About 10 mg of
A Waters Synapt G1 HDMS mass spectrometer was used in combination with a Waters Acquity UPLC system for the study. The Waters Acquity T3 HSS C18 column, measuring 150 mm × 2.1 mm × 1.8 m, was employed for the analysis. The mobile phase constituted of 0.1% (v/v) formic acid (CH₂O₂) in H2O for solvent A and 0.1% (v/v) CH₂O₂ in acetonitrile (C2H3N). The separation of solvent A started with 100% of composition and decreased up to 1% in 16 min and then held constant for 1 min before returning to 100% after 1 min giving a re-equilibrium time of 2 min which resulted in 20 min of total run time. The injection volume was 1 µl and the flow rate was 0.4 ml/min. The temperature in the column was kept at 60°C. The detection and analysis of UHPLC-qTOF-MS2 were carried out in full MS-SIM (mass spectrometry-selected ion monitoring) conditions, followed by data dependent MS2 (dd-MS2) with positive and negative polarity switching throughout the scanning window of mass-to-charge ratio (m/z) 50 to 1,200 Da (Da) with a mass accuracy less than 5 parts per million (ppm). For optimal mass spectroscopy analysis conditions, standardized spectrometric reference parameters were used with a capillary voltage of 2.5 kV, a cone voltage of 30 V, and extraction voltage of 4 V, source temperature of 120°C, and desolvation temperature of 450°C. The following conditions were used to set the overall mass resolution for MS: 550 L/Hr for desolvation gas flow (nitrogen), and 50 L/Hr for cone gas flow (nitrogen). The scan took 0.1 s. The data obtained was analyzed using MassLynx v4.1 SCN 872 software.
Breast cancer cells (MCF-7) (ATCC® HTB 22™) used in this study was obtained from American Type Culture Collection (ATCC). The cells were cultured in T75 culture flask using Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% Fetal Bovine Serum (FBS), 1% Amphotericin B, 1% Penicillin-streptomycin, and incubated at 37°C, 85% humidity, and 5% CO2. Once cells reached 90% confluence, culture flasks containing cells were washed using 10 ml Hank’s balanced salt solution (HBSS) and detached using 3 ml of TrypLE™ Express (TrypLE) (Invitrogen, 12605-028). The cells were seeded at 5 × 105 cell density in 3.4 cm2 diameter culture dishes for experiments.
The ZnPcS4 (sc-264509A) photosensitizer with a molecular weight of 898.15 g/mol was procured from Santa Cruz® Biotechnology and reconstituted to a concentration of 0.0005 M. The stock solution (5 ml) was prepared by dissolving 0.0024 g of ZnPcS4 in 5 ml of 0.001 M 1X phosphate-buffered saline (1X PBS). The reconstituted 0.0005 M was wrapped in aluminum foil and kept at rtp in the dark throughout the study. C1V1 = C2V2 formula was used for the preparation of the stock solution.
About 3 × 105 cells/mL of MCF-7 cells were seeded onto a 3.4 cm2 diameter culture plates with sterile microscopic coverslips. Pre-warmed complete DMEM media (2 ml) was added to the culture plates followed by 1 ml of the cell culture giving a final volume of 3 ml. Cells were then incubated for 4 h and washed three times with 1X PBS and added 3 ml pre-warmed complete media. The cells were then treated with 5 μM of ZnPcS4 and incubated for 24 h post-incubation and washing with 1X PBS, cells were fixed with 1 ml of 4% paraformaldehyde and incubated for 15 min at rtp which was followed by washing with 1X PBS for three times. Cells were permeabilized for 15 min at rtp with 1 ml of 0.5% Triton X-100 and then washed thrice with 1X PBS. Washed cells were then incubated at 4°C for 30 min with 200 μl of pre-warmed probes containing 100 nM mitochondrial tracker (Mito-Tracker), 65 nM lysosomal (Lyso-Tracker), and 65 nM endoplasmic reticulum (ER-Tracker), respectively. Cells were washed three times in 1X PBS after incubation, then counter stained for 5 min with 200 μl of 4′,6-diamidino-2-phenylindole (DAPI) and rinsed three times with 1X PBS. The treated cell coverslips were removed from the culture plates, inverted, and mounted onto sterile glass microscopic slides using aqueous mounting solution. The slides were then sealed with nail polish and examined for ZnPcS4 organelle localization using Alexa Fluor 594, DAPI, and FITC filters under a Carl Zeiss Axio Z1 microscope.
Cellular dose response studies were performed on MCF-7 cells 24 h post-treatment at various concentrations of
Laser irradiation parameters.
| Name | Parameters |
|---|---|
| Laser type | Semiconductor (Diode) |
| Wavelength | 680 nm |
| Emission | Continuous Wave (CW) |
| Spectrum | Red (Visible Light) |
| Power output | 193 mW |
| Fluency | 10 J/cm2 |
| Exposure time | 8 min 6 s |
Experimental models used in dose response studies.
| Experimental model | Experimental groups | Description |
|---|---|---|
| 1 | I | Cells only (control) |
|
|
Concentration (μg/ml) | |
| II | 25 | |
| III | 50 | |
| IV | 100 | |
| II | I | Cells only/cells + 680 nm laser (control) |
| ZnPcS4 | Dark toxicity 680 nm laser (10 J/cm2) | |
| II | Cells + 5 μM Cells +5 μM + laser | |
| III | Cells + 10 μM Cells + 10 μM + laser | |
| IV | Cells + 20 μM Cells + 20 μM + laser | |
| V | Cells + 40 μM Cells + 40 μM + laser | |
| VI | Cells + 60 μM Cells + 60 μM + laser |
The morphology of untreated and treated cells in each experimental models were analysed by comparing the cellular architecture of cells within images captured by a Wirsan Olympus CKX 41 inverted light microscope attached with digital camera (Olympus C5060-ADUS). This was followed by trypsinization cells using 300 μl of TrypLE and later resuspension of cells in HBSS for further cell viability analysis.
The trypan blue dye (Sigma-Aldrich T8154) exclusion assay was performed to determine the percentage of viable cells in cell suspensions of untreated and treated groups. The principle behind this assay stems from the ability of viable cells with undamaged cellular membrane selectively resisting trypan blue staining. On the other hand, non-viable cells with damaged cellular membranes are unable to resist the stain. In order to quantify the percentage of viable and non-viable cells, equal amount (10 μl) cell suspensions and 0.4% trypan blue dye (1:9) were thoroughly mixed and pipetted (10 μl of homogenous mixture) onto each window of the countess slide. Viability was quantitatively read using an automated countess II FL cell counter.
The 50% inhibitory concentration (IC50) determined from dose response studies for
ZnPcS4 and
We further evaluated the cell death mechanism using the established IC50 from dose response studies of two experimental models. The IC50 were administered as individual treatments as well as in combination. After 24 h of treatment, morphology and viability analysis, Annexin V-fluorescein isothiocyanate (FITC)-propidium iodide (PI) flow cytometry, and immunofluorescence (Bax, p53, and caspase 3 expression) experiments were performed.
Annexin V-FITC/PI (BD Pharmingen™) (556570) flow cytometry analysis was used to detect the cell death mechanisms in untreated and treated cells. To assess the proportion of cells undergoing apoptosis or necrosis, the Annexin V-FITC-PI kit was utilized according to the manufacturer’s instructions. Briefly, cells were detached from the 3.4 cm2 culture dishes by adding 300 μl of TrypLE and incubated for 5 min. Cells were later washed thrice and resuspended in 500 μl of 1X binding buffer. About 100 μl of each cell suspension was dispensed into sterile flow cytometry tubes followed by addition of 5 μl Annexin V-FITC and PI in all experimental groups and incubated for 15 min in dark. After incubation, 400 μl 1X binding buffer was added and incubated for 30 min in the dark. Flow cytometry tubes containing controls and experimental samples were read by using a flow cytometer Becton Dickinson (BD) Accuri™ C6.
Immunofluorescence was used to determine the possible cell death pathway induced. Cells were seeded (5 × 105 cells/ml) onto a 3.4 cm2 diameter culture plates with sterile microscopic coverslips at the bottom of the culture plate and incubated. Following the 24 h treatment, culture plates containing cover slips with cells were washed three times using 1X PBS. Cells were then fixed for 15 min by adding 1 ml of 4% paraformaldehyde and later washed thrice with wash buffer to remove excess fixative. Cells were permeabilized by adding 1 ml of 0.5% Triton X100 followed by 15 min incubation at rtp. Excess 0.5% Triton X100 was washed off using 1X PBS for three times. To avoid non-specific binding of antibodies, cells were blocked for 1 h at rtp by adding 1 ml of 1% Bovine Serum Albumin (BSA) which was followed by wash with 1X PBS for three times. Primary and secondary antibodies were subsequently reconstituted as per the manufacturer’s instructions and 200 μl of primary antibodies; anti-Bax (WH0000581M1), anti-p53 (SAB5700047), and anti-caspase 3 (C8487) supplied by Sigma were added to the culture plates and incubated for 2 h at rtp in the dark followed by thrice wash using ice cold 1X PBS. Washed cells were then incubated for 1 h at rtp in the dark with 200 μl of secondary antibodies; goat anti-mouse IgG-FITC (sc-2010) and rabbit anti-goat IgG-FITC (sc-2777) supplied by Santa Cruz® Biotechnology. After 1 h of incubation, the cells were rinsed thrice with ice cold 1X PBS and then stained for 5 min with 200 μl of DAPI. The cells were then washed thrice with ice cold 1X PBS before reinverting coverslips onto sterile microscope slides with aqueous mounting media. Thereafter, the slides were sealed with nail polish and analyzed with a Carl Zeiss Axio Z1 microscopic slide scanner. Filters Alexa Fluor 594, DAPI, and FITC were used to look for apoptotic protein expression on the slides.
The statistical analysis of experimental data was performed by using IBM SPSS version 27. All sets of experiments were repeated four times (