Nano clustering enzyme with hypoxia activation prodrug and preparation method and application of enzyme
A nano-cluster and prodrug technology, applied in the field of medicine, can solve the problems of starvation therapy failure of cancer cells, lethal chain reactions, off-target effects, etc. simple effect
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[0060] The present invention also provides a preparation method of the above-mentioned nano-clustered enzyme containing a hypoxia-activated prodrug, comprising the following steps:
[0061] A) 2-carboxyethyl-3-methylmaleic anhydride is chlorinated and mixed with PEG-b-PHEMA in a pyridine solution to react, and the reaction product is purified to obtain PEG-b-PHEMA CMA ;
[0062] B) Combining the dual enzyme cascade with PEG-b-PHEMA CMA Mix and dissolve in PBS buffer, incubate, centrifuge to obtain inner core;
[0063] C) Resuspend the inner core, add bovine serum albumin and bovine serum albumin grafted hypoxia-activated prodrug and PEG-b-PHEMA in the desired proportions CMA , the reaction is carried out to obtain a nano-clustered enzyme containing a hypoxia-activated prodrug.
[0064] The present invention first prepares PEG-b-PHEMA CMA , the PEG-b-PHEMA CMA It is a polyethylene glycol-b-poly(2-hydroxyethyl methacrylate) block copolymer modified by 2-carboxyethyl-3-methy...
Embodiment 1
[0095] Example 1, block copolymer PEG-b-PHEMA CMA Synthesis:
[0096] (1) First prepare 2-carboxyethyl-3-methyl maleic anhydride (CMA):
[0097] To a suspension of sodium hydride (1.4 g, 58 mmol) in THF (200 mL) was added triethyl 2-phosphonopropionate (19.5 g, 69.5 mmol) under a cooling bath at 0°C, and the reaction was allowed to react until H 2 Gas is no longer produced. Dimethyl 2-oxoglutarate (7.5 g, 43 mmol) was added to the previously reacted mixed solution and stirred at 0° C. for 30 minutes, after which a saturated aqueous ammonium chloride solution (200 mL) was added to the reaction solution. End the reaction. The obtained crude product was extracted with diethyl ether, and the diethyl ether was removed using a rotary evaporator, and the mixture was purified by silica gel column chromatography (ethyl acetate / n-hexane=1 / 10 (V / V), Rf=0.7) to obtain a yellow oil. The resulting yellow oil was dissolved in a mixture of ethanol (180 mL) and 2M aqueous KOH (100 mL) and ...
Embodiment 2
[0102] Embodiment 2, BSA TPZ Synthesis:
[0103] (1) To a solution of BSA (500 mg, 7.58 μmol, 1 equiv.) in 0.2 M borate buffer (60 mL) was added 2 mL of succinic anhydride (25 mg, 0.25 mmol, 33 mL of 1,4-dioxane) as a solvent equiv.) solution, after which the pH of the solution was adjusted to 9.3 using sodium hydroxide solution. After that, the solution was stirred at room temperature for 18 hours, and then dialyzed against 0.01M triethylamine aqueous solution. The samples after dialysis overnight were freeze-dried to obtain fluffy BSA. SA White powder (120 mg).
[0104] (2) at 0 ℃, the BSA SA (50 mg) was reacted with EDC (7.1 mg, 3.7 mmol) dissolved in 5 mL of 0.1 M sodium bicarbonate buffer (pH=8.8). After 5 minutes, TPZ (4.5 mg, 2.5 mmol) was added and stirred at room temperature overnight. BSA was obtained by centrifugal purification of ultrafiltration tube (cut off Mw=10,000) to remove water and free TPZ TPZ . see Figures 7 to 9 , Figure 7 For the BSA in Examp...
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