Scheme 5.1 Schematic representation of the therapeutic approach by combining chemo- and photodynamic therapy
2.2 Experimental Section .1 Chemicals
Selenium dioxide (SeO2), pluronic F-127, paclitaxel (PTX), ascorbic acid, Rhodamine B, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2’,7’-dichlorofluorescein diacetate (DCF-DA), propidium iodide (PI) and 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimi-dazolylcarbocyanine iodide (JC- 1) were purchased from Sigma-Aldrich and used as received. The PE rabbit
anti-active Caspase-3 (Catalog No. 550821) and FITC-Annexin V- PI assay kit were purchased from BD Biosciences.
2.2.2 Cell Culture
Human embryonic kidney cells (HEK-293), lung cells (L-132), cervical cancer cells (HeLa), breast cancer cells (MCF-7), non-small lung carcinoma cells (A549) and colorectal adenocarcinoma cells (HT29) were purchased from National Centre for Cell Science (NCCS), Pune. All the cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% penicillin & streptomycin at 37 °C in humidified air containing 5% CO2.
2.2.3 Synthesis and characterization of selenium nanoparticles (SeNPs)
Synthesis of SeNPs was performed according to the method described by Wen Liu et al1 with slight modification. Stock solutions of Pluronic F-127 (2 mM), selenium dioxide (100 mM) and ascorbic acid (1 M) were prepared freshly just before the experiment. To 8 mL of Milli-Q water, 1 mL of Pluronic F-127, and 0.5 mL of selenium dioxide solution was added. Then, 0.5 mL of ascorbic acid solution was added dropwise with continuous stirring on a magnetic stirrer. As reaction progressed, color of the reaction mixture was changed to orange-red indicating formation of SeNPs. The as-synthesized SeNPs were thoroughly washed with water by repeated centrifugation (20000× g, 20 min).
UV-visible absorption spectra of the SeNPs was recorded with Perkin-Elmer Lambda 25 spectrophotometer. Hydrodynamic diameter (dH) and zeta potential (ζ) measurement were performed in Malvern Zeta Sizer Nano ZS. For TEM analysis, a colloidal suspension of SeNPs was drop cast onto a carbon- coated copper grid and allowed to air dry. TEM images were captured with JEM 2100 (Jeol, Peabody, MA, USA) at 200 kV. Further structural characteristics of SeNPs were evaluated by FESEM and EDX. For this, SeNPs
suspension was drop cast on an aluminum foil coated coverslip and gold coating was done before analysis. Samples were prepared for atomic force microscopy (AFM) by drop-casting SeNPs suspension onto a glass coverslip.
FTIR analysis of lyophilized SeNPs was carried out in a Perkin-Elmer Spectrum One spectrophotometer machine.
2.2.4 Cellular uptake of SeNPs
SeNPs were labeled with fluorescent dye Rhodamine B by adding 100 µg of dye during the synthesis of SeNPs as mentioned above. Rhodamine B-loaded SeNPs were washed with water (centrifugation at 20000 rpm, 20 min) repeatedly to remove unbound dye. To observe cellular uptake, cells were incubated with fluorescent (Rhodamine B-labelled) SeNPs for 4 h and subsequently washed with PBS to remove any bound NPs. Cells were then fixed, counterstained with DAPI and observed under a confocal laser scanning microscope (ZEISS LSM 880) using ZEN 2 software.
2.2.5 Loading of paclitaxel (PTX) onto SeNPs and its release
Paclitaxel was loaded by incubating the drug (180 µg) with 50 mL of SeNPs (5 mM) under continuous stirring for 4 h at room temperature. Paclitaxel- loaded SeNPs (PTX-SeNPs) were separated by centrifugation (15,000 rpm), and the unbound drug was quantified by measuring the drug amount in the supernatant. The amount of paclitaxel in the loading or release media was quantified by measuring absorbance at 232 nm.10 Loading of paclitaxel onto SeNPs was calculated as:
For drug release study, PTX-SeNPs were incubated in phosphate buffer saline (PBS, pH 7.4) and acetate buffer (pH 5) at room temperature. At regular interval, 0.5mL sample was withdrawn and centrifuged at 15, 000 rpm for 20 min to separate the PTX-SeNPs. Absorbance (at 232 nm) of the supernatant was recorded to measure the PTX released in the buffer. Centrifuged PTX- SeNPs were again added into PTX-SeNPs buffer solution.
2.2.6 Cytotoxicity assay
Cytotoxicity of SeNPs and PTX-SeNPs on HEK293, L132, HeLa, MCF-7, A549, and HT29 was studied by MTT assay.11 Briefly, cells were seeded at a density of 5×103 cells/well in 96 well plates. After 48 h of treatment with varying concentration of SeNPs and PTX-SeNPs, 80 µL of MTT (0.5mg/mL in DMEM) was added to each well and incubated for 3 hours. Thereafter, 150µL DMSO was added to each well. Absorbance was measured at 570 nm for dissolved formazon crystals and at 630 nm as reference.
2.2.7 Cell cycle analysis
HeLa and MCF-7 cells were seeded at a density of 105 cells/well in 6 well plates and incubated for overnight. Synchronization of the cells was acquired by incubating them with serum-free media for 48 h. Subsequently, HeLa cells were treated with 4.35 µM (1/2 IC50), 8.7 µM (IC50), and 13 µM (3/2 IC50) of PTX- SeNPs for 48 h. Similarly, MCF-7 cells were treated with 2.7 µM (1/2× IC50), 5.4 µM (IC50), and 8.2 µM (3/2 IC50) of PTX-SeNPs for 48 h. On completion of the treatment, cells were harvested by trypsinization, fixed in 70% ethanol and incubated overnight at -20˚C. Next, the cells were treated with RNase A, stained with PI and analyzed in FACS Calibur (BD Biosciences, NJ, USA).
2.2.8 Annexin V-PI apoptosis assay
HeLa and MCF-7 cells were seeded at a density of 105 cells/well in 6 well plates and incubated for overnight. After 48 h of treatment with PTX-SeNPs at
1/2 IC50, IC50, 3/2 IC50, cells were washed with PBS, trypsinized and were stained with Annexin V-FITC PI kit according to the supplier’s protocol.
Stained cells were incubated for 10 min at room temperature and analyzed by flow cytometer (Cytoflex, Beckman Coulter).
2.2.9 DCF-DA assay
Following treatment with PTX-SeNPs (at 1/2 IC50, IC50, 3/2 IC50) for 12 h, HeLa and MCF-7 cells were washed with PBS, harvested by trypsinization and resuspended into a 0.2 µM DCF-DA in PBS. Thereafter cells were incubated for 30 min at room temperature, pelleted down by centrifugation and resuspended in PBS for further analysis in FACS Calibur (BD Biosciences, NJ, USA). Data analysis was performed using FCS Express 5.
2.2.10 JC-1 staining for mitochondrial membrane potential (MMP) JC-1 is a cationic dye which enters in mitochondria and forms aggregates that fluoresces orange. However, the fluorescence changes to green (due to monomeric JC1) if the membrane potential decreases. HeLa and MCF-7 cells were treated PTX-SeNPs (at 1/2 IC50,IC50, 3/2 IC50) for 48 h, washed with PBS and stained with JC-1 (10µM) in DMEM for 30 min. JC-1 stained cells were observed immediately under a fluorescence microscope (Nikon Eclipse Ti-U, Tokyo, Japan).
2.2.11 Caspase-3 assay
Phycoerythrin (PE) conjugated anti-active caspase-3 antibody (BD Biosciences, Clontech, Palo Alto, CA, USA) was used to estimate the cell population expressing active caspase 3 due to apoptosis. Cells treated with PTX-SeNPs (at 1/2 IC50,IC50, 3/2 IC50) for 48 h were collected by trypsinization and were fixed by using 4% formaldehyde for 10 min. Cells were permeabilized in 90% ice-cold methanol for 30 min. Thereafter cells were washed twice with
with the required (according to manufacturer’s protocol) amount of the PE anti-active caspase-3 antibody for 30 min. After washing the stained cells twice with incubation buffer, they were analyzed in flow-cytometer (FACS Calibur, BD Biosciences, NJ, USA).
2.2.12 Field emission scanning electron microscopy (FESEM)
To observe the morphologies of untreated and treated (with PTX-SeNPs at IC50) cells, cells were harvested, fixed in 70% ethanol, drop cast onto aluminum foil wrapped coverslip and air-dried. Then the cells were observed under FESEM (ZEISS SIGMA).