Cell autophagy inhibitor and its preparation method and application
A type of injection and type of technology, applied in the field of medicine, to achieve the effects of diversified medication methods, wide sources, and broad clinical application prospects
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Embodiment 1
[0030] RA-V(1) inhibits KRAS-dependent protective autophagy in tumor cells:
[0031] Cell viability was measured by MTT assay. The KRAS-independent A549 and H460 and KRAS-dependent H441 and H358 cell lines cultured overnight in 10% FBS medium were digested with trypsin to form a cell suspension, and seeded on a 96-well plate at an appropriate concentration, 100 μl / well , at CO 2 Cultivate in the incubator for 24 hours until the cells are completely attached to the wall, add RA-V(1) at a final concentration of 0, 50, 100, 200 nM, and after 24 hours of action, add 20 μl of MTT solution (5 mg / ml prepared in PBS, pH= 7.4), continue to incubate for 4 hours, terminate the culture, and carefully aspirate and discard the culture supernatant in the well. Add 150 μl DMSO to each well and shake for 10 minutes to fully melt the crystals. Select a wavelength of 490nm, measure the light absorption value of each well on an enzyme-linked immunosorbent assay instrument, record the results, ...
Embodiment 2
[0036] RA-V(1) significantly induces apoptosis of KRAS-dependent tumor cells:
[0037] The KRAS-independent A549 and H460 and KRAS-dependent H441 and H358 cell lines cultured overnight in 10% FBS medium were digested with trypsin to form a cell suspension, which was seeded on a 6-well plate at an appropriate concentration, and added after 24 hours RA-V(1), treated for 24 hours, digested with trypsin, centrifuged at room temperature 2000rpm for 5-10min, collected cells; resuspended cells once in pre-cooled 1×PBS (4°C), centrifuged at 2000rpm for 5-10min, washed cells; added Suspend cells in 300 μL of 1×Binding Buffer; add 5 μL of Annexin V-FITC to mix well, protect from light, and incubate at room temperature for 15 minutes; add 5 μL of PI for staining 5 minutes before going to the machine, and detect cells by flow cytometry.
[0038] The KRAS-independent A549 and H460, KRAS-dependent H441 and H358 cells cultured overnight in 10% FBS medium were planted in a 24-well plate at an...
Embodiment 3
[0041] Preparation method of mPEG200-PDLLA2000 micelles:
[0042] (1) Dissolution: Prepare drug-loaded nanomicelles according to the weight ratio of mPEG2000-PDLLA2000 and RA-V(1) 30-10:1, accurately weigh mPEG2000-PDLLA2000 and RA-V(1) according to Table 1, and mix them Dissolve in a pear-shaped bottle containing 100mL of dichloromethane, shake continuously until the drug and materials are dissolved, then add 25mL of methanol, shake continuously until the drug and materials are completely dissolved, and obtain a clear solution after about 5 minutes.
[0043] (2) Evaporation of solvent: place the pear-shaped bottle on a rotary evaporator, vacuum rotary evaporation, the rotating speed is 100 rpm, the control temperature is 60 ° C, and the organic solvent is removed. After it is completely removed, the temperature is reduced to 40 °C, continue vacuum rotary evaporation for 3 hours to remove the residual organic solvent to obtain a transparent gel-like mixed drug film of RA-V (1)...
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