Full Papers
doi.org/10.1002/ejic.202001174
This solution was then added to the reaction mixture and stirred for
Prism 6). The experiment was repeated with cells that were
pretreated with biotin (2 mM) 1 h prior to treatment with the
complexes.
5 h. The solution was filtered, and slow evaporation of the solvent
yielded a dark brown solid that was isolated and washed with ether
and dried in vacuum over P4O10.
For cellular incorporation assay, ~0.4×106 cells/well were seeded in
12-well plates. Cells were allowed to grow undisturbed for 24 h.
Subsequently, the cellular media was replaced by fresh media,
complexes were added (2% DMSO) and incubated for 4 h. The cells
were then washed with PBS and trypsinized. They were centrifuged
to obtain cell pellets which were resuspended in 400 μL PBS. They
were acquired on the BD FACS VerseTM flow cytometer (BD
Biosciences, USA). Singlet gate (forward scatter-area versus forward
scatter-height) and live gate (forward scatter-area versus side
scatter-area) was used to select the viable cells and ~10,000 events
were recorded. The analysis of results and construction of histo-
grams was done using the flow cytometry analysis software, BD
FACSDiva™.
[Fe(L1)(cur)Cl]Cl (1): Yield: 85% (0.62 g) C35H37FeN4O6Cl2 (MW:
736.4425): calcd. C 57.08, H 5.06, N 7.61; found C 57.01, H 5.09, N
7.59. ESI-MS in CH3CN calculated for [MÀ Cl]+ (m/z): 700.1751; found:
700.2156 IR data: ν=417 (w), 735 (m), 1160 (m), 1290 (s), 1500 (vs),
3075 (br) cmÀ 1 (vs, very strong; s, strong; m, medium; w, weak; br,
broad). UV/Vis (1:1 DMSO/DPBS): λmax/nm [ɛ/MÀ 1 cmÀ 1]=437 [3.25×
104]. Molar conductivity in DMF at 298 K (ΛM): 58 Sm2 MÀ 1. μeff at
298 K: 5.83 μB.
[Fe(L2)(cur)Cl]Cl (2): Yield: 75% (0.7 g) C45H51FeN6O8SCl2 (MW:
962.7378): calcd. C 56.14, H 5.34, N 8.73; found C 56.12, H 5.37, N
8.68. ESI-MS in CH3CN calculated for [MÀ Cl]+ (m/z): 926.2527; found:
926.2710 [MÀ Cl]+. IR data: ν=420 (w), 745 (m), 1080 (s), 1330 (vs),
1505 (s), 1590 (m), 3400 (br) cmÀ 1. UV/Vis (1:1 DMSO/DPBS): λmax
/
The fluorogenic dye, 2’,7’-dichlorofluorescein diacetate (DCFDA)
was used to measure the intracellular oxidative stress. In 12-well
plates, the cells were seeded and incubated for 24 h. Subsequently,
nm [(ɛ/MÀ 1 cmÀ 1)]=440 [3.07×104], 450 (sh, shoulder) [2.64×104].
Molar conductivity in DMF at 298 K (ΛM): 63 Sm2 MÀ 1. μeff at 298 K:
5.87 μB.
°
the cells were treated with the compounds for 4 h at 37 C in dark.
This was followed by 1 h visible light photo-irradiation during
which DMEM was replaced by colourless saline buffer. Post
exposure, the cells were washed, trypsinized and re-suspended in
DPBS. Trypan blue exclusion assay was used to count viable cell
numbers using a hemocytometer. About 0.25×106 viable cells were
Theoretical Calculations
The geometries of the complexes 1 and 2 were optimized by
density functional theory (DFT) using B3LYP level of theory and
LanL2DZ basis set as implemented in Gaussian 09 program.[39] The
electronic transitions with their transition probability were obtained
using linear response time dependent density functional theory
(TDDFT). The coordinates of the energy minimized structures and
selected transitions in the visible region are listed in Table S1,
Table S2 (vide Supporting Information). Some estimated bond
distances and angles for the complexes 1 and 2 from the DFT
calculations are given in Table S3 and Table S4 (vide Supporting
Information).
°
stained with 10 μM of DCFDA and incubated at 37 C for 30 min in
dark. The stain was then washed off and the cells were re-
suspended in PBS. Data was acquired (1.0×104 cells in the live
singlet gate) on the BD FACS VerseTM flow cytometer (BD
Biosciences, USA). The results were analyzed and the representative
histograms were constructed using the flow cytometry analysis
software, BD FACSDiva™.
The cells were stained with Annexin V-FITC and PI to determine the
mode of cell death. About 0.5×106 cells were seeded and
incubated for 24 h in 6-well plates, post which, the cells were
°
treated with the compounds for 4 h at 37 C in dark. This was
Biological Experiments
followed by 1 h photo-exposure. The cells were subsequently
washed, trypsinized and re-suspended in PBS buffer. Viable cells
were stained with Annexin V-FITC and PI in the binding buffer
solution (APOAF, Sigma Aldrich) for 20 min. The cells were acquired
(10000 events in the singlet gate) on the BD FACS VerseTM flow
cytometer (BD Biosciences, USA). Construction of the Annexin V-
FITC versus PI dot-plots, analysis, and quantification of the results
was done using BD FACSDiva™ software.
HeLa, MCF-7 and HepG2 cells were grown in 100 mm polymer
culture dishes (SPL Life sciences, Korea) in DMEM. The media was
supplemented with 10% FBS, 100 U penicillin/mL, 100 μg strepto-
mycin/mL and incubated in a humidified 5% CO2 incubator (Sanyo,
°
UK) at 37 C. MTT assay was done to study the photocytotoxic
behavior of the complexes in these cells using a broad-band visible
light (400–700 nm) photoreactor (Luzchem Model LZC-1, Ontario,
Canada; light fluence rate: 2.4 mWcmÀ 2; light dose: 10 JcmÀ 2).
Approximately 8×103 cells were seeded for each of the cell line in
two 96 well plates in 100 μL media per well. The cells were grown
Quantification of Cellular Iron by ICPMS
About 2×106 cells were seeded in complete culture medium
supplemented with 10% heat-inactivated fetal bovine serum and
maintained at 37 C in a humidified atmosphere containing 5%
CO2. The complexes were added for final concentration of 15 μM
and allowed to incubate for 4 h. After drug exposure, the cells were
washed with DPBS solution. The wells were treated with 0.5 mL
°
for 24 h at 37 C in a CO2 incubator. The complexes were dissolved
in 2% DMSO and were added to the cells at different concen-
trations. Incubation was continued for another 4 h and then the
media of one plate was replaced with DPBS and photo-irradiated
for 1 h in broad-band visible light (400–700 nm). This was followed
by removal of DPBS and addition of fresh media. The media of the
other plate was discarded right after 4 h and replaced with fresh
media. Both the plates were incubated further for 20 h in the dark.
Then 25 μL MTT (4 mgmLÀ 1) was added in each well and incubated
in dark for an additional 3 h. Finally, the culture media was
discarded from each well of the plates and 200 μL DMSO was
added to each well to solubilize the purple formazan crystals.
Molecular Devices Spectra Max M5 plate reader was used to
measure the absorbance at 540 nm. The cytotoxicity of the
complexes was measured as the percentage ratio of absorbance of
treated cells to the untreated controls. The IC50 values were
determined by using nonlinear regression analysis (Graph Pad
°
°
65 C nitric acid for 1.5 h and left to digest at room temperature.
Afterwards, solutions were quantitatively transferred into 15 mL
tubes and analysed by ICP-MS. The total iron content was
calculated as ng/mL of protein or ppb.[57]
Confocal microscopy study
To study the cellular localization pattern of the fluorescent complex
(20 μM) inside HeLa cells, confocal microscopy was used (Olympus
FV 3000). ~0.5×105 cells were seeded in each glass bottom dish
Eur. J. Inorg. Chem. 2021, 1640–1650
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