet...
Embodiment 2
[0053] Example 2: The steps of this example are the same as in Example 1, except that step (6) prepares the coating reaction: dilute the apoferritin mother solution with the coating buffer to a final concentration of 2 mg / mL, take 10 mL and put it into the pre-used In a dialysis bag (MWCO 12000-14000) wetted with coating buffer, put it into a beaker containing coating buffer, and place it in the dark for 12 hours at 4°C for dialysis; in addition, use coating buffer to prepare a final concentration of 10mM Benzothiazole compound solution and add the sodium hypochlorite that final concentration is 0.02mmol / L, stand-by, wherein the mol ratio of apoferritin and benzothiazole compound is 1000:1; Benzothiazole compound is respectively 2-(4-amino -3-methylphenyl)-5-fluorobenzene (compound 1), 2-(4-methoxy-3-methoxyphenyl)-5-fluorobenzene (compound 2), L-lysine -(4-(5-fluorobenzo[d]thiazol-2-yl)-2-methoxy)-phenyl ester (compound 4);
[0054] Table 2 Loading efficiency (%) of apoferri...
Embodiment 3
[0056] Embodiment 3: The compound of embodiment 1 and embodiment 2 inhibits tumor growth experiment
[0057] Tumor cells MKN-45, NCI-N87, KATO-III, SGC-7901 and BGC-823 cell lines were cultured in RPMI-1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2 box.
[0058] Take the above cells in the logarithmic growth phase (cell confluency 80%) for experiments; plant 5000-6000 cells in a 96-well plate, set up a drug-dosing group and a control group in each well plate, and add the concentration of the reagent in terms of the compound For 0.001 μM, 0.01 μM, 0.1 μM, 1 μM and 50 μM, place the 96-well plate in the incubator and incubate for 72 h, then add 25 μL of 5 mg / mL thiazolyl blue (MTT) into the incubator and incubate for 4 h. After the MTT incubation, take out the 96-well plate and carefully remove the liquid in the 96-well plate with a vacuum pump to avoid sucking away the blue formazan crystals formed by the MTT incubation at the bottom; after pipetting, add 150 ...
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