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Size-dependent in vitro cytotoxicityassay of gold nanoparticlesS. Vijayakumar a & S. Ganesan ba Department of Physics, Sri Ramakrishna Institute of Technology,Tamilnadu, Indiab Department of Physics, Government College of Technology,Tamilnadu, IndiaAccepted author version posted online: 29 Jan 2013.Version ofrecord first published: 22 Feb 2013.

To cite this article: S. Vijayakumar & S. Ganesan (2013): Size-dependent in vitro cytotoxicity assayof gold nanoparticles, Toxicological & Environmental Chemistry, 95:2, 277-287

To link to this article: http://dx.doi.org/10.1080/02772248.2013.770858

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Size-dependent in vitro cytotoxicity assay of gold nanoparticles

S. Vijayakumara� and S. Ganesanb

aDepartment of Physics, Sri Ramakrishna Institute of Technology, Tamilnadu, India;bDepartment of Physics, Government College of Technology, Tamilnadu, India

(Received 3 May 2012; final version received 24 January 2013)

Gold nanoparticles (AuNps) may serve as a promising model to address the size-dependent biological response of cell lines. Their size can be controlled with greatprecision during chemical synthesis. AuNps have potential applications in drugdelivery, cancer diagnosis, and therapy, in the food industry, and for environmentalremediation. However, some of the recent literature contains conflicting data regardingthe cytotoxicity of gold nanoparticles. Against this background, a systematic study ofwater soluble gold nanoparticles stabilized by citrate ranging in size from 3 nm to45 nm were synthesized. The cytotoxicity of these particles were tested by employingthe (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction assay),the neutral red cellular uptake assay, and lactate dehydrogenase release assay.Noticeable differences in the cytotoxic effects depending on the assay, and thenanoparticle size have been found. Citrate stabilized gold nanoparticles with sizes of3 nm, 8 nm, and 30 nm were more sensitive to the cell lines and caused gradual celldeath within 24 h at higher concentrations. This results in IC50 values rangingfrom 57 to 78mgmL�1 depending on the particle size, and cell line combinations. Incontrast, AuNps with diameters of 5 nm, 6 nm, 10 nm, 17 nm, and 45 nm were nontoxicup to three to four fold higher concentrations, and at long-term exposure.

Keywords: gold nanoparticles; citrate; MTT; LDH; cytotoxicity

Introduction

Over the past few years the use of nanoparticles has brought about the new era of nano-

medicine altering the foundations of disease diagnosis, treatment, and prevention because

of their peculiar physical, and chemical properties. There is a wide array of fascinating

nanoparticulate technologies capable of targeting different cells, and extracellular ele-

ments for drug delivery, genetic materials, and diagnostic agents specific to these loca-

tions (Brigger, Dubernet, and Couvreur 2002; Paciotti et al. 2004; Jain 2005; Moghimi,

Hunter, and Murray 2005; Arvizo et al. 2011; Cuenca et al. 2006; Kam, Liu, and Dai

2006; Zhang et al. 2006; Bhattacharya et al. 2007).

Gold nanoparticles (AuNps) may serve as a promising model to address size-

dependent toxicity, since gold is extraordinarily biocompatible. Recently, elevated toxicity

of nanoparticles due to their physical dimensions has been recognized (Born and

Muller-Schulte 2006). AuNps can enter cells efficiently and most studies show that they are

almost harmless to cultured cells (Connor et al. 2005; Hauck, Ghazani, and Chan 2008;

*Corresponding author. E-mail: vijaysk.research@gmail.com

� 2013 Taylor & Francis

Toxicological & Environmental Chemistry, 2013

Vol. 95, No. 2, 277–287, http://dx.doi.org/10.1080/02772248.2013.770858

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Paciotti et al. 2004). Amine and thiol groups bind strongly to AuNps enabling their surface

modification with amino acids, and proteins for biomedical applications (Dani et al. 2008;

Shukla et al. 2005; Xu and Han 2004).

In vitro studies have proved the potential of AuNps for targeting, imaging, and thera-

py of breast cancer cells (Li et al. 2009), for enhancing the radiation sensitivity of prostate

cancer cells (Zhang et al. 2008), and for noninvasive ablation of rat hepatoma cells

(Cardinal et al. 2008). Many non-toxic bulk materials become poisonous when their size

is reduced to the nanoscale. The toxicity of AuNps has been investigated at the cellular

level showing that they enter cells in a size, and shape dependent manner (Chithrani,

Ghazani, and Chan 2006; Pan et al. 2007). Most studies indicate that AuNps are harmless

to cells (Hauck, Ghazani, and Chan 2008; Dani et al. 2008, Shukla et al. 2005; Chithrani,

Ghazani, and Chan 2006).

There are studies suggesting that nanoparticles may elicit adverse health effects

(Goodman et al. 2004) but fundamental cause–effect relationships are ill defined. Thus,

investigations to understand the molecular basics of the interaction of nanoparticles with

biological systems such as living cell is a most urgent areas of collaborative research in

material science and biology (Pan et al. 2007). Therefore, a systematic cytotoxicity study

of water soluble AuNps ranging in size from 3nm to 45nm and stabilized by citrate was

performed.

Materials and methods

All chemicals were obtained from Sigma-Aldrich (Coimbatore, India) and used as re-

ceived. Double distilled water was used in all experiments. The human prostate cancer

cell line PC-3 and human breast cancer cell line MCF-7 were obtained from the American

Type Culture Collection (ATCC) through the Department of Microbiology, PSG Institute

of Medical Sciences and Research, Coimbatore, India. The Chinese ovary hamster cell

line (CHO22) was obtained from R&D Bio Industries (Tamilnadu, India).

Preparation of gold nanoparticles

Gold nanoparticles of diameters from 3 nm to 45 nm were synthesized as reported earlier

(Brown, Walter, and Natan 2000; Liu et al. 2003). The seed colloids were prepared by

adding 1mL of 0.25mmol L�1 HAuCl4 to 90mL H2O and stirring for 1min at 25�C.Two mL of 38.8mmol L�1 sodium citrate was added, the solution was stirred for another

1min, followed by the addition of 0.6mL freshly prepared 0.1mol L�1 NaBH4 in

38.8mmol L�1 sodium citrate. AuNps of diameters ranging from 3 nm to 45 nm were pre-

pared by using different volumes of seed colloid suspension. The solution was stirred for

an additional 5–10min.

Characterization

AuNps capped with citrate were characterized by UV–visible absorption spectroscopy

using a UV spectrophotometer (model 1700, Shimadzu, Kyoto, Japan), (Figure 1). Trans-

mission electron microscopic (TEM) images were taken with an electron microscope

(CM200, Phillips, Eindhoven, the Netherlands) with an operating voltage range of

20–200 kV and at 2.4 A resolution. TEM images of different particle sizes are shown in

Figure 2, and size histograms are shown in Figure 3.

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Cytotoxicity assay

MTT assay

Cytotoxicity evaluation of citrate stabilized AuNps was performed using the MTT reduc-

tion assay as described by Mosmann (1983). About 1�105mL�1 cells per well (MCF-7

and PC-3) in their exponential growth phase were seeded into a flat-bottom 96 well plate

and were incubated for 24 h at 37�C in a 5% CO2 incubator. A dilution series of (10, 40,

70, 100, and 130 mgmL�1) of AuNps in the medium was added to the plate. After 24 h of

incubation, 10mL of MTT reagent was added to each well and was incubated for four

hours. Formazan crystals formed after four hours in each well was dissolved in 150mL of

detergent and the plates were read immediately in a microplate reader (microplate reader-

550, Bio-Rad, Gurgaon, Haryana, India) at 570 nm. Untreated PC-3 and MCF-7 cells as

well as the cells treated with different concentrations (10, 40, 70, 100, and 130mgmL�1)

of AuNps for 24 h were subjected to the MTT reduction assay for cell viability

determination.

LDH assay

Cytotoxicity was assessed using an LDH cytotoxicity detection kit (Roche Applied Sci-

ence, Basel, Switzerland). This assay measures the release of the cytoplasmic (LDH)

damaged cells. Cells cultured in 96 well plates were treated with increasing concentra-

tions of AuNps (10, 40, 70, 100 and 130mgmL�1). After 48 h of treatment, culture super-

natant was collected and incubated. The LDH catalyzed conversion results in the

Figure 1. Optical absorption spectra of AuNps synthesized at different citrate-to-gold ratios.

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Figure 2. TEM micrographs of citrate capped AuNps (A) 3 nm, (B) 5 nm, (C) 6 nm, (D) 8 nm,(E) 10 nm, (F) 17 nm, (G) 30 nm, and (H) 45 nm.

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Figure 3. Size distribution analysis of the citrate capped AuNps.

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reduction of 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) salt

to formazan, which can be read at the absorbance of 490 nm. Any significant increase

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

Neutral red uptake cytotoxicity assay

CHO22 cells were seeded in a population of 1.5�104 cells per well in a 96 well plate.

The cells were incubated for 24 h and reached 80–90% confluence. The spent medium

was removed and the cells were washed with PBS (0.01mol L�1 phosphate buffer,

0.0027mol L�1 KCl, and 0.137mol L�1 NaCl) and 1mL fresh medium was added. The

media were then replaced with AuNps of different concentrations (10, 40, 70, 100, and

130mgmL�1) and mixed with fresh medium. The plates were then incubated for 24 h at

37�C in a humidified incubator with 5% CO2 environment. Following the incubation

period, the cells were washed twice with PBS (0.01mol L�1 phosphate buffer,

0.0027mol L�1 KCl, and 0.137mol L�1 NaCl) and 100mL serum free medium containing

neutral red (100mgmL�1) was added to each well and incubated for 2–3 h.

After the incubation, the cells were washed twice with PBS (0.01mol L�1 phosphate

buffer, 0.0027mol L�1 KCl, and 0.137mol L�1 NaCl) thereafter 50mL of dye release

agent (a solution of 1% acetic acid in 50% ethanol) was added to each well and the plates

were incubated for ten minutes. The plate was placed on a shaker (Vortex Genie, Scientif-

ic Industries, Inc., New York, USA) for 30min after which the optical density was deter-

mined at 540 nm on a multiwall spectrophotometer (SCINCO, Seoul, Korea).

Results and discussion

To examine the cytotoxicity of AuNps, about 1�105mL�1 cells (MCF-7 and PC-3) in

their exponential growth phase were incubated with increasing amounts of AuNps for

24 h and the cell viability expressed as percentage of the untreated control (100% cell

viability) was investigated by MTT assay. Figures 4(A) and 5(A) shows that the AuNps

with sizes 5 nm, 6 nm, 10 nm, 17 nm, and 45 nm did not have any effect on the viability of

the MCF-7 and, PC-3 cell lines, while 3 nm, 8 nm, and 30 nm which present a mild toxici-

ty above 70mgm/L�1 and also in long term exposure.

Interestingly by the MTT assay gold induced cytotoxicity in 5 nm, 6 nm, 10 nm,

17 nm, and 45 nm could not be detected. To further investigate the possible cytotoxicity

induced by AuNps on PC-3 and MCF-7, the amount of LDH released was analyzed in

this cellular toxicity study. Figures 4(B) and 5(B) clearly shows that after 24 h exposure

to AuNps, a mild LDH release in the PC-3 and MCF-7 was observed. In addition up to

72 h exposure to AuNps in a dose dependent manner, a release of LDH in the supernatant

was observed and the amount was significantly higher in 3 nm, 8 nm, and 30 nm AuNps.

We observed a comparable LDH release after 12 h treatment with AuNps for sizes 3 nm,

8 nm, and 30 nm, and there is no release of LDH even after 24 h exposure of AuNps with

sizes 5 nm, 6 nm, 8 nm, 10 nm, 17 nm, and 45 nm.

The mammalian Chinese hamster ovary CHO22 cell line was also used in the elucida-

tion of cytotoxicity effects of the AuNps by neutral red uptake assay. This cell line has

been termed as the mammalian equivalent of the model bacterium E. coli (Puck and Kao

1968). For the elucidation of the cytotoxicity of the AuNps, the CHO22 cells were treated

with the nanoparticles for 24 h. The spectrophotometric measurements were done for the

neutral red dye uptake and release. The viability assay data for the comparison is pre-

sented in Figures 4(c) and 5(C) in which 3 nm, 8 nm, and 30 nm AuNps exert signs of

toxicity.

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Figure 4. Cell lines in the exponential growth phase were exposed to different concentrations ofgold nanoparticles. Cell viability was determined by the MTT (A), LDH (B), and neutral red (C) as-say as described in the experimental section. Each result represents the mean viability � standarddeviation (SD) of three independent experiments and each of these was performed in triplicate. Cellviability was calculated as the percentage of viable cells compared to untreated controls.

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The results are consistent with previous investigations performed with dermal fibro-

blasts (Pernodet et al. 2006) and leukemic cells (Shukla et al. 2005). Perdodet et al (2006)

demonstrated that citrate-capped AuNps impaired the proliferation of dermal fibroblasts

and induced an abnormal formation of actin filaments, causing the reduced cellular

Figure 5. Cell lines in the exponential growth phase were exposed to different sizes of gold nano-particles for 24 h, 48 h, and 72 h. Cell viability was measured by the MTT (A), LDH (B), and neutralred (C) assay 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 tripli-cate. Cell viability was calculated as percentage of viable cells compared to the untreated controls.

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motility and influencing the cell morphology. On the contrary, Connor et al. (2005) and

Shukla et al. (2005) reported that citrated and biotinylated 18 nm AuNps did not induce

toxicity in leukemic cells (cell line K562), whereas smaller particles were more toxic.

AuNps have been found to be almost nontoxic to cell culture compare to gold nanorods

(Johnston et al. 2010; Cui et al. 2012; Hao et al. 2012; Jiang, Wang, and Chen 2011;

Qiu et al. 2010; Schaeublin et al. 2012). This is because the cytotoxicity depends on the

particle size, shape, surface modification and the mechanisms of cellular uptake

(Nikoobakht and El-Sayed 2003; Johnson et al. 2002; Jana, Gearheart, and Murphy

2001).

In addition, AuNps did not exert any toxic effect in the human gastrointestinal cancer

cells Panc-1 and HepB3 (Gannon et al. 2008) nor in HeLa cells (Shukla et al. 2005).

Hauck et al. (2008) have demonstrated that the use of different surface coatings does not

influence the toxicity induced by AuNps in HeLa cells and suggested that the lack of

toxicity was because AuNps were stored intracellularly in membrane bound vesicles, and

therefore particles could not directly interfere with the nuclei or with other cytoplasmic

organelles and induce toxic events.

Nevertheless, there is evidence that the size of the AuNps induces in vitro cytotoxicity

in HeLa cells. In fact, Pan et al. (2007) and Hauck et al. (2008) have shown that the size,

not the particle chemistry, is responsible for determining the toxicity of the gold

nanoparticles. It was shown that AuNps of different sizes had a different toxic effect. In

addition, they demonstrated that using different particle stabilizers, the cytotoxicity

observed was almost indistinguishable, leading the authors to conclude that it is the size

of the particles to play a pivotal role in inducing in vitro cytotoxicity (Hauck, Ghazani,

and Chan 2008).

Conclusion

In conclusion, we found that the AuNps with different sizes stabilized with citrate were

viable to different cell lines through different assays. The cell viability of the treated cells

with AuNps depending on the particle size. The cell viability test shows distinguishable

cytotoxic effect for AuNps of sizes 3 nm, 8 nm, and 30 nm but the nanoparticles 5 nm,

6 nm, 10 nm, 17 nm, and 45 nm are three to four fold viable to cell lines even at higher

concentrations and long term exposure. The viability data lead to the conclusion that

AuNps exert only a mild toxicity or in some cases no toxicity at all in the cell lines MCF-

7, PC-3, and CHO22 cell lines. The toxicity due to their nanometer dimensions must be a

major concern since AuNps have been widely used in biomedical applications.

Table 1. Summary of results in the citrate-capped AuNps investigation.

Sample code SPR absorption Average particle size IC50 values (mgmL�1)

C1 516 nm 3 nm 65 ± 5C2 518 nm 5 nm 182 ± 4C3 520 nm 6 nm 198 ± 4C4 521 nm 8 nm 78 ± 4C5 522 nm 10 nm 216 ± 6C6 523 nm 17 nm 287 ± 6C7 527 nm 30 nm 57 ± 2C8 533 nm 45 nm 192 ± 4

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