Synthesis of nanoparticles
Gold nanoparticle (AuNP) was synthesized according to Turkevich’s method [-]. 0.256 mM Chloroauric acid was reduced by 1.2 mM sodium citrate in the boiling condition with continuous stirring. Brick red color colloidal suspension of AuNP was formed. Similarly Iron oxide nanoparticle (IONP) was synthesized by double reduction of ferrous and ferric salts []. The nanoparticles were then characterised by dynamic light scattering (DTS Nano zeta-sizer), scanning electron microscopy (SEM) and UV–vis Spectroscopy (see Additional file 1: Figure S1).
Blood was collected from healthy human subjects with informed consent and the matter was cleared by ethics committee of Nilratan Sarkar Medical College, Kolkata.
PBMCs isolation and culture
Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of healthy human volunteers by venipuncture in a sodium citrate vial. The protocol by Ivan J. Fuss [] was followed with few modifications. Diluted blood (1:1 diluted with PBS) was carefully overlaid on the layer of ficoll-hypaque (Histopaque-1077 from SIGMA Aldrich) and centrifuged at 1600 rpm for 30 min at room temperature. After centrifugation, the buffy coat layer of PBMCs was carefully aspirated to a fresh tube, which was then washed with PBS and centrifuged at 1200 rpm for 10 min. This step was repeated twice to remove the trace of platelets and obtained an almost pure population of PBMCs. The viability count was performed using trypan blue exclusion test in a haemocytometer. This method yields approximately more than 95% viable and PBMCs. PBMCs were then suspended at 1 × 106 cells/ml in RPMI 1640 complete medium (GIBCO BRL) supplemented with 10% FBS, 1% Pen-strep and 2 mM L-glutamine. PBMCs were then incubated in a 5% CO2 incubator at 37°C.
Treatment of PBMCs (normal cells) and U937 (cancer cells) with different nanoparticles (GNP and IONP) both in presence and absence of an external static magnetic field (70 mT)
PBMCs were treated with GNP and IONP separately in a 12 well tissue culture plate. Three different doses (100 μg/ml, 10 μg/ml and 1 μg/ml) of both nanoparticle (NP) were given to cells in RPMI complete medium and incubated in 5% CO2 incubator at 37°C. In a duplicate plate and separate incubator, Static magnetic field (SMF) exposure was given to cells so that unexposed cells were not affected by such external perturbation.
Similarly U937 cell lines was cultured and plated in a 12 well tissue culture in RPMI 1640 complete medium at a concentration of 1×106 cells/ml at 37°C in 5% CO2 incubator. Three different doses of GNP and IONP were also given to cells. SMF exposure was given at a separate incubator in a duplicate plate. The work had been repeated more than thrice and results shown were mean of all the experiments.
DNA damage assay by single cell Gel electrophoresis (SCGE)
SCGE or Comet assay was performed on both normal and cancer cells after treatment with respective controls (cells not exposed to nanoparticles and SMF). Detection of DNA damage in the PBMCs was done by SCGE or Comet assay using protocol developed by Singh and Tice et al. method []. To brief, PBMCs were mixed with low melting agarose (LMA) and layered over pre-coated glass slides. Then a third layer of LMA was applied on it. Then slides were dipped in lysis buffer and incubated overnight at 4ºC. Afterwards slides were kept in alkaline electrophoresis buffer for 20 min and then electric field 24 V or 300 mA is applied. After that slides were neutralised using neutralising buffer and stained with EtBr. The slides were then manually scanned under fluorescence microscope. The images of comets were scored by the comet program COMET SCORE.
Flow cytometry analysis of cells treated with Nanoparticles
After treatment with nanoparticles and exposure to SMF, normal and cancer cells were assayed by flow cytometry in BD Verse instrument. Mitochondrial membrane potential and cellular viability of the cells were analyzed using JC-1 [] and Propidium Iodide (PI) probe [] respectively.
Alteration in mitochondrial membrane potential (ΔΨ) using JC-1 probe
JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide) is a lipophilic fluorescent probe which is used to evaluate the status of ΔΨ in a cellular system. It is a cationic lipophilic membrane permeable dye that emits fluorescence in both FITC and PE channel. JC-1 is a monomeric molecule that forms aggregates (J-aggregates) at high dye concentration. J-aggregates emit fluorescence in both FITC and PE channel but JC-1 monomers give fluorescence only in FITC. In a live cell JC-1 penetrates the plasma membrane and accumulates within mitochondria as J-aggregates. ΔΨ of such cells are polarised and give fluorescence at both FITC and PE channels. In a depolarised cell, JC-1 monomers start to leak from mitochondria to the cytosol and thus give only FITC fluorescence. Cells with high PE and high FITC intensity are polarised and cells with only high FITC fluorescence are depolarised.
Cellular viability assessment using propidium iodide dye
Propidium Iodide (PI) is a DNA staining dye and it is widely used to determine cell viability by flow cytometry. After treatment with two type of nanoparticles separately, PI was given to about 50,000 cells at concentration of 5 μg/ml and incubated for 5 min at room temperature and analyzed immediately by BD Verse. PI has absorption maximum at 535 nm and gives maximum fluorescence at 617 nm and is captured at PE channel. The data was analyzed by Flowjo, software version 10.0.6.
Flow cytometry data were analyzed in flowjo software. In each sample 10,000 cells were acquired which contain desired population of more than 50%. The desired population was the lymphocyte enriched viable population in case of PBMC cells. This lymphocyte enriched population was referred to as L1. Similarly in case of cancer cells (U937) 10,000 cells were acquired which mainly contains viable population. In order to exclude cellular debris, viable population was gated as P1. This LI and P1 population was further sub-divided into L2 and L3, and P2 and P3 in the analysis of JC-1 data. L2 and P2 are population of polarised cells and L3 and P3 were population of depolarised cells in normal and cancer cells respectively.
FCS files were transformed into csv format by flowjo program. Then the csv files were analyzed in a MATLAB platform. The 2D data (e.g. FSC, SSC) was converted to a 2D grid in which the matrix elements indicate the events occurrence density. To compare the effects of field (or any other particles) the control and the experimental matrices were dumped into two independent colour planes. While pure colours represented either control or experimental (red and green in case of SMF). The superposed regions represented in the grid points having overlap data (points where experimental data is similar to control). The result is represented as RGB image. The image is rotated in such a way that the XY-axes are the conventional XY co-ordinates and not the image axes. The scale of image is represented along a normalized logscale.
Determination of ΔΨ from JC-1 data using matlab program
ΔΨ of the cells are determined using matlab computer program with the help of the following MATLAB function (MATLAB 9.0, MATHWORKS USA):
Function psi = jyoti_potcal(x,y)
% x -- > array of monomer counts (FITC Fluorescence)
% y -- > array of dimer counts (PE Fluorescence)
% the function returns the single cell membrane potential
% the dye dimer is sensitive to membrane potential
% x + x →y
% dimensionalize to express fractional concentration
x = x/sum(x);
y = y/sum(y);
keq = y./(x.^2);
R = 8.314462; F = 9.6485e + 004; T = 300;
% assume the dye is in Nernst equilibrium
% Electrochemical potential = 0
% The membrane potential is equivalent to chemical potential difference of the dye
% This is the free energy (delta G = −RT ln keq) scaled by Faraday’s constant
psi = (R*T/F) * log(keq);
% express psi in millivolts
psi = psi*1000;