Method for detecting drug loading capacity of drug-loaded vesicles
A detection method and technology of drug loading, applied in measurement devices, instruments, scientific instruments, etc., can solve the problems of long time, affecting the detection limit of the method, and complicated steps of the pretreatment method, so as to simplify the sample pretreatment steps and shorten the processing time. time, the effect of shortening the detection time
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Embodiment 1
[0021] Example 1. Preparation of methotrexate drug-loaded vesicles
[0022] Take sterile, mycoplasma-free, fast-growing human lung cancer cells, wash them twice with PBS, centrifuge at 1000 rpm, 4°C to 25°C for 8min, and count. Resuspend the cells with 1640 culture medium, place in a 150×25mm culture dish, and shake well. Open the lid of the petri dish, and place it directly under the ultraviolet lamp and irradiate it in the middle for 50 minutes. Add methotrexate solution and an appropriate amount of 1640 culture medium to make the final concentration of methotrexate 2mg / ml, place at 37°C, 5% CO 2 Incubator cultivation.
[0023] After the above-mentioned apoptotic cells incubated with the drug were cultured for 16-20 hours, all the liquid in the culture dish was collected in a 50ml centrifuge tube, and the culture dish was washed with 20ml PBS, and all the liquid was combined in a centrifuge tube to make the total volume reach 40ml. Centrifuge the supernatant at 1500 rpm, ...
Embodiment 2
[0024] Example 2 Detection of methotrexate vesicle drug loading
[0025] First, take 100-300 μL of the concentrated drug-loaded vesicle suspension to be tested, add three times the volume of acetonitrile: chloroform (1:2, V:V) solution, mix on a vortex shaker for 20 seconds, and place it in a high-speed centrifuge at 10000 g Centrifuge for 5 min, and take the upper layer solution as a test sample.
[0026] After filtering the above upper layer solution, inject it into a high performance liquid chromatograph Thermo UltiMate 3000 for analysis and detection. The phase is: 0.01M potassium dihydrogen phosphate solution-acetonitrile 9:1 (pH 6.60), the injection volume is 40μL, the detection wavelength is 304nm, and the single-needle run time is 10min. At the same time, the external standard method was used for quantitative analysis, and then the instrument analysis software was used for data collection and calculation processing to obtain the methotrexate vesicle drug loading.
Embodiment 3
[0027] Example 3 Comparison of pretreatment methods for drug-loaded vesicle detection
[0028] In order to test the effectiveness of the vesicle pretreatment method of the present invention, the homogeneous vesicle suspension sample was divided into two parts, and the detection results of the commonly used cell lysis method and the method of the present invention were compared. Specifically, take 300 μL of concentrated vesicle suspension, add 400 μL of cell lysate (mainly composed of SDS and EDTA), pipette evenly, and incubate on ice for 20 min; use an ultrasonic cell crusher for 1 min to destroy the membrane structure of vesicles, and centrifuge at 10,000 g Take 500 μL of the supernatant and add it to 1 mL of acetonitrile, shake vigorously, and centrifuge at 10,000 g for 5 minutes; take 1.4 mL of the supernatant and add it to 6 mL of chloroform, shake vigorously, and centrifuge at 2000 rpm for 10 minutes; the supernatant is the sample A to be tested; At the same time, treat the...
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