chapter 2 materials and methods -...

Materials and Methods

40

CHAPTER 2


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.

(CH3COO)2 Zn.2H2O + 2NaOH → Zn(OH)2+2CH3COONa +2H2O

Zn(OH)2 + 2MeOH → Zn2++ 2OH-+ 2MeOH

→ Zn(OH)42- + 2Me+

Zn(OH)42- → 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


41

pre-cursor solutions were mixed well by stirring for about 30 min in order to get

the fine ZnO nano particles. Size tuning of these synthesized nano particles can

be done by repeating the same procedure by varying the molar concentration of

NaOH in the range 0.025 to .225 with a step increase of .025 each experiment.

2.1.2 Preparation of 200 nm size ZnO:

Forced hydrolysis of zinc acetate in diethylene glycol (DEG) at 160°C was

done as previously described (2). In a typical synthesis, 0.03 M zinc acetate was

added to 300 ml DEG. This solution was heated under reflux to 160°C. The

particle size and shape were controlled by optimising the precursor concentration

and hydrolysis ratio. The possible chemical reaction is given below.

Zn(CH3COO)2 + xH2O → Zn(OH-)x (CH3COO-)2-X + xCH3COOH (1)

Zn(OH-)X (CH3COO-)2-X→ Zno + (x-1) H2O+(2-x) CH3COOH (2)

(Adapted from Ref. 3)

In equation (1), Above 110º C the zinc acetate dehydrated and remove

acetic acid to form zinc hydroxyde. In equation (2), As zinc hydroxide phase is

not stable in higher temparature and further converted in to pure ZnO. Then the

reaction temperature was increased to 160 ºC and maintained for aging for 1 h.

After 1 hr the reaction mixture was cooled down to room temparature and washed

by repeated centfigugation and resuspended in distilled water for further analysis.

2.1.3 Starch coated ZnO NCs:

For starch coated samples, we followed aqueous phase synthesis as

reported by Vigneshwaran et al (4). In a typical preparation, 0.1 M zinc nitrate

hexahydrate was dissolved in 500 ml of 0.5% soluble starch solution by stirring.

After complete dissolution, 500 ml of 0.2 M sodium hydroxide solution was added

drop-wise under constant stirring. The reaction was allowed to proceed for 2 h

after the complete addition of sodium hydroxide. The solution was centrifuged

and washed with distilled water. After complete washing, ZnO NCs were dried at

80°C for 3 h. All synthesized particles were washed with ethanol and water to


42

remove unreacted molecules and by-products. The obtained precipitate was

redispersed in distilled water for toxicity studies. The possible chemical reaction

is given below.

Zn(NO3)2·6H2O + 2NaOH→ Zn(OH)2 + 2NaNO3 + 6H2O (1)

Zn(OH)2 + 2H2O →Zn2+ + 2OH− + 2H2O→Zn(OH)2−

4 + 2H+ (2)

Zn(OH)2− 4 →ZnO + H2O + 2OH−. (3)

(Adapted from Ref. 4)

2.1.4 Silica capped ZnO

In order to obtain silica capping on the surface of these NCs, a modified

Stöber method was followed (5) . Typically, ~ 200 µl of tetra-ethyl-ortho-silicate

(TEOS) solution was added to the as prepared ZnO nanocrystal solution with

stirring, followed by a step-wise addition of 500 µl water and 200 µl ammonia.

Formation of silica layer over ZnO was confirmed by FTIR.

2.1.5 PEGylated ZnO

Spherical nanoparticles of PEGylated ZnO NCs were synthesized by

reacting 0.1 M of zinc acetate dihydrate and 0.025-0.2 M of NaOH in methanol.

The reaction mixture was vigorously stirred for 20 min at ambient temperature

with polyethylene glycol (PEG) as a surfactant during synthesis. The amount of

added PEG per unit gm of ZnO was selected so as to get approximate monolayer

coverage over 40 nm size nanoparticles. A rough calculation will show that this

corresponds approximately to 0.1 g of PEG per gm of ZnO. There are reports that

PEG molecules link with ZnO through hydrogen bonding (6). In the present case,

PEG molecules with average molecular weight 8000 has a chain length of ~ 87

nm. The average surface area of a molecule of this length is about 19 nm2. The

surface area of a ZnO particle of size 40 nm is about 500 nm2, so that about 25

molecules of PEG is required to provide full surface coverage. This corresponds

to about 0.1 g PEG per gm ZnO. In order to remove the byproducts (sodium

acetate) the precipitate was washed several times with de-ionized water and then

re-dispersed in de-ionized water by ultrasonication.


43

2.1.6 ZnO nanorods

The ZnO nanorods were synthesized according to the method documented

in reference (7) with slight modification. Typically, 0.5M Zinc nitrate

hexahydrate was dissolved in 0.5 % soluble starch solution and 1M NaOH was

dissolved in deionized water. Under constant stirring, the zinc nitrate solution was

added slowly (drop wise for 30 min) to NaOH solution which was maintained at ~

70oC. After 2 h reaction time, the white precipitate deposited at the bottom of the

flask was collected and washed several times with absolute ethanol and distilled

water. ZnO samples were obtained by centrifugation and dehydration of the

precipitate in vacuum at 60–70 °C and finally re-dispersed in de-ionized water by

ultrasonication.

Zn(NO3)2·6H2O + 2NaOH = Zn(OH)2 (gel) + 2NaNO3 + 6H2O (1)

Zn(OH)2(gel)+ 2H2O →Zn2+ + 2OH− + 2H2O→Zn(OH)2−

4 + 2H+ (2)

Zn(OH)2− 4 = ZnO + H2O + 2OH−. (3)

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).


44

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 .

45

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.


46

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


47

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

membrane integrity studies, 2, 7-dichlorofluorescein diacetate (DCFH-DA) assay

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


48

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]


49

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.


50

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).


51

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

triphenylphosphonium 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

(5, 5, 0, 6, 6, 0 -tetrachloro-1, 1, 0, 3, 3, 0 – tetraethyl - benzimidazol

carbocyanine iodide) assay (BD Biosciences, CA, USA) for the flow cytometric

and confocal microscopic estimation of mitochondrial membrane potential (17).


52

JC-1 can exist in two different forms aggregates or monomers, with different

emission spectra.

Figure 2.6. Schematic diagram depiting the mechanism of the ΔΨm dependent

accumulation of JC-1 in to healthy mitochondria. (Adapted from

http://lcbim.epfl.ch /research).

Once the healthy cells are incubated with JC-1, JC-1 enters in to the cyosol

as a monomer. Accumulation of JC-1 into mitochondria is specefically mediated

by the Δψ. The cells with heatly mitochondria with polarized Δψ are rapidly

taken up the JC-1 in to mitochondria and leading to the formation of JC-1

aggregates and shows red fluorescence. The cells with depolarized mitochondria,

JC-1 exist as monomer in the cytosol and exhibit green fluorescence. The

mitochondrial membrane integrity can be studied by measuring the red to green

fluorescence ratio by flow cytometry.

The procedure is as following: After 24 h incubation with different

concentrations of ZnO NCs, the cells (HUVECs and KB) were washed and

stained with 500 µl JC-1 according to the manufacturer´s protocol, at room


53

temperature in the dark. After incubation for 15 minutes, cells were washed twice

with 1X assay buffer and resuspended in the same buffer. The stained cells were

analyzed in FACS. For confocal microscopic analysis HUVEC and KB cells were

cultured on 13 mm coverslips and treated with ZnO NCs. The cells were stained

with JC-1 and fixed using 3.7% paraformaldehyde.

2.4.7 Detection of apoptosis

Figure 2.7. Schematic diagram depiting the mechanism of Annexin V/PI staining

[Adapted from http://sky.lifesci.dundee.ac.uk].

Annexin V–FITC and PI assay (BD Biosciences, CA, USA) was employed

to detect apoptotic and necrotic cells. The assay consists of FITC conjugated

Annexin V antibody, which is a 35 - 36 kDa Ca2+ dependent phospholipid-binding

protein that has a high affinity for the membrane phospholipid phosphatidylserine

(PS) and binds to cells with exposed PS (18). In normal viable cells the PS is

directed into the cytoplasm and upon initiation of programmed cell death, the PS

flips and get expressed on outer leaflet of the plasma membrane. In addition, cells

are incubated with propidium iodide (PI), which differentiates apoptotic and


54

necrotic cells. Using both dyes in combination with two-wavelength flow

cytometry, they identify the apoptotic (AnV+) and necrotic cells (PI+) separately.

Following 12 h and 24 h incubation with different concentration of ZnO

NCs, cells were washed and stained with Annexin V and PI. Typically 1 X 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 minutes,

stained cells were diluted with 400 µl of binding buffer and directly analyzed in

FACS, measuring the fluorescence emission at 530 nm and 575 nm. Apoptotic

cells were visualized by confocal microscopy (He-Ne and Ar laser).

2.4.8 Cell cycle analysis

Figure 2.8. Schematic diagram depiting the different stages in cell cycle[Adapted

from http://www.nature.com].

The influence of ZnO NCs on the cell cycle was analyzed by staining the

DNA with propidium iodide (PI; Invitrogen, CA, USA) using flow cytometry as

previously described (19) with slight modification. Typically 1 X 105 cells after

24 h of exposure to ZnO NCs were trypsinized, washed in PBS, fixed in ice-cold

ethanol (70%) and stored at -20°C. Before flow cytometry analysis, cells were

washed in PBS and stained with PI in RNase (40 µg/mL PI and 100 µg/mL RNase


55

A) and incubated at 37°C for 30 mins, followed by incubation at 4°C until

analysis.

2.4.9 Detection of intracellular and extra cellular Zn2+ ions

The cell permeable Zn2+ selective indicator Newport Green DCF

(Invitrogen, CA, USA) was used to study whether the toxicity of ZnO NCs is

associated with the intracellular release of Zn2+. Newport Green DCF is a cell

permeant acetate ester molecule, Once inside the cells the intracellular estrase

cleave the diacetate moiety and the dye molecule become charged, inhibiting the

escape from the cells and allowing specifically bind to the Zn2+. For Newport

green DCF, excitation was set at 490 nm and emission at 530 nm. Typically 1 X

105 HUVECs and KB cells after 24 h of exposure to nanoparticles were re-

suspended in phenol red free culture medium loaded with 5 µM Newport Green

for 30 mins and intracellular Zn2+ release was studied using FACS. We confirmed

no interference from the fluorescence of ZnO NCs in the measurements as the

undissoluted ZnO give emission only by exciting at 365 nm and not at 490 nm.

Confocal microscopy (He-Ne laser) was used for the direct visualisation of

intracellular ionised zinc.

For detecting ZnO dissolution at different pH conditions that mimic the

extracellular pH, Inductively Coupled Plasma (ICP) technique was employed.

Different concentrations of ZnCl2 (0 - 1 mM) were used to create standard curve.

For dissolution studies, varied concentration of ZnO NCs were incubated at pH 5

and 7.4 over 24 h at 37 °C and the supernatant is collected after centrifugation at

25000 rpm for 30 mins. No additional digestion step is used for detecting the Zn2+

ions that is spontaneously dissoluted due to the effect of pH.

2.4.9.1 Estimation of extracellular pH

The extracellular pH of normal primary cells and cancer cells were

measured by monitoring the pH near to the outer membrane of the cells in a

culture plate using a microelectrode set-up (Thermo Scientific; Orion). The

primary HUVEC and cancer cells (KB) were cultured in 6 well plates and the pH


56

was measured after 24 h. The initial pH recorded was 7.4 which served as the 0th

reading.

2.4.9.2 Statistical analysis

Statistical analyses of the values for all experiments are expressed as mean

± standard deviation of three independent experiments. The data were analyzed

using Student’s t-test (Microsoft Excel, Microscoft Corporation, USA) where

statistical significance was calculated using untreated and ZnO NCs treated

samples. * denotes p < 0.05 compared with control.

2.5 Graphene toxicity analysis

2.5.1 Kidney epithelial cells (Vero) cells

2.5.1.1 Cellular uptake studies using flow cytometry

We have adopted a flow cytometric light scatter based method for the

intracellular uptake of nanoparticles. In this method make use of the forward

scatter (FSC) and side scatter (SSC). Forward scatter (FSC) correlates to the size

of the cells and side scatter (SSC) depends on the inner complexity of the cells.

Typically, the cells were treated with different doses of nanoparticles and

incubated for 24h. After treatment th cells were trypsinized and washed with PBS

and suspended in PBS. The intracellular uptake was further quantified by flow

cytometry using 488 nm laser, and measured the forward and side scattering

intensities.

2.5.1.2 Detection of cell viability (Alamar blue Assay)

Alamar blue (Invitrogen, CA, USA) assay was used to evaluate the cell

viability. When cells reached 80% confluency, they were harvested and 3 × 104

cells/ml were seeded in 24 well plates and incubated for 24 h at 37 °C. The cells

were then treated with different concentrations of p-G and f-G for 24 h at 37 °C

and Alamar Blue assay was performed. Fluorescence was recorded using a

fluorescence microplate reader (Beckman Coulter DTX 880 Multimode Detector,

USA) using 560/590 nm ex/em filter settings.


57

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


58

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.


59

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


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


60

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


61

fluorescence at 530 nm and 575 nm. Apoptotic cells were imaged using confocal


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


62

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.


63

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

(IL-1β), Interleukin-6 (IL-6), Interleukin-10 (IL-10), Tumor Necrosis Factor

(TNF), and Interleukin-12p70 (IL-12p70) were quantified. The isolated PBMCs

were treated with various concentrations of both graphene systems and incubated

for 24 h. PBS and 1 µg mL-1 lipopolysacharide (LPS; Sigma, St.Louis, USA)

served as negative (0% activation) and positive controls (100% activation)

respectively. After incubation the cells were centrifuged at 4000 rpm for 10 min

and the supernatant was collected for the quantification of cytokines using flow

cytometry. The percentage activation was calculated according to the following

equation.

Where Ct, Cn, and Cp are the cytokine concentrations obtained from test, PBS and

LPS respectively.

2.5.3.5 Lymphocyte isolation

10 ml of blood was collected in a Lithium Heparin Vaccutainer. This was

brought to the biosafety cabinet and was transferred aseptically to a 50 ml Falcon

tube. The separation was done by density gradient centrifugation. 10 ml of

Histopaque was added to the blood and was centrifuged at 600g for 20 minutes.

The buffy layer containing the leukocytes was collected and transferred to another

50 ml Falcon tube. This was washed twice by adding 10 ml of RPMI cell culture

medium and centrifuging at 400g for 10 minutes each (this washing reduces the

number of monocytes in the sample, considerably). The supernatant was

discarded at the end of the wash and the pellet was resuspended in 10ml of fresh

RPMI medium.


64

2.5.3.6 Lymphocyte proliferation analysis

Lymphocytes were isolated from anticoagulated peripheral blood by

density gradient centrifugation. The isolated PBMCs were washed twice with

HBSS at 250 g for 10 min. PBS and 100 µg mL-1 PHA-M (Phytohemagglutinin;

Sigma, St.Louis, USA) served as the negative (0 % proliferation) and positive

control (100% proliferation) respectively. After incubation for 72 h, Alamar Blue

assay was used to study the lymphocyte proliferation. Immunosuppression

analysis was studied using lymphocytes cultured in the presence of 100 µg mL-1

PHA-M and different concentrations of both graphene systems for 72 h. After

incubation Alamar Blue assay was performed to evaluate the possibility of the

interferences of both graphene systems in lymphocyte proliferation induced by a

mitogen, PHA-M.

2.5.4 Genotoxicity evaluations of Graphene

2.5.4.1 Comet Assay

We have adopted single cell gel electrophoresis assay to detect the DNA

damages in single cell level. Comet analysis was done according to the protocol

devoloped by Alok Dhawan’s group (22) with slight modification.

2.5.4.2 Materials

Low melting point agarose (LMPA), normal melting agarose (NMA),

phosphate buffered saline-PBS (Ca2+, Mg2+ free), ethylene diamine tetraacetic

acid disodium salt (EDTA), propidium iodide, sodium chloride (NaCl), sodium

hydroxide (NaOH), Triton X-100, trizma base.

Microscope slides (frosted end, 75mm x 25mm), coverslips (24 x 60 mm),

eppendorf tubes, micropipettors and tips, Coplin jars.

2.5.4.3 Preparation of reagents

1. Prepare Ca2+, Mg2+ free PBS 1000 ML (PH 7.4).

2. Prepare 0.75% LMPA and 0.75% NMA in PBS.

3. Prepare lysing solution Lysing solution (2.5 M NaCl, 100 mM EDTA, 10

mM Trizma base). Add all the above ingredients to about 70 mL dH2O

with 0.8 g NaOH and dissolved. NaO is using for dissolving the EDTA.


65

Final solution should be prepared freshly which containg 10% DMSO and

1% Triton and refirgerate for at least 1 hr.

4. Prepare electrophoresis buffer (1X solution: 300 mM NaOH/1 mM

EDTA). For the experiment, 1X Buffer is made freshly before each

electrophoretic analysis. Mix 30 mL NaOH and 5 mL EDTA stock

solutions and make up to 1000mL with chilled dH2O, the final pH of the

electrophoresis buffer has to be >13.

5. Prepare neutralisation buffer (0.4M Tris). 6. Staining solution: Propidium iodide (10 µg/ml).

2.5.4.4 Preparation of base slides The NMA (0.75%) is hot (45°C), the slides are dipped up to one-third the

frosted area and gently removed. The bottom of the slide is wiped to remove

agarose and the slides kept on a flat surface to dry.

2.5.4.5 Prepartion of cells

Following the nanoparticle treatment, the cells were washed with PBS and

detach the cells by trpsinization. The cells wre suspended in cold FBS containg

medium and collect the cell pellet by centrifugation and resuspend the cells in

cold fbs containg medium and adjust the cell density at 4 x 106 cells/ml. Remove

two 10 µl aliquots from each tube and add to separate eppendorf tubes. Kept the

both tubes on ice.

2.5.4.6 Prepartion of microgel slides

Add 75 µl of LMP agarose to the cell suspension, mix by pipetting up and

down a fewt imes then layer the agarose/cell mix on to predipped 0.75% NMP

agarose. Allow the agarose to solidify,then place the slides on ice for ~5mins.

Take the slides off the ice and allow them to retrn to room temparature. Remove

the coverslips then add a further layer of LMP agarose (85 µl) and leave to

solidify as before. Remove the coverslips. Place all the slides in cold lysing

soultion at 4° C for 2 hrs.


66

2.5.4.7 Electrophoresis of microgel slides for comet assay

After the lysis step, remove slides and rinse in elctrophoresis buffere.

Place the slides in a bath of electrophoresis buffer for 15 mins then transfer to the

rig with fresh electrophoresis buffer for a further 25 minutes under cold

conditions. Electrophoresis at 25 V, 300mA, for 20 minutes.

2.5.4.8 Neutralization and staining

After electrophoresis, neutralise the slides with neutralisation buffer for 5

mins and repeat twice. After neutralization place the slides in staing solution

containg 2.5 µg/ml propidium iodide in distilled wated for 20 mins. Rinse the

slides with distilled water to remove th unbounded dye and place coverslip on

each slide. Score 50 cells per slide using the fluorescent microscope.

2.5.4.9 Toxicogenomic analysis using microarray

Gene expression studies were carried out in HUVECs treated with

10µg/ml of both p-G and f-G. Microarray technique was employed to carry out

these studies. Genespring software was used to analyze the results obtained using

affymatrix exon expression human gene chip. The filter probeset was set at 20

percentile and the initial significance level for choosing the genes was set at a very

stringent p<0.001. All three samples i.e. Control, p-G and f-G samples were

compared for differential gene expression. At the end of this, fold change was set

at 1.4 and the biological functions affected were detected by setting p < 0.1. At the

end of this, the significantly affected pathways were obtained at p < 0.05.

2.6 In vivo toxicological analysis

2.6.1 Housing of animals

The animals were bred and maintained at Central Lab Animal Facility,

Amrita institute of Medical Sciences & Research Centre, Kochi and were housed

in groups of 5 in polypropelene cages under identical environmental conditions

(temperature controlled room (22 ± 2◦C) with a photoperiod of 12hr light and 12hr

dark cycle). They were fed with balanced standard mash feed and filtered water ad

lib. The experiments were designed and conducted in strict accordance with the

ethical norms approved by the institutional Animal Ethics Committee guidelines.


67

2.6.2 Injection in mice and sample preparation

Mice were anesthetized by intramuscular injection of Ketamine &

Xylazine in the ratio 4:1. Graphene samples are injected intravenously by lateral

tail vein injection. Controls were given normal saline. The volume of sample was

calculated based on bodyweights of mice so that the final concentration of

graphene injected in each mouse is 20 mg/kg. Undisturbed behavior as well as

response to external stimuli was recorded. Body weight, temperature, food and

water intake were checked periodically. Mice were euthanized by over dose

anesthesia. Blood was collected by cardiac puncture. All the major organs like

brain, heart, lungs, liver, kidney, spleen, intestine, testis and bone marrow were

harvested.

2.6.3 Histopathology

All the tissues are stored in 10 % buffered formalin immediately after

harvesting The tissues are processed in Leica automated tissue processor and wax

blocks are prepare by embedding with paraffin wax (paraffin wax dispenser SLEE

MPS/P1) and paraffin sections of 4 µ thickness are prepared using microtome

(Leica microsystems). The slides containing the paraffin sections are

deparaffinized in xylene for three changes ten minutes each and rehydrated in

descending grades of alcohol i.e. 100%, 95%, 80%, 70% three minutes each and

finally in distilled water for 5 minutes. Then slides are stained with Harris

haematoxylin for 10 minutes followed by rinsing with tap water and dipping in

1% acid alcohol for 2 seconds. Then the slides are placed in 0.2% ammonia water

for bluing. Slides are then stained with eosin for 5 minutes, rinsed in distilled

water and dehydrated in ascending grades of alcohol and cleared in xylene. Cover

slips are placed over slides using DPX mountant and dried overnight in the hood.

Qualitative and quantitative analysis of slides was performed.

2.6.4 RT-PCR studies on mouse blood

2.6 5 RNA isolation Ambion kit

After collection of blood from mice the blood cells are pelleted down by

spinning at 15000 rpm for three minutes. Supernatant is discarded and lysis

solution is added to the pellet and vortexed until the pellet dissolves completely.


68

Then 200 µl of 3M sodium acetate solutions and 1.5 ml of acid phenol +

chloroform solution are added to cell lysate and shaken vigorously for thirty

seconds and stored at room temperature for five minutes. After centrifuging this

again for 10 minutes at 2000 g the aqueous phase containing the RNA is separated

from the organic phase . The aqueous phase is transferred to a fresh tube and

100% ethanol is added to precipitate RNA. The precipitate is separated using a

filter cartridge and eluted in to a fresh collection tube and labeled.

2.6 6 Biochemical analysis of mouse blood

Blood was collected from mice after intravenous injection of Pristine and

functionalized graphene and sent for biochemical analysis to clinical chemistry

lab, Amrita institute of Medical sciences and Research centre, Kochi. Analysis

was done for hepatic and renal function apart from plasma proteins.


69

References:

1. Patra MK, Manoth M, Singh VK, Siddaramana Gowd G, Choudhry VS,

Vadera SR, et al. Synthesis of stable dispersion of ZnO quantum dots in

aqueous medium showing visible emission from bluish green to yellow.

Journal of Luminescence .2009;129(3):320–4.

2. Feldmann C, Jungk H-O. Polyol-Mediated Preparation of Nanoscale Oxide

Particles We thank Jacqueline Merikhi and Gerd Much for carrying out the

scanning electron microscopy (SEM) and the atomic force microscopy

(AFM) investigations, respectively. Angewandte Chemie (International ed.

in English) . 2001;40(2):359–62.

3. Cheng H-M, Hsu H-C, Chen S-L, Wu W-T, Kao C-C, Lin L-J, et al.

Efficient UV photoluminescence from monodispersed secondary ZnO

colloidal spheres synthesized by sol–gel method. Journal of Crystal Growth

. 2005;277(1-4):192–9.

4. Vigneshwaran N, Kumar S, Kathe AA, Varadarajan PV, Prasad V.

Functional finishing of cotton fabrics using zinc oxide–soluble starch

nanocomposites. Nanotechnology . 2006;17(20):5087–95.

5. STOBER W. Controlled growth of monodisperse silica spheres in the

micron size range*1. Journal of Colloid and Interface Science .

1968;26(1):62–9.

6. Liufu S. Investigation of PEG adsorption on the surface of zinc oxide

nanoparticles. Powder Technology . 2004;145(1):20–4.

7. Wu C, Qiao X, Chen J, Wang H, Tan F, Li S. A novel chemical route to

prepare ZnO nanoparticles. Materials Letters . 2006;60(15):1828–32.

8. Subrahmanyam KS, Panchakarla LS, Govindaraj A, Rao CNR. Simple

Method of Preparing Graphene Flakes by an Arc-Discharge Method. The

Journal of Physical Chemistry C . 2009;113(11):4257–9.


70

9. Subrahmanyam K, Vivekchand S, Govindaraj A, Rao C. A study of

graphenes prepared by different methods: characterization, properties and

solubilization. Journal of Materials Chemistry . 2008;18(13):1517–23.

10. Baudin B, Bruneel A, Bosselut N, Vaubourdolle M. A protocol for isolation

and culture of human umbilical vein endothelial cells. Nature Protocols .

2007 Mar 1;2(3):481–5.

11. Mosmann T. Rapid colorimetric assay for cellular growth and survival:

Application to proliferation and cytotoxicity assays. Journal of

Immunological Methods . 1983;65(1-2):55–63.

12. Koh JY, Choi DW. Quantitative determination of glutamate mediated

cortical neuronal injury in cell culture by lactate dehydrogenase efflux

assay. Journal of Neuroscience Methods . 1987;20(1):83–90.

13. Hotulainen P, Llano O, Smirnov S, Tanhuanpää K, Faix J, Rivera C, et al.

Defining mechanisms of actin polymerization and depolymerization during

dendritic spine morphogenesis. The Journal of cell biology .

2009;185(2):323–39.

14. Wang H, Joseph JA. Quantifying cellular oxidative stress by

dichlorofluorescein assay using microplate reader11Mention of a trade

name, proprietary product, or specific equipment does not constitute a

guarantee by the United States Department of Agriculture and does not

imp. Free Radical Biology and Medicine . 1999;27(5-6):612–6.

15. Crow JP. Dichlorodihydrofluorescein and dihydrorhodamine 123 are

sensitive indicators of peroxynitrite in vitro: implications for intracellular

measurement of reactive nitrogen and oxygen species. Nitric oxide biology

and chemistry official journal of the Nitric Oxide Society . 1997;1(2):145–

57.

16. Johnson I, Spence MTZ. Molecular Probes Handbook, A Guide to

Fluorescent Probes and Labeling Technologies . Life Technologies; 2010.


71

17. Guthrie HD, Welch GR. Determination of high mitochondrial membrane

potential in spermatozoa loaded with the mitochondrial probe 5,5’,6,6'-

tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1)

by using fluorescence-activated flow cytometry. Methods in molecular

biology (Clifton, N.J.) . 2008;477:89–97.

18. van Engeland M, Nieland LJ, Ramaekers FC, Schutte B, Reutelingsperger

CP. Annexin V-affinity assay: a review on an apoptosis detection system

based on phosphatidylserine exposure. Cytometry . 1998;31(1):1–9.

19. Krishan A. Rapid flow cytofluorometric analysis of mammalian cell cycle

by propidium iodide staining. The Journal of cell biology . 1975;66(1):188–

93.

20. Chandran P, Sasidharan A, Ashokan A, Menon D, Nair S, Koyakutty M.

Highly biocompatible TiO�:Gd3+ nano-contrast agent with enhanced

longitudinal relaxivity for targeted cancer imaging. Nanoscale .

2011;3(10):4150–61.

21. Ashokan A, Chandran P, Sadanandan AR, Koduri CK, Retnakumari AP,

Menon D, et al. Development and haematotoxicological evaluation of

doped hydroxyapatite based multimodal nanocontrast agent for near-

infrared, magnetic resonance and X-ray contrast imaging. Nanotoxicology .

2011.

22. Dhawan A, Bajpayee M, Parmar D, Singh NP, Lai HC, Hartmann A, et al.

Comet Assay in Toxicology . Cambridge: Royal Society of Chemistry;

2009.

chapter 2 materials and methods -...

Documents