Materials and Methods 40 CHAPTER 2 Materials and Methods 2.1 Synthesis and physico-chemical characterization of of ZnO NCs All chemicals used for the synthesis of ZnO Nanocrystals (NCs) were of reagent grade and procured from Sigma Aldrich, USA. The synthesis of ZnO NCs involves mainly the reaction of zinc salt with an alkali hydroxide in alcoholic or aqueous medium. Depending on the size, shape and surface-chemistry properties of NCs needed for the present study, we optimized six different methods of synthesis. 2.1.1 Preparation of ~ 5 nm size NCs: An ethoxyethanol route is selected for making fluorescent, 5 nm sized crystalline ZnO quantum dots (1). In a typical preparation, 50 ml of 0.1 M of zinc acetate dihydrate is made to react with 50 ml of 0.1 M of NaOH in ethoxyethanol medium. The reaction mixture was stirred for 30 mins at ambient temperature. The clear solution thus obtained was found to show bright fluorescence under UV excitation, thereby indicating formation of ZnO NCs. The possible chemical reaction is given below. (CH 3 COO) 2 Zn. 2 H 2 O + 2NaOH → Zn(OH) 2 +2CH 3 COONa +2H2O Zn(OH) 2 + 2MeOH → Zn 2 ++ 2OH - + 2MeOH → Zn(OH) 4 2- + 2Me+ Zn(OH)4 2- → ZnO + H2O + 2OH - Typically, 0.1M of zinc acetate was stirred in 25 ml of alcoholic solution for 15-20 minutes and then .025 M of NaOH were allowed to dissolve completely in 25 ml of alcoholic solution by constant stirring. After that both the
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Materials and Methods
40
CHAPTER 2
Materials and Methods
2.1 Synthesis and physico-chemical characterization of of ZnO NCs
All chemicals used for the synthesis of ZnO Nanocrystals (NCs) were of
reagent grade and procured from Sigma Aldrich, USA. The synthesis of ZnO
NCs involves mainly the reaction of zinc salt with an alkali hydroxide in alcoholic
or aqueous medium. Depending on the size, shape and surface-chemistry
properties of NCs needed for the present study, we optimized six different
methods of synthesis.
2.1.1 Preparation of ~ 5 nm size NCs:
An ethoxyethanol route is selected for making fluorescent, 5 nm sized
crystalline ZnO quantum dots (1). In a typical preparation, 50 ml of 0.1 M of zinc
acetate dihydrate is made to react with 50 ml of 0.1 M of NaOH in ethoxyethanol
medium. The reaction mixture was stirred for 30 mins at ambient temperature.
The clear solution thus obtained was found to show bright fluorescence under UV
excitation, thereby indicating formation of ZnO NCs. The possible chemical
In the above first reaction zinc nitrate reacted with sodium hydroxide and
form Zn(OH)2 colloids. In the second reaction scheme Zn(OH)2 get dissolved
and form Zn2+ and OH−. In the third reaction, when the concentration attained in
the supersaturation level, ZnO nuclei will form.
Figure 2.1. Schematic diagram depicting the growth scheme of ZnO nano rod by
the hydrothermal process (Adapted from Ref. 7).
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2.1.7 physicochemical characterization
Nanoparticle size, shape and structure were characterized using scanning
electron microscope (JEOL, JSM-6490LA) and high resolution Transmission
electron microscope (JEOL-JEM-200CX). Crystallinity of the samples was
studied using an X–ray diffractometer [Rigaku Dmax-C] fitted with a Cu-Kα
source. The phase identification was carried out with the help of standard JCPDS
database. A Nicomp Particle Size Analyser (Nicomp 380, Particle Sizing
Systems, USA) was utilized for the particle size analysis, employing the technique
of Dynamic Light Scattering. The average particle size as well as dispersion in
size could be noted from this measurement. Zn2+ concentration and
microelectrode was used to detect extracellular pH variations.
2.2 Synthesis and physico-chemical characterization of graphene
2.2.1 Synthesis of graphene by arc-discharge method:
To prepare pristine graphene (p-G), direct current arc discharge of graphite
evaporation was carried out in a water-cooled stainless steel chamber filled with a
mixture of hydrogen and helium in different proportions without using any
catalyst (8). The proportion of H2:He used in our experiments is 200:500 torr. In a
typical experiment, a 6 mm wide and 50 mm long graphite rod (Alfa Aesar;
99.99% purity) was used as the anode and a 13 mm wide and 60 mm long graphite
rod was used as the cathode. The discharge current was 125 A, with an open
circuit voltage of 60 V. The arc was maintained by continuously translating the
cathode to maintain a constant distance of 2 mm from the anode. After the
chamber has cooled down to room temperature, p-G was collected from the inner
walls of the chamber and used for further characterization.
2.2.2 Surface functionalization:
The surface functionalization was carried out according to the method
documented in reference (9). Typically, as prepared graphene (25 mg) was
refluxed with dilute nitric acid (2 M) for ~12 h. The product was washed with
distilled water and centrifuged repeatedly to remove traces of acid. Graphene thus
obtained, functionalized with hydrophilic groups, could be dispersed in water or
physiological medium .
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2.2.3 Labeling of f-G with Tc-DTPA Typically 4mg of f-G was dispersed in 2 ml of distilled water using probe sonication for 10 min. 2ml of 8mCi 99mTc-DTPA was added and again probe sonicated for 5 min and boiled this sol in water bath (90-95oC) for 1 hour. After this the solution was vortexed for 2 min. then centrifuged @14500 rpm/5min. Then the settled sample pellet was re-dispersed in 2 ml of distilled water and repeated the centrifugation to wash of the free Tc-DTPA. Finally the above obtained 99mTc-DTPA tagged FG pellet sample is dispersed in 2 ml of distilled water. 2.2.4 Physico-chemical characterization: Raman spectra was recorded using LabRAM HR High Resolution Raman spectrometer (Horiba Jobin Yvon, USA), with a He–Ne Laser (λ=632.8 nm). HR-TEM images were obtained with JEOL JEM 3010 (JEOL, Japan). AFM measurements were performed using a Dimension 3100 Nanoman AFM (Veeco, NY).
2.2.5 Contact angle measurement: Hydrophobic/hydrophilicity of pristine and COOH-functionalized graphene samples were obtained by measuring the contact angle of spreading sessile drops, with distilled water as the contacting solvent. A drop shape analyzing system (DSA 100 EasyDrop Contact Angle Measuring System, KRÜSS, Germany) was used to determine the surface contact angles. A 0.5-1.0 µl droplet of distilled water was suspended from the tip of the micro liter syringe. The syringe tip was advanced toward the disk surface until the droplets made contact with the disk surface. Images were collected using the attached CCD camera and contact angle between the drop and the substrate was measured from the magnified image. Three samples each from the different surface modification processes were used to collect the contact angle data. 2.3 Antibacterial activity studies The wild type Escherichia coli (W3110) was obtained from E.coli genetic stock center (Yale) and Staphylococcus aureus (ATCC 25923) was from the Microbiology Lab of Amrita Institute of Medical Sciences, Kochi, India. Luria-Bertani (LB) medium was used for growing and maintaining E.coli, while Brain Heart Infusion (BHI) broth (Himedia Laboratories, Mumbai, India) was used for S.aureus.
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concentration of 106 colony-forming units per milliliter (CFU/ml) was inoculated
to 10 ml media. The culture tubes containing the nanoparticles were incubated
with shaking (200 rpm) in a water bath at 37 ºC. After 24 h, cell viability was
measured by serial dilution of the culture in 10 mM MgSO4, followed by plating
on solid media. The viable cell number was recorded by counting the number of
bacterial colonies grown on the plate multiplied by the dilution factor and
expressed as CFU/ml. The surface morphology of both treated and untreated
E.coli was studied using SEM.
2.3 Cell culture experiments
Human umbilical vein endothelial cells (HUVECs) were isolated and
cultured from the umbilical cord veins using the method of Baudin et al (10).
Umbilical cord was obtained from female donors after informed consent and
approval by the Institute Ethical Committee (IEC) at Amrita Institute of Medical
Sciences and Research Centre, Kochi, Kerala, India. In brief, the cords were
obtained in sterile Hanks Balanced Salt Solutions (HBSS) and washed thoroughly
with HBSS, cannulating the vein to wash out the blood within the lumen with
HBSS. Subsequently, the vein was filled with 0.1% Type I collagenase
(Invitrogen, USA) in HBSS (GIBCO, Invitrogen, USA) and incubated for 15 min
at 37 °C. Subsequently, the separated cells were collected by perfusion with HBSS
and washed in HBSS. The harvested cells resuspended in complete medium MI99
(GIBCO, Invitrogen) supplemented with 20 % Fetal Bovine Serum (GIBCO,
FBS), 50 IU/ml penicillin 50 µg/ml streptomycin and 50 µg/ml amphotericin and
50 µg/ml ECGF respectively. The HUVECs were culturing on the tissue flask
precoated with 2% gelatin (Sigma Aldrich, USA). The culture were incubated at
37oC under a humidified atmosphere with 5% CO2, confluent cells in the 3-4
passage having a typical cobble stone morphology were used for all the studies.
The phenotype of the isolated cells was confirmed by the analysis of the
expression of the endothelial cell specific markers such as CD62E and CD31 by
using flow cytometry. Typically, 1 X 104 cells/ml were resupended in 100 µl of
PBS, 5 µl of PE conjugated mouse anti-human CD62E and 5 µl FITC conjugated
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mouse anti-human CD31 were incubated for 15 min at room temperature in dark
condition and analysed by flow cytometry.
Peripheral blood mononuclear cells (PBMC) were isolated from human
peripheral blood after obtaining approval from IEC, using the method Ficoll-
Hypaque (Histopaque- 1077, Sigma, St Louis, MO) density gradient
centrifugation. Cells were washed twice with HBSS and resuspended in RPMI
supplemented with 10 % FBS. Normal Human Dermal Fibroblasts (NHDF) were
procured from PromoCell, Germany and cultured in fibroblast growth medium
kindly provided by them. Breast adenocarcinoma cell line (MCF-7),
nasopharyngeal carcinoma cell line and MDA-MB-231cell line were acquired
from National Centre for Cell Science (NCCS), Pune, India. HUVEC cells
maintained in Iscove's Modified Dulbecco's Medium (IMDM; Invitrogen, CA,
USA) supplemented with Endothelial Cell Growth Supplement (ECGS; Sigma, St.
Louis, USA). Eagles’ Minimal Essential Medium (MEM; Invitrogen, CA, USA)
supplemented with 10% FBS was used to culture cancer cells. Both the media
were supplemented with 50 IU ml−1 penicillin and 50 µg ml−1 streptomycin
(Invitrogen, CA, USA). Cells were incubated in a humidified atmosphere of 5%
CO2 at 37 ºC. Murine macrophage cell line (RAW 264.7) was procured from
National Centre for Cell Science (NCCS), Pune, India, and maintained in DMEM
(Invitrogen, CA, USA). Both media were supplemented with 10% fetal bovine
serum (FBS; Invitrogen, CA, USA), 50 IU mL-1 penicillin and 50 µg mL-1
streptomycin (Invitrogen, CA, USA). Cells were incubated in a humidified
atmosphere of 5% CO2 at 37 °C.
2.4 ZnO NCs toxicity analysis
We employed seven different cell function assays such as MTT, Alamar
blue and lactate dehydrogenase (LDH) assays for cell viability and plasma
to detect intracellular levels of ROS, MitoSOX Red assay to register
mitochondrial superoxide generation, JC-1 assay to assess alteration of
mitochondrial membrane potential, Newport Green DCF assay to determine the
intracellular Zn2+ concentration and Annexin V/Propidium iodide assay to detect
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apoptosis. For flow cytometric analysis, ten thousand events gated on size
(forward scatter; FSC) and granularity (side scatter; SSC) were acquired and
analyzed, and the percentage of positively stained cells was determined by
comparing with the negative controls. The possibility of interference from the
fluorescence of ZnO NCs, in FACS and confocal measurements, were excluded
by invoking appropriate gating measures or background subtraction (ZnO
excitation: 365 nm, emission: 555 nm).
2.4.1 Detection of cell viability
MTT assay was used to evaluate the mitochondrial activity according to
the protocol developed by Mossman (11). When cells reached 80% confluency,
they were harvested and 104 cells/ml were seeded in 96 well plates and incubated
for 24 h. Triton X-100 (1%) was used as positive control for toxicity and NCs-free
culture media served as the negative control. Cells were then treated with
different concentrations of ZnO NCs. The final concentrations of ZnO NCs in
each well were 0, 10, 25, 50, 100, 300 and 500 µM, (dissolved in appropriate
medium) respectively. The cells were then incubated for 12 and 24 h and MTT
assay was performed. Optical absorbance was measured in a microplate
spectrophotometer (Biotek PowerWave XS, USA) at 570 nm with 660 nm set as
the reference wavelength. Cell viability was calculated by the following equation:
[A]test / [A]control × 100, where [A]test was the absorbance of the test cells treated
with ZnO NCs and [A]control was the absorbance of cells without ZnO NCs. The
results were expressed as percentage viability compared to the untreated controls.
Figure 2.2. Schematic diagrom depicting the reduction of MTT by mitochondrial
dehydrogenase enzyme and form formzan crystals. [Adapted from
http://www.biocompare.com]
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2.4.2 Detection of LDH release
Cytoplasmic membrane integrity assays are particularly important as a
measure of cellular damage. LDH is a stable cytoplasmic enzyme that is normally
released upon cell membrane disruption or cell death (12). The LDH assay,
therefore, is a measure of cytoplasmic membrane integrity. The amount of LDH
released is proportional to the number of damaged or dead cells. Presence of
LDH in extracellular medium was assessed using a commercial test kit using
manufacturer’s protocol.
Figure 2.3. Schematic diagram depicting the enzymatic conversion of the
tetrazolium salt [iodonitrotetrazolium (INT)]in to puprle cloured formazan.
[Adapated from http://www. gbiosciences.com]
The detection principle was based on the NADH consumption during the
conversion of pyruvate into lactate, which promotes conversion of tetrazolium
salt, INT to water-soluble formazan crystals, which is detected
spectrophotometrically. After incubation with nanoparticles for 12 h and 24 h the
cell culture medium was collected for LDH measurement. After the incubation
with nanoparticle the cell culture medium was collected and centrifuged at 10000
rpm for 10 min. An aliquot of 50 µl culture medium was used to measure LDH
leakage and absorbance was measured in a microplate spectrophotometer (Biotek
PowerWave XS, USA) at 490 nm with 690 nm set as the reference wavelength.
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2.4.3 Cytoskletal imaging The cytoskeleton is a crucial component of the cell’s structure. After treatment with different concentrations of ZnO [0 - 200µM] the cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were then stained with Alexa Fluor 488 conjugated Phalloidin (Invitrogen, CA, USA) specific for F-actin filaments (13). Cytoskeletal alignment was visualized using confocal laser scanning microscopy (He-Ne and Ar laser). 2.4.4 Detection of intracellular ROS In order to determine the role of ROS generation in cytotoxicity, intracellular ROS generation was measured using an oxidation sensitive dye 2,7-dichlorofluorescein diacetate (DCFH-DA; Invitrogen, CA, USA) according to the procedure as reported (14). DCFH-DA is a non-fluorescent compound that diffuse through the plasma membrane, then enzymatically hydrolyzed by intracellular estrase to form DCFH. The deacetylated DCFH is rapidly reacted with intracellular ROS and form fluorescent dichlorofluorescin (DCF). The oxidation product of DCFH-DA has excitation/emission maxima of 495 nm/529 nm enabling detection using flow cytometry (FACS Aria; BD Biosciences, CA, USA) and Confocal laser scanning microscopy (Leica TCS SP5 II; He-Ne laser).
Figure 2.4. Schematic diagram of depicting the mechanism of the oxidation of
DCFH-DA (adapted from Ref. 15).
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Typically 1 X 105 cells after 24 h of exposure to ZnO NCs were re-
suspended in PBS buffer containing 5 µM of DCFH-DA for 30 minutes and the
cells were washed twice with PBS buffer. The level of intracellular ROS
generation was evaluated using flow cytometry and confocal microscopy. The
cells without nanoparticle treatment and 5 mM DEM (Diethyl maleate) were used
as negative control and positive control respectively.
2.4.5 Detection of mitochondrial superoxide
Mitosox red is a cell permeant cationic derivative of dihydroethidium dye
and is selectively translocated to mitochondria of live cells (15). The cationic
triphenylphos- phonium substituent of MitoSOX Red indicator is responsible for
the electrophoretically mediated uptake of the MitoSox Red indicator to actively
respiring mitochondria. Once inside the mitochondria, MitoSOX gets oxidised
rapidly by superoxide and emits red fluorescence (ex/em: 400/580 nm). Typically
1 X 105 cells after 24 h of exposure to ZnO NCs were re-suspended in HBSS
containing 5 µM of MitoSOX red dye for 10 minutes and the level of
mitochondrial superoxide was evaluated using flow cytometry.
Figure 2.5. Schematic diagram depiting oxidation of MitoSox Red in to 2-
hydroxy- 5-(triphenylphosphonium) hexylethidium. (Adapted from Ref. 16).
2.4.6 Assessment of mitochondrial membrane potential
To further assess ZnO NCs interaction with mitochondria, we used JC-1
Figure 2.9. Schematic diagram represent the Alamar blue assay priniciple. Non
fluorescent Resazurin get converted to highly fluorescent Resorufin. [Adapated
from http://www.bmglabtech.com]
2.5.1.3 Detection of LDH release:
After incubation with different concentrations of graphene for 24 h at 37 °C the cell culture medium was collected and centrifuged at 10000 rpm for 10 min.
LDH level in the extracellular medium was assessed using a commercial test kit
(Sigma, St. Louis, USA) using manufacturer’s protocol. An aliquot of 50 µl
culture medium was used to measure LDH leakage and optical absorbance was
measured in a microplate spectrophotometer (Biotek PowerWave XS, USA) at
490 nm with 690 nm set as the reference wavelength.
2.5.1.4 Cytoskletal imaging:
After treatment with p-G and f-G the Vero cells were fixed with 4%
paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were then
stained with Alexa Fluor 488 conjugated Phalloidin (Invitrogen, CA, USA)
specific for F-actin filaments. Nuclei were further stained with propidium iodide
(PI). Cytoskeletal alignment was visualized using confocal laser scanning
microscopy (He-Ne and Ar laser).
2.5.1.5 Detection of apoptosis:
Annexin V–FITC and PI assay (BD Biosciences, CA, USA) was employed
to detect apoptotic and necrotic cells. After incubation with a dose range of
graphene for 24 hr at 37 °C, the cells were washed and stained with Annexin V
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and PI. Typically 2 × 105 cells were resuspended in 100 µl of binding buffer and 5
µl of FITC-conjugated Annexin V (Annexin V–FITC) and 5 µl of propidium
iodide (PI) were added sequentially at room temperature in the dark. After
incubation for 15 min, stained cells were diluted with 400 µl of binding buffer and
directly analyzed in flowcytometry (BD FACSAria; BD Biosciences, CA, USA),
measuring the fluorescence emission at 530 nm and 575 nm.
2.5.1.6 Detection of intracellular ROS:
Intracellular ROS generation was measured using an oxidation sensitive
dye 2,7-dichlorofluorescin diacetate (DCFH-DA; Invitrogen, CA, USA). DCFH-
DA is a non-fluorescent dye that undergoes intracellular de-acetylation, followed
by ROS mediated oxidation to a fluorescent dichlorofluorescin (DCF) which has
an excitation/emission maxima of 495 nm/ 529 nm. Typically 2 × 105 Vero cells
after 24 h at 37 °C of exposure to graphene were re-suspended in HBSS containing
5 µM of DCFH-DA for 30 min and intracellular ROS generation was evaluated
using flow cytometry.
2.5.2 Murine macrophage cells (RAW 264.7)
2.5.2.1 cell culture
Peripheral blood samples were obtained from healthy volunteers after
informed consent and approval by the Institute Ethical Committee (IEC) at Amrita
Institute of Medical Sciences and Research Centre, Kochi, Kerala, India.
Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient
centrifugation (Histopaque-1077; Sigma, St Louis, USA) from anticoagulated
blood samples. The isolated PBMCs were washed thrice with Hanks balanced salt
solution (HBSS; Sigma, St.Louis, USA) and cultured in RPMI medium
(Invitrogen, CA, USA). Murine macrophage cell line (RAW 264.7) was procured
from National Centre for Cell Science (NCCS), Pune, India, and maintained in
DMEM (Invitrogen, CA, USA). Both media were supplemented with 10% fetal
bovine serum (FBS; Invitrogen, CA, USA), 50 IU mL-1 penicillin and 50 µg mL-1
streptomycin (Invitrogen, CA, USA). Cells were incubated in a humidified
atmosphere of 5% CO2 at 37 °C.
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2.5.2.2 Intracellular uptake studies
To investigate intracellular uptake of both graphene systems in
macrophage cells, we have employed a laser scanning confocal microscopy (TCS
SP5 II, Leica Microsystems, USA) and confocal Raman spectral mapping (Alpha
300RA, Witec, Germany). RAW 264.7 cells were grown on 13 mm cover slips,
treated with both p-G and f-G and analyzed. Z-plane stacks were acquired with
confocal Raman microscope to create 3-D Raman spectral image. The surface
morphology of both p-G and f-G treated cells were studied using scanning
electron microscopy (JSM-6490 LA, JEOL, Japan).
2.5.2.3 Cytoskeletal imaging
After 24h incubation with p-G and f-G, RAW 264.7 cells were
immunostained for F-actin as previously reported by our group.[41] Briefly, the
cells were fixed with 4% paraformaldehyde (Polysciences, USA) in phosphate
buffer saline (PBS) for 10 min and permeabilized with 0.1% Triton X-100 in PBS
for 3 min. Cells were then incubated with Alexa Fluor 488 conjugated Phalloidin
(Invitrogen, CA, USA) for 30 min. Subsequently, cells were washed thrice with
PBS and the cytoskeletal alignment was analyzed using confocal laser scanning
microscopy (He-Ne and Ar laser).
2.5.2.4 Cell viability (Alamar Blue Assay)
Alamar blue assay was employed to evaluate the cell viability. When
RAW 264.7 cells reached 80% confluency, they were harvested and 1 x 105 cells
were seeded in 24 well plates and incubated for 24 h at 37 °C with 5% CO2. After
treating with graphene for 48 h, cells were washed twice with PBS, and Alamar
Blue was added and further incubated for 4 h. The relative cell viability was
calculated using the following equation.
Where [F]test was the fluorescence of graphene treated cells and [F]control was the
fluorescence of untreated cells. Fluorescence was recorded using a fluorescence
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microplate reader (Beckman Coulter DTX 880 Multimode Detector, USA) using
560/590 nm ex/em filter settings.
2.5.2.5 Detection of plasma membrane integrity (LDH Assay)
Lactate dehydrogenase leakage (LDH) was evaluated to determine the
integrity of the plasma membrane using a commercial kit (Sigma, St. Louis, USA)
according to manufacturer’s protocols. Briefly, after incubation with various
concentrations of graphene for 24 h, cell culture medium was collected and
centrifuged at 10,000 rpm for 10 min. An aliquot of 50 µl culture medium was
used to quantify the LDH level. Absorbance was measured in a microplate
spectrophotometer (Biotek PowerWave XS, USA) at 490 nm with 690 nm set as
the reference wavelength.
2.5.2.6 Detection of ROS (DCFH-DA Assay)
Intracellular ROS generation was detected using 2,7-dichlorofluorescin
diacetate (DCFH-DA; Invitrogen, CA, USA). Typically 3 x 105 cells treated with
graphene for 24 h were resuspended in HBSS containing 5 µM of DCFH-DA for
30 min and intracellular ROS generation was evaluated using flow cytometry (BD
FACSAria; BD Biosciences, CA, USA) as described previously. For flow
cytometric analysis, ten thousand events gated on size (forward scatter; FSC) and
granularity (side scatter; SSC) were acquired and analyzed, and the percentage of
positively stained cells were determined by comparing with untreated cells.
2.5.2.7 Detection of Apoptosis (Annexin V-FITC/PI Assay)
Annexin V–FITC/PI dual staining (BD Biosciences, CA, USA) was
employed to detect apoptotic and necrotic cells. After incubation with various
concentrations of both graphene for 24 h, the cells were washed and stained with
Annexin V and PI. Typically 3 x 105 cells were resuspended in 100 µl binding
buffer followed by the sequential addition of 2.5 µL of FITC-conjugated Annexin
V (Annexin V–FITC) and 2.5 µL of propidium iodide (PI). After incubation for
15 min in the dark at room temperature, stained cells were resuspended in 400 µL
binding buffer and directly analyzed in flow cytometry measuring the
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fluorescence at 530 nm and 575 nm. Apoptotic cells were imaged using confocal
microscopy (He-Ne and Ar laser).
2.5.3 Human primary blood cells
Blood was drawn from healthy human donors who were not under any
medications, and collected in tubes containing citrate phosphate dextrose solution
with adenine (CPDA; Sigma, St. Louis, USA) at ratio of 9:1 blood:anticoagulant.
In order to rule out endotoxin contamination, all experiments were performed
under aseptic conditions. Endotoxin level in as prepared graphene was quantified
using Limulus Amoebocyte Lysate (LAL) endotoxin assay kit (Genscript, USA)
as per the manufacturers’ protocol.
2.5.3.1 Hemolysis
Hemolytic potential of graphene was detected by Soret band based
absorption of free hemoglobin (Hb) at 415 nm in the plasma using
spectrophotometer. Whole blood was collected into tube containing
anticoagulant. 450 µL whole blood was treated with 50 µL of graphene sample for
3 h at 37 °C on a shaker. Diluted blood incubated with normal saline (0% lysis)
and 1% Triton X-100 (100% lysis) served as negative and positive controls
respectively and the analysis was done as reported earlier (20),(21). Samples were
centrifuged at 4000 rpm for 15 min to collect plasma. Plasma obtained was diluted
with 0.01% sodium carbonate and absorbance was measured at 380 nm, 415 nm
and 450 nm using UV–Vis spectrophotometer (Shimadzu, Japan). Amount of
plasma hemoglobin (Hb) was calculated using the following equation.
where A415, A380, A450 are the absorbance values at 415, 380 and 450
nm. A415 is the Soret band absorption of Hb and A380, A450 are correction
factors of uroporphyrin whose absorption falls under the same wavelength range.
E is the molar absorptivity of oxyhemoglobin at 415nm which is 79.46. 1.635 is
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the correction factor applied for the turbidity of plasma sample. Hemolytic
property of graphene samples was plotted as percentage hemolysis of various
sample concentrations as per the following equation.
2.5.3.2 Platelet activation and aggregation study:
Platelet rich plasma (PRP) was obtained by centrifuging whole blood at
150 g for 10 min at 20 °C. PRP was diluted ten times using normal saline and
the mixture was equilibrated for 30 min at 37 °C in a water bath. 450 µL of
diluted PRP was treated with 50 µL of sample for 20 min. Saline and 50 µM
Adenosine diphosphate (ADP; Sigma, St. Louis, USA) served as negative and
positive controls respectively. 100 µL of treated PRP was incubated with 20 µL of
PerCP–Cy5 labeled CD62P and FITC labeled CD42b (BD Biosciences, CA,
USA) antibodies and incubated for 30 min after which the sample was diluted
with PBS and analyzed using flow cytometry. In platelet aggregation analysis,
PRP was treated with both graphene systems for 30 min and platelet count was
done using hematology analyzer (Abbott CELL-DYN 3700).
2.5.3.3 Plasma coagulation studies
Peripheral blood was centrifuged at 4000 rpm for 15 min at 19 °C to obtain
platelet poor plasma (PPP). 50 µL sample was treated with 450 µL of PPP for 30
min at 37 °C. 100 µL of prothrombin reagent (Diagnostica Stago, France) was
added to 50 µL of treated plasma and time taken for the plasma to coagulate was
measured as prothrombin time (PT). In activated partial thromboplastin time
(aPTT) analysis 50 µL of aPTT activator (Diagnostica Stago; France) was added
to 50 µL of treated plasma and incubated for 180 sec followed by the addition of
0.025 M CaCl2 and analyzed. aPTT value was expressed as a ratio, calculated by
the following equation.
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2.5.3.4 Inflammation analysis
The effect of graphene treatment on cytokine secretion by PBMCs was
studied using human inflammation kit (BD Biosciences, CA, USA) as per the
manufacturer’s protocol. Cytokines such as Interleukin-8 (IL-8), Interleukin-1β