Chemical compounds and measurements
All chemical reagents and solvents were of analytical grade and were obtained from Sigma-Aldrich. Microanalyses (C, H, N) data were obtained using a Carlo Erba model 1106 elemental analyzer. Infrared spectra (4000-400 cm-1, KBr discs) were recorded on a Perkin Elmer 65 spectrometer. Magnetic susceptibilities of the complex (Gouy’s method) were measured using a Sherwood Scientific magnetic balance. Electronic spectra were measured on an Agilent-Carry 60 spectrophotometer using matched quartz cuvettes were used for measurements. Nuclear magnetic resonance (NMR) measurements were carried out on a Bruker-300 MHz spectrometer using DMSO-d6 solvent. Mass spectra of solid complexes (70 eV, EI) were performed on a Bruker microflex LT MALDI-TOF MS spectrometer. Thermogravimetric (TG) analyses were done under stream of nitrogen gas (10 °C/min) using a Shimadzu DT-50 thermal analyzer.
Synthesis and characterization of Schiff base-Zn(II) complex
(E)-4-[(2-hydroxybenzylideneamino)pyrimidin-2(1H)-one (L) (0.34 g, 1.58 mmoL in 15 mL methanol) was mixed with ZnCI2 (0.31 g, 3.15 mmol in 15 mL methanol). The resulted mixture was stirred and heated under reflux for 5 h. The separated complex was collected by filtration, washed thoroughly with methanol, ether. It was then crystallized in methanol/chloroform (1/2) and finally dried in a vacuum over fused CaCl2. The structure of Zn(II) complex was shown in Fig. 1. Yield 82%, mp 305 °C. Color: White. Anal. Calcd. for (C22H18N6O5Zn2Cl2) (FW: 648.08 g/mol) (%): C; 40.77, H; 2.79, N; 12.96. Found: C; 40.74, H; 2.75, N; 13.00. FT-IR (KBr, υ max (cm-1)): 3430, 3373, 3320 (OH), 3165 (NH), 3088, 3032 (Ar–CH), 2963, 2899 (Alip–CH), 1720 (C=O), 1636 (CH=N), 1655 (CH=Npyrimidine ring), 1564, 1537, 1454 (Ar–C=C), 1266 (C–O), 584-523 (M–O), 506, 470, 461 (M–N). 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 8.34 (s, H, N=CH), 8.04 (s, H, NH), 7.52-5.81 (m, 5H, Ar–H), 3.86 (d, Ar–Hpyrimidine ring). 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 163.94 (C=O), 161.41 (CH=N), 156.29 (C–OH), 157.16 (C–N), 140.17-100.01 (Ar–C). UV-Vis bands (λmax, nm) (ε, M-1 cm-1): 220 (3463), 230 (3504), 275 (3456), 335 (95), 430 (53), 495 (39). Mass spectrum [ESI], m/z: 629.06 (calcd.), 629.16 (found) [M-H2O-H]+.
Cell lines and culture
U118 (RRID: CVCL_0633) and T98G (RRID: CVCL_0556) originated a human brain glioma and HDF (Human Dermal Fibroblast) were obtained from the American Type Culture Collection (ATCC, Rockville, USA). The cells were kept in 75 cm2 flasks of Dulbecco’s Modified Eagle’s Medium (DMEM) containing 100 U/mL Penstrep and 10% FBS at 37 °C with 5% CO2, 95% air, and complete humidity.
Once the cells reached ~80% confluency, they were detached using trypsin/EDTA and counted by hemocytometer and 0.4% Trypan Blue was used to determine the viability rate. All the experiments were carried out at the viability rate of 90% or more. 1×104 cells for U118 and T98G and 5x103 cells for HDF were plated into each well of 96 well-plate and incubated at 37 °C for 24 h. Then, the medium were removed and 100 μL of Zn(II) complex and 5-FU solution (at the concentrations of 50, 100, 200, 300, 400, and 500 µM) prepared in the culture medium were added to the wells, and the cells were incubated for 24 h again. The concentrations were kept the same in all the MTT tests. Only 100 μL of Dulbecco's Modified Eagle Medium (culture medium) was added to the control wells .
Establishment of efficient electroporation (EP) protocols
Cell viability and PI uptake determine how efficient an EP protocol is. Cell viability refers to the ratio of the cells able to remain undamaged and metabolically active after the electrical field is applied. After the T98G and U118 cells reached a confluence of 70%-80%, they were removed using Trypsin/EDTA solution (Invitrogen) and centrifuged for 5 min at 1000 rpm for precipitation. 10 μL propidium iodide (PI) was added to the cell suspension (90 μL) with a density of 1x106 cell/mL and put into EP cuvettes with 4 mm gap (BTX) for determining the cell electropermeabilization. Depending on how permeable the cells are, propidium iodide permeates the cells and emits red fluorescent. With the purpose of determining the cell mortality caused by the electric pulses, 400 μL cell suspension at the density of 1x106 cell/mL was added into the 4 mm EP cuvettes, which were put separately into the chamber of BTX Gemini X2 EP device (Harvard Apparatus, USA). Then, 8 square-wave pulses (electric field intensity: 0-1500 V/cm, frequency: 1 Hz, pulse duration: 100 μs) were applied to all cells except the control cells. After the electric field application, the cells were left for incubation at room temperature for 15 min. A fluorescence microscopy was used to observe the electropermeabilization and the MTT analysis method was used to determine the cell viability.
After reaching a confluence of 70%-80%, the T98G and U118 cells were harvested using 0.05% trypsin and counted, then 1x106 cells/mL were suspended again in 400 µL of Dulbecco's Modified Eagle Medium (DMEM) with/without 5-FU and Zn(II) complex in 4 mm EP cuvettes (BTX). 5-FU and Zn(II) complex were added into the cell suspension before the EP at the concentrations of 25, 50, 100, and 200 µM. 8 square-wave electric pulses (frequency:1 Hz, pulse duration: 100 𝜇s) were applied to the T98G cells at 1000 V/cm and the U118 cells at 1250 V/cm in line with the ECT therapy clinical practices [22,23]. The EP parameters used in the ECT were optimized to reach a high level of permeabilization and a low level of cellular mortality by EP alone using the PI uptake and MTT assay. The cells that were subjected to the compound applications alone were also put in the EP cuvettes for the same duration and under the same conditions, but no voltage was applied. All the experiments were carried out in 4 replications.
After the EP, the cells were left for incubation for 15 min at room temperature and seeded on the plates with 96 wells and again left for incubation. After a 24 hours incubation, the ECT efficiency was assessed based on a cell viability analysis carried out using an MTT assay .
Zn(II) complex and 5-FU were applied to the cells alone or in combination with EP for 24 hours, and the cell viability was measured using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. After the chemotherapy and ECT, the cells were seeded into the plates and incubated with 5% CO2 at a humidity of 95% for 24 h at 37 °C. After the incubation, the substance solutions in the wells were taken, 90 μL DMEM and 10 μL MTT solution were added, and the cells were incubated again for another 4 h. After the second incubation, the growth medium with MTT was removed from the medium, and 100 μL dimethyl sulfoxide (DMSO) was added to the wells to dissolve the formazan crystals in the wells. The optical density (OD) of the color intensity was measured using a spectrophotometer at 570 nm . Only the fresh medium was used as the control. Because light affects MTT, the experiments were carried out in dark. The absorbance values of the groups subjected to the compounds were proportioned to the absorbance value of the control, and % viability was computed using the formula below.
% Viability = (OD of the group subjected to the compound / OD of the control group) x 100
Median inhibitory concentration (IC50) refers to the concentration that exerts an inhibitory effect of 50% on the cell proliferation. IC50 values were estimated via the regression equation created using the dose-response curve. The cytotoxicity of the 5-FU and Zn(II) complex against the cancer cells and their safety toward the normal cells were calculated using the ratio of the IC50 for the normal cells (HDF) to the IC50 for the cancer cells (T98G and U118). All the analyses were carried out in 3 parallels. Mean±standard deviation was used to express the results.
Graph Pad Prism software (Graph Pad Software Inc., San Diego, USA) was used to conduct the statistical analyses. The data are given as mean ± standard deviation (SD), and all of the experiments were carried out in triplicate. p<0.05 was chosen as the statistical significance level.