Neutrophile granulocyte drug-loading preparation and applications thereof
A neutrophil and drug-loading technology, applied in the direction of non-central analgesics, antipyretics, anti-inflammatory agents, etc., can solve the problem of reduced targeting efficiency, reduced targeting efficiency, and difficulty in screening out optimal structures, etc. problem, to achieve precise targeting and improve targeting efficiency
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Embodiment 1
[0026] Isolation of neutrophils: Draw out heparin with a 10mL syringe, and push out the heparin after wetting the wall of the syringe. 10 mL of venous blood was drawn from the mice. Take 2 mL of erythrocyte lysate (purchased from BD Company), mix it with venous blood, lyse the red blood cells at 4°C for 3 min, centrifuge at 1500 rpm for 3 min, and redisperse the pelleted cells with 1 mL of PBS (pH 7.4) to obtain a cell suspension. Take 0.6ml of 65% (V / V) cell separation solution (purchased from BD Company) in a centrifuge tube, add 0.3ml of 55% (V / V) cell separation solution on top, and finally add 0.3ml of cell suspension , 2500 rpm, centrifuged for 30 minutes, and collected the cell layer between the concentration of 65% cell separation liquid and the concentration of 55% cell separation liquid to obtain neutrophils. cells by 1.5 x 10 6 Each bottle was inoculated in a culture bottle containing RPMI-1640 medium, placed at 37°C, 5% CO 2 incubator culture, such as figure 1 ...
Embodiment 2
[0027] Preparation of antineoplastic drug-loaded cells: the neutrophils prepared in Example 1 were mixed with 1×10 6 cells / well were inoculated in 24-well cell culture plates (containing RPMI-1640 medium), placed at 37°C, 5% CO 2 After pre-incubating in the incubator for 0.5 h, the medium was aspirated, and 100 μL of paclitaxel albumin nanoparticles (purchased from Abraxis BioScience) at a concentration of 2 mg / mL was added to each well for 2 h, and washed 3 times with RPMI-1640 medium preheated at 37 °C. Scrape the cells with a cell scraper and centrifuge at 1200rpm for 10min. Discard the supernatant, add 10mL PBS, repeat 3 times, collect the drug-loaded cells, which is the neutrophil drug-loaded preparation; set aside.
Embodiment 3
[0029] Preparation of antineoplastic drug-loaded cells: the neutrophils prepared in Example 1 were mixed with 1×10 6 cells / well were inoculated in 24-well cell culture plates (containing RPMI-1640 medium), placed at 37°C, 5% CO 2 After pre-incubating in the incubator for 0.5 h, suck out the medium, add 100 μL of 2 mg / mL doxorubicin liposome (purchased from Roche MabThera) to each well and incubate for 2 h, wash with RPMI-1640 medium preheated at 37 °C for 3 times , scrape the cells with a cell scraper, and centrifuge at 1200rpm for 10min. Discard the supernatant, add 10mL PBS, repeat 3 times, collect the drug-loaded cells, measure the concentration, and set aside.
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