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CHAPTER 4

IN VITRO CYTOTOXICITY ASSAY ON GOLD

NANOPARTICLES WITH DIFFERENT

STABILIZING AGENT

4.1 INTRODUCTION

The nanoparticles have been shown to adhere to cell membranes

(Ghitescu and Fixman 1984) and be ingested by cells (Parak et al 2002). The

breaching of the cell membrane and the intracellular storage may have a

negative effect on the cells regardless of the toxicity of the particles and their

subsequent functionality. In this study, gold nanoparticles stabilized with

citrate, starch and gum arabic are used for cytotoxicity studies. The assays

used are based on different modes of detection like (3-[4,5-dimethylthiazol-2-

yl] -2,5-diphenyltetrazolium bromide reduction assay) (MTT), the neutral red

cellular uptake assay and lactate dehydrogenase (LDH) release assay. We

found that the gold nanoparticles stabilized with citrate, starch and gum arabic

are viable to different cells through different assays with different

concentrations and time of exposure of gold nanoparticles. It was found that

the viability depends on the stabilizing agent and the types of cytotoxicity

assay used.

4.2 MATERIALS

All Chemicals were obtained from Sigma-Aldrich and used as

received. Deionized water was used in all experiments. The prostate cancer

cell lines (PC-3) and breast cancer cell lines (MC-7) were obtained from the

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American Type Culture Collection (ATCC) through the Department of

Microbology, PSG Institute of Medical Sciences and Research, Coimbatore,

Tamilnadu, India. Gold nanoparticles stabilized with citrate, starch and gum

arabic were prepared and characterization studies were performed before

inducting into cytotoxicity studies as referred in chapter 2.

4.3 EXPERIMENTAL METHODS

4.3.1 MTT Assay

4.3.1.1 Gold Nanoparticles with Different Concentrations

Cytotoxicity evaluation of citrate (CAuNPs), starch (SAuNPs) and

gum Arabic (GAuNPs) stabilized gold nanoparticles were performed using

MTT assay as described by Mosman (1983). About 1×105 mL

-1 cells (MCF-7

and PC-3) in their exponential growth phase were seeded in a flat-bottomed

96-well plate and were incubated for 24 hr at 370 C in 5% CO2 incubator.

Series of dilution (30, 60, 90, 120 and 150 g/mL) of gold nanoparticles

stabilized with citrate, starch and gum arabic in medium were separately

added to the plate. After 24 hr of incubation, 10 L of MTT reagent was

added to each well and was further incubated for 4 hr. Formazan crystals

formed after 4 hr in each well were dissolved in 150 l of detergent and the

plates were read immediately in a microplate reader (BIO-RAD microplate

reader-550) at 570nm. Untreated PC-3 and MCF-7 cells as well as the cell

treated with (30, 60, 90, 120 and 150 g/mL) concentration of AuNPs for

24 hr were subjected to the MTT assay for cell viability determination.

4.3.1.2 Time of exposure assay

Cytotoxicity was also assessed using MTT assay at different time

period. About 1×105 mL

-1 cells lines in their exponential growth phase were

seeded in a flat-bottomed 96-well polystyrene coated plate. Gold

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nanoparticles stabilized with citrate, starch and gum arabic with concentration

50 g/mL were diluted in the growth medium and added to the plate.

Incubations carried out for various times (12, 24, 36, 48, 60 and 72 hr) at 37oC

in an atmosphere of 5% CO2 in air. At the end of incubation 10 µL of MTT

reagent was added to each well and further incubated for 4 hr. Formazan

crystals formed after 4 hr in each well were dissolved in 150 µL of detergent

and the plates were read immediately in a micro plate reader

(BIO-RAD micro plate reader-550) at 570nm. Untreated cell lines as well as

the cell treated with gold nanoparticles at different time were subjected to the

MTT assay for cell viability determination.

4.3.2 LDH Assay

4.3.2.1 Gold nanoparticles with different concentrations

Cytotoxicity was assessed using a LDH cytotoxicity detection kit

(Roche applied sciences). This assay measures the release of cytoplasm

enzyme lactate dehydrogenase (LDH) by damaged cells. Cells cultured in 96

plates were treated with increasing concentrations of gold nanoparticles (30,

60, 90, 120 and 150 g/mL) stabilized with citrate, starch and gum arabic.

After 48 hr of treatment, culture supernatant was collected and incubated with

reaction mixture. The LDH catalyzed conversion results in the reduction of

the tetrazolium salt to formazan, that can be read absorbance 490nm. These

data are measured in LDH activity as a percentage of the control. Any

significant increase in LDH levels would indicate cellular disruption or death

due to the treatment.

4.3.2.2 Time of exposure assay

Cytotoxicity was assessed using an LDH detection kit (Roche

Applied Science). This assay measures the release of cytoplasm enzyme

lactate dehydrogenase (LDH) by damaged cells. Cells cultured in 96 plates

84

were treated with 50 g/mL concentrations of gold nanoparticles stabilized

with citrate, starch and gum arabic. Incubations were carried out for various

times (12, 24, 36, 48, 60 and 72 hr). After the treatment, culture supernatant

was collected and incubated with reaction mixture. The LDH catalyzed

conversion results in the reduction of the tetrazolium salt to formazan, which

can be read at the absorbance of 490nm. Any significant increase in LDH

levels would indicate cellular disruption or cell death due to the treatment.

4.3.3 Neutral Red Cell Uptake Assay

4.3.3.1 Gold nanoparticles with different concentrations

PC-3 and MC-7 cells were seeded at a population of 1.5×104 cells

per well in a 96 well plate. The cells were incubated for 24 hr and reached

80-90% confluence. The spent media was removed and the cells were washed

with PBS (0.01 M phosphate buffer, 0.0027 M KCl and 0.137 M NaCl) and

1 L fresh media was added. Gold nanoparticles stabilized with citrate, starch

and gum arabic with different concentrations (30, 60, 90, 120 and 150 g/mL)

were mixed with fresh media. The plates were then incubated for 24 hr at 370

in a humidified incubator with a 5% CO2 environment. After the incubation,

the cells were washed twice with PBS (0.01 M Phosphate buffer 0.0027M

KCl and 0.137M NaCl) thereafter 50 L of dye release agent (a solution of

1% acetic acid: 50% ethanol) added to each well and the plates were

incubated for further 10 minutes. The plate was placed on a shaker (Vortex

Genie) for 30 minutes after that the optical density at 540nm was determined

on a multiwall spectrophotometer.

4.3.3.2 Time of exposure assay

The cells were seeded at a population of 1.5×104 cells per well in a

96 well plate. The cells were incubated for 24 hr and reached 80-90%

confluence. The spent media were removed and the cells were washed with

85

PBS (0.01 M phosphate buffer, 0.0027 M KCl and 0.137 M NaCl) and 1 L

fresh media was added. The gold nanoparticles stabilized with citrate, starch

and gum arabic with 50 g/mL concentration is taken and mixed with fresh

media. The plates were then incubated for various times (12, 24, 36, 48, 60

and 72 hr) at 370 C in a humidifier incubator with 5% CO2 environment. After

the incubation, the cells were washed twice with PBS (0.01 M phosphate

buffer, 0.0027 M KCl and 0.137 M NaCl) thereafter 50 L of dye release

agent (A solution of 1% acetic acid:50% ethanol) was added to each well and

the plates were incubated for further 10 minutes. The plate was placed on a

shaker (Vortex Genie) for 30 minutes after that the optical density was

determined at 540nm on a multiwall spectrophotometer.

4.4 RESULTS

The PC-3 cell lines in the exponential growth phase were exposed

to different concentrations of citrate, starch and gum arabic stabilized gold

nanoparticles. The cell viability was measured as described in the

experimental section. Each result represents the mean viability + standard

deviation (SD) of three independent experiments and each of these was

performed in triplicate. Cell viability was calculated as the percentage of the

viable cells compared to the untreated controls.

In MTT assay, cells that are viable after 24 hr exposed to the

sample were capable of metabolizing a dye (3-(4,-dimethylthiozol-2-yl)-2,5-

disphenyl tetrazolium bromide) efficiently and the purple coloured precipitate

which is dissolved in a detergent was analyzed spectro photometrically. After

24 hr of post treatment of PC-3 cell lines showed excellent viability even up

to the concentration of 150 g of citrate, starch and gum arabic capped gold

nanoparticles, Figure 4.1(a). These results clearly demonstrate that the

stabilizing agents provide non-toxic coating on AuNPs and corroborate the

results of the internalization studies discussed above. But the data shows that

there is a marginal cytotoxic effect of citrate stabilized gold nanoparticles

w

N

an

st

ex

F

F

with differe

Neutral red

nd gum ar

tarch and

xperienced

Figure 4.1(b

Figure 4.1

ent cell lin

cell assay

rabic. In a

gum arabi

d in the ca

b) and Figu

The PC

exposed

starch (

nanopar

assay (b

nes used. A

for the go

addition, th

ic stabilize

ase of citra

ure 4.1(c).

C-3 cell lin

d to differ

(SAuNps)

rticles. Th

b) LDH ass

Also we h

old nanopar

he cell viab

ed gold na

ate stabiliz

nes in the

rent conc

and gum

he cell via

say and (c

had a simil

rticles stab

bility is ap

anoparticle

zed gold n

e exponent

centrations

Arabic (G

bility was

c) Neutral

lar result w

bilized with

ppreciable

es, but fee

anoparticle

tial growt

s of citra

GAuNps) s

s measured

Red Cell u

with LDH

h citrate, st

in the cas

eble toxici

es as show

th phase w

ate (CAuN

stabilized

d by (a) M

uptake ass

86

H and

tarch

se of

ty is

wn in

were

Nps),

gold

MTT

say

87

We have investigated the cell viability using the cell line MCF-7.

The cell lines in the exponential growth phase were exposed to different

concentrations of gold nanoparticles. The cell viability was measured by MTT

assay, LDH assay and Neutral Red assay and the results are shown in

Figure 4.2 (a), Figure 4.2 (b) and Figure 4.2 (c) respectively. In the cell

viability assay experiments like MTT, LDH and Neutral red cell, the

nanoparticles stabilized with starch and gum arabic were showing more viable

to cell lines. The citrate stabilized gold nanoparticles exerts little toxic effect

on the cell lines used.

Cytotoxicity was also assessed using MTT assay, LDH assay and

Neutral red cell assay at different time period (12, 24, 36, 48, 60 and 72 hr)

along with PC-3 and MCF-3. The PC-3 cell line in the exponential growth

phase was exposed to 50 g/mL concentration of gold nanoparticles for 12h,

24h, 36h, 48h, 60h and 72h. The cell viability was measured by MTT assay,

LDH assay and Neutral Red assay as described in the experimental section.

Cell viability was calculated as the percentage of the viable cells compared to

the untreated controls. The result shows, after 12 hour exposure to gold

nanoparticles, the viability started to decline. Figure 4.3(a), Figure 4.3 (b) and

Figure 4.3 (c) shows the result of MTT, LDH and Neutral Red cell assays

respectively. It was observed that the cell viability was marginally lower in

citrate stabilized gold nanoparticles. The gold nanoparticles stabilized with

starch and gum arabic were shows more than 90% of viability even after 72 hr

of exposure.

FFigure 4.2 The MC

exposed

starch (

nanopar

assay (b

CF-7 cell l

d to differ

(SAuNps)

rticles. Th

b) LDH ass

lines in th

rent conc

and gum

he cell via

say and (c

he exponen

centrations

Arabic (G

bility was

c) Neutral

ntial grow

s of citra

GAuNps) s

s measured

Red assay

wth phase w

ate (CAuN

stabilized

d by (a) M

y

88

were

Nps),

gold

MTT

F

to

an

Figure 4.3

T

o 50 g/mL

nd 72h. Th

The PC

exposed

Arabic

36h, 48h

(a) MTT

The MCF-7

L concentr

he cell via

C-3 cell lin

d to citrat

(GAuNps)

h, 60h an

T assay (b)

7 cell line i

ration of go

ability was

nes in the

te (CAuN

) stabilized

d 72h. Th

) LDH ass

in the expo

old nanopa

measured

e exponent

Nps), starc

d gold nan

he cell via

say and (c)

onential gr

articles for

d by (a) M

tial growt

ch (SAuN

noparticles

bility was

) Neutral R

rowth phas

r 12h, 24h,

TT assay

th phase w

Nps) and

s for 12h,

s measured

Red assay

se was exp

, 36h, 48h,

(b) LDH a

89

were

gum

24h,

d by

.

posed

, 60h

assay

90

and (c) Neutral Red Assay as described in the experimental section. Figure

4.4(a), Figure 4.4(b) and Figure 4.4(c) shows the results of MTT, LDH and

Neutral Red cell assays using MCF-7 cell line. We found that the cell viability

was slightly lower in the case of citrate stabilized gold nanoparticles and the

gold nanoparticles stabilized with starch and gum arabic were three to four

fold viable to cell line used.

Table 4.1 Average size, plasmon wavelength, plasmon width and IC50

of gold nanoparticles stabilized with citrate (CAuNps),

starch (SAuNps) and gum Arabic (GAuNps)

Sample name Average size

(nm)

Plasmon

wavelength

( max) nm

Plasmon

width ( )

nm

IC50values

(µg/mL)

CAuNp 21±1.4 523 90 63

SAuNp 21±1.5 525 90 220

GAuNp 20±2.3 528 85 239

We found that the gold nanoparticles stabilized with citrate, starch,

and gum arabic are viable to different cells through different assays with

different concentrations and time of exposure of gold nanoparticles. The

viability of the cell lines are depending on the stabilizing agent and the types

of cytotoxicity assay used. The cell viability test shows distinguishable

cytotoxic effect for citrate stabilized gold nanoparticles at a higher

concentration and this is may be the surface coating is acidic in nature

compared to starch and gum arabic. The IC50 values for citrate, starch and

gum arabic stabilized gold nanoparticles were 63 g/mL, 220 g/mL and

239 g/mL depending on the particle stabilizer used. The possibility of size

effect is ignored since we have used the same size of gold nanoparticles.

Interestingly the gold nanoparticles stabilized with starch and gum arabic are

three-to-four fold viable than citrate at higher concentrations and in long time

ex

g

el

T

F

xposure. T

old nanop

lucidation

Table 4.1.

Figure.4.4

The average

particles st

of stabili

The MC

exposed

Arabic

36h,48h

(a) MTT

e size, plas

tabilized w

izer based

CF-7 cell l

d to citrat

(GAuNps)

h, 60h and

T assay (b)

smon wave

with citrate

d cytotoxic

line in the

te (CAuN

) stabilized

d 72h. Th

) LDH ass

elength, pl

e, starch a

city studie

e exponen

Nps), starc

d gold nan

e cell viab

say and (c)

lasmon wid

and gum

es were s

ntial growt

ch (SAuN

noparticles

bility was

) Neutral R

dth and IC

arabic for

summarize

th phase w

Nps) and

s for 12h,

measured

Red assay

91

C50 of

r the

ed in

were

gum

24h,

d by

92

4.5 DISCUSSION

Comparison of cytotoxicity studies based on stabilizing agents

revealed that citrate produced little toxic as compared to starch and gum

arabic stabilized gold nanoparticles in different concentration and at different

time. Even though the citrate has a feeble cytotoxicity, it was found to be

viable because, it has more than 80% cell viability. Cell viability was also

determined by a LDH release assay which employed to measure the

cytotoxicity of the gold nanoparticles at different concentrations and time.

The absorbance of the produced formazan at 490nm is proportional

to the number of damaged or dying cells. The cytotoxicity of various cell lines

exposed to increasing concentrations of nanoparticles stabilized with three

different stabilizing agents were analyzed for 24 hr. At each concentration,

there were no significant cytotoxicity effect is produced. The cell viability

results indicate that gold nanoparticles are non-toxic to the array of cells

tested. The incorporation of surface functionalities via citrate, starch and gum

arabic renders these nanoparticles highly biocompatible. Noble metal

particles, such as gold are generally non-toxic due to their inert nature. The

cell survival at different concentrations of gold nanoparticles stabilized with

different capping agent shows that there is a small variation in cell viability

with the increase in concentration and at the long time exposure of

nanoparticles with cell lines.

Cell based cytotoxic assay with different concentrations of gold

nanoparticles shows very small variation among citrate, starch and gum

arabic. The gold nanoparticles used were having the same size, it differs only

by its stabilizing agent. In comparison the citrate stabilized gold nanoparticles

show less viability than starch and gum arabic. This is may be the citrate is

acidic in nature because the size dependent cytotoxicity is ruled out since in

all the three cases, the particles sizes were same.

93

These results are consistent with previous investigations performed

with dermal fibroblasts (Pernodet et al 2006) that demonstrated the gold and

citrate nanoparticles impaired the proliferation of dermal fibroblasts and

induced an abnormal formation of actin filaments, causing a reduced cellular

morphology. On the contrary (Connor et al 2005) reported that the gold and

citrate nanoparticles impaired the proliferation of dermal fibroblasts and

induced an abnormal formation of actin filaments, causing therefore a reduced

cellular motility and influencing the cell morphology. In addition Connor

(2005) reported that the citrate and biotinylated 18nm gold nanoparticles did

not induce any toxicity in leukemia cells.

4.6 CONCLUSION

In conclusion, we have found that the nanoparticles stabilized with

citrate, starch and gum arabic are viable to different cells through different

assays used. It was found that, the viability of the treated cell lines were

depending on the stabilizing agent. The cell viability studies show that a

feeble cytotoxic effect of citrate stabilized gold nanoparticles at higher

concentrations and long time exposure. It was concluded that the surface

coating is acidic in nature compared to starch and gum arabic. The IC50 values

for citrate, starch and gum arabic stabilized gold nanoparticles were 63

g/mL, 220 g/mL and 239 g/mL respectively depending on the particle

stabilizer used. The possibility of size dependent cytotoxicity is ignored since

we have used the same size of gold nanoparticles. Interestingly the gold

nanoparticles stabilized with starch and gum arabic were three-to-four fold

viable than citrate at higher concentrations and in long time exposure.