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  • br HPLC analyses on the

    2019-09-24


    4.7. HPLC analyses on the stability and releasing ability
    profiles were recorded on UV detection at 280 nm. Mobile phase con-sisted of ACN/water (v:v = 80:20), flow rate was 1.0 mL/min. Samples were taken for HPLC analysis after filtered by 0.45 μm filter.
    GSTs activity was spectrophotometrically assayed at 340 nm by measuring the rate of CDNB conjugation with GSH as a function of time at 25 °C, as reported previously. The assay mixture contained 1 mM GSH, 1 mM CDNB and 0.1 mM EDTA in 1 mL (final volume) of 0.1 M potassium phosphate buffer, pH 6.5. In brief, NBDHEX, compound 2 and complex 1 at different concentrations (1, 5, 10 μM) were added to GSTs which was extracted from cancer Nifurtimox and mixed well. After in-cubation at room temperature for 5 min, CDNB and GSH were added and quickly mixed well. Absorbance was measured at 340 nm for 5 min. Prior to each experiment, the baseline of the UV spectrometer was corrected by replacing GSTs solution with phosphate buffer. All ex-periments were performed in triplicate.
    4.9. Cellular uptake and DNA platination
    A549 or A549/DDP cells were cultured in 6-well plates until the cells reached about 90% confluence. Complex 1 or cisplatin was added at a concentration of 10 μM or 20 μM. After incubated for 12 h, cells were collected and washed three times with cold PBS, followed by
    centrifugation for 10 min and resuspension in 1 mL PBS. Then, 100 μL suspended cells were taken to measure the cell density. The rest of the cells were spun down and digested at 65 °C in 200 μL 65% HNO3 for 24 h. The concentrations of platinum were detected by ICP-MS and all experiments were performed in triplicate. For the measurement of Pt concentration in cellular DNA in A549 and A549/DDP cells, cellular DNA was isolated using Genomic DNA Mini Preparation Kit (KeyGEN, China) according to the manufacturer's instructions. Following de-termination of concentration, the DNA in solution was digested with 70% HNO3 and Pt content in DNA was analyzed using ICP-MS. The DNA-Pt adduct was represented as ng of platinum per mg of DNA.
    The log P values were obtained by using the shake-flask method and UV–vis spectroscopy. Stock solutions of 1, 2 and NBDHEX were prepared in n-octanol that was pre-saturated with 0.9% NaCl (w/v) solution. Stock solutions of cisplatin were prepared in 0.9% NaCl (w/v) solution that was presaturated with n-octanol. Subsequently, the stock solution was added to an equal volume of n-octanol or 0.9% NaCl (w/v) solution. The het-erogeneous mixture was shaken vigorously for 24 h before centrifuging to achieve phase separation. The concentrations of the compounds in the organic and aqueous phases were then determined using UV absorbance spectroscopy. log P was defined as the logarithm of the ratio of the concentrations of the compound in the organic and aqueous phases.
    A549 or A549/DDP cells were plated into 6-well culture plates and treated with cisplatin, NBDHEX or complex 1 at a concentration of 20 μM for 24 h. Cells were harvested, mixed with molten LM Agarose (Trevigen) and immediately pipetted onto Comet Slide (Trevigen). Slides were immobilized at 4 °C for 10 min, and immersed in prechilled Lysis buffer at 4 °C for 30 min. Then the slides were immersed in al-kaline unwinding solution (1 mM EDTA, 200 mM NaOH) for 40 min at room temperature. Electrophoresis was done using alkaline electro-phoresis solution (1 mM EDTA, 200 mM NaOH) at 25 V for 30 min. Finally, slides were washed with water and stained with propidium iodide (PI) and visualized by microscopy.
    Cancer cells were plated into 6-well culture plates and cultured in
    5% CO2 at 37 °C overnight. Complex 1, cisplatin or NBDHEX was added at a concentration of 20 μM. After 24 h, cells were digested with trypsin and washed twice with cold PBS, then collected by centrifugation (2000 rpm, 5 min). Cell apoptosis was determined by flow cytometry using an Annexin V-FITC/PI Apoptosis Detection Kit (Keygen, China) according to the manufacturer's protocol. Detailed operation as follows: cells were stained with 5 μL Annexin V-FITC for 5 min in Annexin-binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4). PI was added to cells with 5 μL before incubated at room temperature for 15 min and fluorescence was measured by flow cytometer.