2.1 Chemicals and reagents
Phosphonomethyl iminodiacetic acid (PMIDA), propidium iodide, cobalt chloride (CoCl2·3H2O), RNaseA, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT reagent), Histopaque 1077, and Rhodamine isothiocyanate (RITC) were procured from Sigma (St. Louis, MO, USA). Minimum essential medium (MEM), RPMI 1640, fetal bovine serum (FBS), penicillin, streptomycin, doxorubicin, sodium chloride (Nacl), sodium carbonate (Na2CO3), sucrose, Hanks balanced salt solution, and ethylene diamine tetra acetate were purchased from Himedia, India. Tris–HCl, Tris buffer, KH2PO4, K2HPO4, Hcl, formaldehyde, alcohol, and other chemicals were procured from Merck Ltd., SRL Pvt. Ltd., Mumbai, India. Commercially available dimethyl sulfoxide was procured from Himedia, India, and was purified by vacuum distillation over KOH. All other chemicals were from Merck Ltd., SRL Pvt., Ltd., Mumbai, and were of the highest purity grade available.
2.2 Synthesis of cobalt oxide nanoparticles
Cobalt oxide NPs were prepared by thermal decomposition method. The starting materials were CoCl2·3H2O, Na2CO3. Two grams of starting material (CoCl2·3H2O) was taken in a beaker and mixed with Na2CO3 as a molar ratio 1:1. Then the solution was rotated 1 h at room temperature. After that the precipitations were collected by centrifugation to obtain the nanoparticle precursor. The precursor was calcinated at 300 °C in air in a porcelain crucible for 2 h to get cobalt oxide NPs.
2.3 Surface modification of synthesized cobalt oxide nanoparticles
Thirty milligrams of synthesized cobalt oxide NPs was dispersed in 10 ml of milli-Q (Millipore) to make a solution 3 mg/ml. Another solution of PMIDA was prepared by dissolving 27 mg PMIDA in 10 ml of distilled water. Then this solution was mixed with above solution. The pH of the medium was maintained at 10 and stirring for 12 h. After that particles were collected by centrifugation. Then the recovered particles were washed three times with milli-Q (Millipore).
2.4 Characterization of synthesized surface modified cobalt nanoparticles
2.4.1 X-ray diffraction study
The phase formation and crystallographic state of cobalt oxide NPs were determined by XRD with an Expert Pro (Phillips) X-ray diffractometer using CoKα radiation (¥ = 0.178 nm). Samples were scanned from 20° to 80° of 2θ increment of 0.04° with 2 s counting time. Hydrodynamic size of the cobalt oxide nanoparticles aggregates was measured in a Brookhaven 90 Plus particle size analyzer (Chakraborty et al. 2010).
2.4.2 Dynamic light scattering
Dynamic light scattering (DLS) analysis was done by Zetasizer Nano ZS (Malvern Instruments) according to the method of Chakraborty et al. (2010) with some modifications. The concentration of the cobalt oxide nanoparticles was 100 μg/ml and was sonicated for 2 min and dynamic particle sizes were measured suspending two drops of aqueous suspension of nanoparticles in 10 ml of Millipore water. When particle was completely dispersed in water then particle was analyzed with a dynamic light scattering analyzer. The experiments were repeated several times to get average size of nanoparticles.
2.4.3 Transmission electron micrography
The particle size and microstructure were studied by high-resolution transmission electron microscopy in a JEOL 3010, Japan operating at 200 kV according to the method of Chakraborty et al. (2010) with some modifications. In brief, cobalt oxide nanoparticles was suspended in deionized at a concentration of 1 mg/ml then the sample was sonicate using a sonicator bath until sample form a homogenous suspension. For size measurement, sonicated stock solution of all cobalt oxide nanoparticles (0.5 mg/ml) was diluted 20 times. TEM was used to characterize the size and shape of the cobalt oxide nanoparticles. A drop of aqueous cobalt oxide nanoparticles suspension was placed on to carbon-coated copper grid and this was dried in air to get TEM image.
2.4.4 Fourier transform infrared spectroscopy
The conjugation of PMIDA with CoO nanoparticles was investigated by the FTIR with a model Thermo Nicolet Nexux-model 870 according to the method of Chakraborty et al. (2010) with some modifications. In brief, 1.0 mg sticky mass of PMIDA-coated CoO nanoparticles with 100 μl KBr medium and a thin film was prepared on the Nacl plate by drop casting method and under atmosphere separately. The FTIR value was taken within 500 to 4,000 wave numbers (cm−1).
2.5 Selection of human subjects for collection of lymphocytes and oral squamous epithelial cell
Six healthy subjects were chosen to collect the blood sample for separation of lymphocytes and collection of squamous epithelial cell by scraping. All subjects enrolled in this study were asymptomatic and none of them had abnormality on physical examinations and routine laboratory tests. All the subjects were from same geographical area and same economic status, non-smokers and non-alcoholic, and having same food habit. These subjects received no medication, including vitamin E and vitamin C. All subjects gave informed consent. The selection excluded not only individuals with acute infections or chronic diseases, but also excluded healthy individuals undergoing supplementation with antioxidative substances. The study protocol was in accordance with the declaration of Helsinki, and was approved by the ethical committee of Vidyasagar University.
2.5.1 Separation of lymphocytes
Fasting blood samples were collected from all groups of individuals satisfying the Helsinki protocol. The lymphocytes were isolated from heparinized blood samples according to the method of Hudson and Hay (Hudson et al. 1991). Blood was taken and diluted with phosphate-buffered saline (pH 7.0) in equal ratio and then layered very carefully on the density gradient (histopaque) in 1:2 ratio, centrifuged at 500×g for 40 min and the white milky layer of mononuclear cells, i.e., lymphocytes were carefully removed. The layer was washed twice with the same buffer and then centrifuged at 2400 rpm for 10 min to get the required pellet of lymphocytes.
2.5.2 Collection of oral squamous epithelial cells
Mucosal scrapings were placed in medium MEM supplemented with 10 % fetal bovine serum and then passed through a sterile 100-mm nylon mesh followed by further washes with MEM complete medium. Filtrate was then centrifuged at 2,200–2,400 rpm for 5 min and the supernatant discarded. The cell pellet is suspended in MEM supplemented with 10 % fetal bovine serum, 100 μg/ml streptomycin, and 100 U/ml penicillin (Steele and Fidel 2002).
2.6 Cell culture
The normal human lymphocytes and oral squamous epithelial cells, T-cell lymphoma (Jurkat) and oral epithelial cancer (KB) cell lines were cultivated for in vitro experiments. Cell lines were obtained from the National Centre for Cell Sciences, Pune, India. It was cultured in RPMI 1640 medium and minimal essential medium supplemented with 10 % fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin, 4 mM l-glutamine under 5 % CO2, and 95 % humidified atmosphere at 37 °C.
2.7 Preparation of drug
Several doses of PMIDA-coated CoO nanoparticles and doxorubicin (1–25 μg/ml) were prepared using sterile phosphate buffer saline (pH 7.4). In this study, all these doses were charged against normal and cancer cell line for evaluation of in vitro anti-cancer activity.
2.8 Experimental design
Each type of cells was divided into nine groups. Each group contained six Petri dishes (2 × 105 cells in each). The cells of each Petri dishes of control and experimental groups were maintained in MEM and RPMI 1640 media, where as it is applicable, supplemented with 10 % FBS, 50 μg/ml gentamycin, 50 μg/ml penicillin, and 50 μg/ml streptomycin at 37 °C in a 95 % air/5 % CO2 atmosphere in CO2 incubator. The following groups were considered for the experiment and cultured for 48 h:
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Group I
Control, i.e., cells in culture media
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Group II
Cells + 1 μg/ml doxorubicin in culture media
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Group III
Cells + 1 μg/ml PMIDA-coated CoO nanoparticles in culture media
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Group IV
Cells + 5 μg/ml doxorubicin in culture media
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Group V
Cells + 5 μg/ml PMIDA-coated CoO nanoparticles in culture media
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Group VI
Cells + 10 μg/ml doxorubicin in culture media
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Group VII
Cells + 10 μg/ml PMIDA-coated CoO nanoparticles in culture media
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Group VIII
Cells + 25 μg/ml doxorubicin in culture media
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Group IX
Cells + 25 μg/ml PMIDA-coated CoO nanoparticles in culture media
After the treatment schedule, the cells were collected from the Petri dishes separately and centrifuged at 2,200 rpm for 10 min at 4 °C. The cells were washed twice with 50 mM PBS, pH 7.4 and then processed for the biochemical estimation (Sandhu and Kaur 2002).
2.9 In vitro cytotoxicity
Normal human lymphocytes and oral squamous epithelial cell, Jurkat and KB cell lines were seeded into 96 wells of tissue culture plates having 180 μl of complete media and were incubated for 48 h. Doxorubicin and PMIDA-coated CoO nanoparticles were added to the cells at different concentrations (1, 5, 10, and 25 μg/ml), were incubated for 48 h at 37 °C in a humidified incubator (NBS) maintained with 5 % CO2. The cell viability was estimated by 3-(4,5-dimethylthiazol)-2-diphenyltetrazolium bromide according to the method of our previous laboratory report Chakraborty et al. 2011.
2.10 Detection of cellular apoptosis by flow cytometric analysis using propidium iodide
Cell apoptosis was measured using propidium iodide staining and analysis by flow cytometry (Roa et al. 2009). After the treatment schedule; the cells were scraped and centrifuged with the supernatant medium at 3,500 rpm for 5 min. Following washes, cells were resuspended in PBS and fixed in 70 % ethanol for 1 h on ice. Fixed cells were washed with PBS and stained with propidium iodide (5 μg/mL) solution containing ribonuclease (RNase; 50 μmol/L) for 30 min at room temperature. Then, cells were analyzed on a Becton Dickinson FACS Calibur flow cytometer. The sub-G0 population of the cells was determined by CellQuest software.
2.11 Alteration of cellular morphology by scanning electron microscope
After the treatment schedule, Jurkat and KB cells were examined under electron microscope according to the method of Papis et al. (2009) to visualize whether the cell undergoes for morphological alteration or not. In brief, treated cells were harvested, fixed in 2 % glutaraldehyde in 0.1 M Na-cacodylate buffer (pH 7.2) for 1 h at room temperature, washed in the same buffer, and post fixed for 1 h with 1 % osmium tetroxide in 0.1 M Na-cacodylate buffer (pH 7.2) at room temperature and dehydrated in graded ethanol and observed under electron microscope.
2.12 Intracellular uptake
Nanoparticle uptake by Jurkat and KB cells was studied by fluorescence microscopy methods according to our previous laboratory report (Sahu et al. 2010). After the treatment schedule, 2 × 105 cells were seeded into 35 mm cell culture plates. It was incubated in a humidified incubator maintained with 5 % CO2 and 37 °C. After 8 h the cells were washed with incomplete media and were incubated with 25 μg/ml DOX/DOX-RITC and PMIDA-coated CoO NP/PMIDA-coated CoO NP-RITC for fluorescence microscopy. Then the cells were allowed to adhere to a glass cover slip in 35-mm Petriplates, followed by incubation for 4 h at 37 °C in a humidified incubator maintained with 5 % CO2. Fluorescence images were acquired with 488 nm laser for differential interference contrast microscopy and 543 nm lasers for RITC excitation on an Olympus research phase contrast with fluorescence microscope (model: CX41; Olympus Singapore Pvt. Ltd., Valley Point Office Tower, Singapore).
2.13 Statistical analysis
The data were expressed as mean±SEM, n = 6. Comparisons between the means of control and treated group were made by one-way ANOVA test (using a statistical package, Origin 6.1, Northampton, MA 01060 USA) with multiple comparison t tests, p < 0.05 as a limit of significance.
