Inorganic Chemistry
Article
for C14H24Cl2N2O5Pt: C 29.69, H 4.27, N 4.95%. Found: C 29.43, H
4.36, N 4.70%.
subtracted as background from each sample. The IC50 values were
calculated by SPSS software after three parallel experiments, and the
results were expressed as the mean SD.
Preparation of 3. Complex 3 was prepared in 49% yield using the
same method of complex 1. ESI-HRMS: calcd for m/z [M − H]−
Stimuli-Responsive Property of 1−3 Treated with NQO1 or
VC. The activation of Pt(IV) complexes was determined by HPLC
using VC or NQO1 as the stimuli. The final concentration of NQO1
was set to 100 μg/mL and 1 mM for VC. Complexes 1−3 were used
at the concentration of 0.1 mM. Reversed phase HPLC was
implemented on a 250 × 4.5 mm ODS column. The chromatograms
were recorded on UV detection at 210 nm. The flow rate used for the
study was set to 1.0 mL/min. Before the HPLC analysis, the samples
were filtrated by 0.45 μm filter.
Confocal Microscopy. Confocal microscopy was performed to
investigate the cellular localization of tested compounds (5 μM) in
A549 and LO2 cells. Generally, 0.5 × 106 cells were seeded on the 35
mm glass bottom dishes (MatTek). Then the tested compounds were
added, and the cells were co-incubated at 37 °C under 5% CO2 for 3
h. Then 15 min before the end of the co-incubation, DAPI was added
for nucleus staining. After washed three times with PBS, the cells were
further incubated in colorless serum-free media for 15 min and then
imaged by a confocal laser scanning microscope (Zeiss LSM 700,
Zeiss, Germany. Ex: 335 nm; Em: 490−520 nm). Fluorescence from
DAPI appears as blue signals (Ex: 340 nm; Em: 450−470 nm).
Determination of Quantum Yields. In this study, Rhodamine B
(Φ = 0.59) was used as a standard, and the test was performed at 25
°C. The absorption of Rhodamine B was adjusted to the same value
(abs <0.1) as that of fluorescent molecules. Excitation wavelength was
set at 320 nm, and the emission spectra were recorded in the range
from 340 to 520 nm. The quantum yields were calculated by the
equation: Φsample = Φstandard (Fsample/Fstandard) (Asample/Astandard), where
Φ is the quantum yield, F is the integration of emission intensity, and
A is the absorbance value at excitation wavelength.
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767.0892, found 767.0916. H NMR (300 MHz, DMSO-d6): δ 1.34
(s, 6H), 1.88 (s, 3H), 1.92 (s, 3H), 2.05 (s, 3H), 2.84 (s, 2H), 4.73 (s,
2H), 6.26−6.29 (d, 2H, J = 10.0 Hz), 6.46 (m, 6H), 6.91−6.94 (dd,
1H, J = 2.3, 8.7 Hz), 6.98 (d, 1H, J = 2.0 Hz), 7.57−7.60 (d, 1H, J =
8.6 Hz), 7.96−7.99 (d, 1H, J = 9.6 Hz). 13C NMR (75 MHz, DMSO-
d6): δ 12.32, 13.17, 14.31, 28.98, 38.73, 40.00, 49.53, 65.22, 102.12,
112.97, 113.35, 113.42, 129.68, 137.59, 137.66, 143.43, 144.72,
153.50, 155.67, 160.74, 161.72, 175.34, 180.25, 187.39, 190.68. 195Pt-
NMR (129 MHz, DMSO-d6): δ 1260 ppm. Anal. calcd for
C25H30Cl2N2O9Pt: C 39.07, H 3.93, N 3.65%. Found: C 38.75, H
3.99, N 3.48%.
UV−vis Experiments. Complexes 1−3 were dissolved in PBS
under different pH values, and the final concentration was set to 0.1
mM. Then the UV absorption of the samples was recorded on a
Shimadzu UV2600 instrument equipped with a thermostatically
controlled cell holder with wavelength ranging from 190 to 450 nm.
All the experiments were carried out at 37 0.1 °C.
Stability of 1−3 in Medium RPMI-1640 Containing 10%
Fetal Bovine Serum. A HPLC study was performed to investigate
the stability of Pt(IV) complexes in RPMI-1640 (containing 10% fetal
bovine serum (FBS)). Complexes 1−3 were dissolved in PBS with 5%
DMF, and the concentration was set to 20 mM. The complexes were
added into RPMI-1640 and incubated for 0, 6, 12, 18, and 24 h at 37
°C, respectively. Reversed phase HPLC was implemented on a 250 ×
4.5 mm ODS column. The chromatograms were recorded on UV
detection at 210 nm. The flow rate used for the study was set to 1.0
mL/min. Before the HPLC analysis, the samples were filtrated by 0.45
μm filter. For the calculation of the relative ratio, the HPLC peak at 0
h was used as the reference (i.e., 100%), then the HPLC peak at a
different time point was compared with the reference peak to calculate
the relative ratio.
Cyclic Voltammetry. The CV was carried out on PGSTAT101
(Autolab, Metrohm) with a three-electrode setup comprising a glassy
carbon working electrode, platinum wire auxiliary electrode, and a
calomel reference electrode. The scan started from −1.0 V to +1.0 V.
The tested sample was prepared by dissolving the complex in PBS
containing 5% DMF, and the final concentration was 20 mM. The
scan rate was 100 mV·s−1 at 25 °C. KCl solution (0.1 M, pH 7.0) was
used as a background electrolyte.
Preparation of Stock Solutions for Cellular Studies. The
stock solution was prepared by dissolving the organic compounds or
the platinum(IV) complex in DMF to a final concentration of 20 mM.
The stock solution was serially diluted before the test. The final DMF
concentration in culture medium was <0.4%. For cisplatin, the stock
solution was prepared by dissolving cisplatin in water and was diluted
directly into culture medium when used.
Cell Culture. All adherent cell lines including HepG-2 (human
hepatocellular carcinoma cell line), A549 (human lung carcinoma cell
line), LO2 (human hepatic cell line), and A549/CDDP (human lung
carcinoma cell line-cisplatin resistance) were cultured in a humidified,
5% CO2 atmosphere at 37 °C and maintained in monolayer culture in
RPMI-1640 medium supplemented with 10% FBS, 100 μg/mL of
penicillin and 100 μg/mL of streptomycin.
Cytotoxicity Measurement. The cell viability was determined by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay. The tested compounds were dissolved in DMF and diluted to
the required concentration with culture medium (DMF final
concentration <0.4%). The suspension of 5000 cells/well was plated
in 96-well culture plates with culture medium and was incubated
overnight at 37 °C, in a 5% CO2 incubator. Then the tested
compound solution was added. Cells were incubated at 37 °C for 72
h, respectively. After that, the cells were treated with 10 mL MTT dye
solution (5 mg/mL) for 4 h cultivation. After the media with MTT
solution were removed with 100 mL of DMSO solution, the
absorbance of formazane solution was measured with an ELISA
reader at 570 nm. Wells without cells were used as blanks and were
Cellular Uptake Test. A549 and A549/CDDP cells were seeded
in 6-well plates at 37 °C under 5% CO2, respectively. After the cell
density reached 80%, cisplatin or complexes 1−3 (final concentration
5 μM) was added and then co-incubated for further 24 h. After that,
cells were collected, washed three times with ice-cold PBS,
centrifuged for 10 min, and resuspended in PBS. The obtained
suspension (100 μL) was used for determining the cell density. HNO3
(200 μL, 65%) was used to digest the remaining cells. Finally, ICP-
MS was employed to quantify the results via three parallel
experiments.
Comet Assay. A549 cells (1 × 105) and cisplatin or complexes 1−
3 were co-incubated for 24 h, respectively, and then molten LM
Agarose (Trevigen) at a ratio of 1/10 (v/v) was added. Thereafter,
the mixture was quickly transferred onto Comet Slide (Trevigen).
The samples were incubated at 4 °C for another 10 min in the dark
and then immersed in Lysis buffer and stood at 4 °C for 0.5 h. Slides
were then treated with alkaline unwinding solution prepared by
mixing 1 mM EDTA and 200 mM NaOH for 20 min at 25 °C in the
dark. For the electrophoresis, alkaline electrophoresis solution (1 mM
EDTA, 200 mM NaOH) was used at 21 V for 0.5 h. The slides were
immersed in water twice for 5 min and then washed once by 70%
EtOH. After drying overnight, the slides were visualized by
microscopy.
Cell Cycle Measurement. A549 cells (10 000 per well) were
cultured in 6-well plates overnight at 37 °C. Then, 5 μM of the tested
compounds were added and co-incubated with cells for 24 h. All cells,
including adherent and floating cells, were collected and washed twice
with PBS and then fixed by 70% EtOH at 4 °C for 24 h. After that,
fixed cells were washed with PBS. Thereafter, the cells were
centrifuged, stained with 50 μg/mL propidium iodide (PI) solution
containing 100 μg/mL RNase at 37 °C for 0.5 h. Finally, the sample
was measured by flow cytometry using Cell Quest software and
recording PI in the FL2 channel.
Western Blot Analysis. The A549 cells and 5 μM of the
compounds were co-incubated for 24 h at 37 °C. Then the proteins
were extracted, and the concentration of protein was measured by the
bicinchoninic acid (BCA) assay with a Varioskan multimode
microplate spectrophotometer (Thermo, Waltham, MA). After the
H
Inorg. Chem. XXXX, XXX, XXX−XXX