Nanotoxicology, March 2010; 4(1): 120137

Gold nanoparticles cellular toxicity and recovery: Effect of size,

concentration and exposure time
TATSIANA MIRONAVA1, MICHAEL HADJIARGYROU2, MARCIA SIMON3,
VLADIMIR JURUKOVSKI3, & MIRIAM H. RAFAILOVICH1
1

Department of Materials Science and Engineering, 2Department of Biomedical Engineering, and 3Department of Oral
Biology and Pathology, School of Dental Medicine, State University of New York at Stony Brook, New York, USA

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(Received 19 May 2009; accepted 5 November 2009)

Abstract
Gold nanoparticles (AuNPs) are used in many applications; however, their interactions with cells and potential health risk
(s) are not fully known. In this manuscript, we describe the interactions of AuNPs with human dermal broblasts and
show that they can penetrate the plasma membrane and accumulate in large vacuoles. We also demonstrate that the uptake
of the AuNPs is a function of time, their size and concentration. Specically, we demonstrate that 45 nm AuNPs penetrate
cells via clathrin-mediated endocytosis, while the smaller 13 nm enter mostly via phagocytosis. Furthermore, we provide
evidence of cytoskeleton lament disruption as a result of AuNPs exposure and reconstitution during recovery (following
AuNP removal), despite no changes in actin or beta-tubulin protein levels. In contrast, the expression of the extracellular
matrix (ECM) proteins, collagen and bronectin, was diminished in the cells exposed to AuNPs. We also examined the
proliferation rates of cells exposed to AuNPs and show that its diminution is a function of apoptosis and speculate that
apoptosis results from the number of vacuoles present in the cells, which is probably the main factor that disrupts the
cytoskeleton causing cell area contraction and decreases in motility. Lastly, we also present data that indicates that AuNPs
damage to cells is not permanent and that the cells can completely recover as a function of AuNPs size, concentration and
exposure time. Taken together, our data suggest that AuNPs exert detrimental effects on cell function that could reverse
following AuNPs removal.

Keywords: Gold nanoparticles, human dermal broblast, endocytosis, apoptosis, recovery

Introduction
Nanotechnology is becoming an increasingly common technique for the fabrication of products ranging
from consumer items, and electronics to biomedical
devices. Because of their large surface to volume ratio,
nanometer-sized metallic and semiconductor particles are central to many of these applications.
Although, nanoparticles are currently and widely
used, their effects on cells are still under intense
investigation. Over the last decade, many reports
brought forth the fact that such nanoparticles exhibit
physical properties that allow them to penetrate
unusually deep into skin and other organs
(Lademann et al. 1999; Kreilgaard 2002; Hostynek
2003; Kato et al. 2003; Tinkle et al. 2003; Hoet et al.
2004). A fundamental question exists, whether the
toxicity of these particles arises from known chemical

interactions, whose effectiveness is magnied by the

increased surface area or from new phenomena which
arise simply due to the decreased particle size.
AuNPs play important roles in the fabrication of
nanowires, in nanomedicine, and nanoelectronics,
where the tolerance for oxidative reactions is very
low (Chan 2007; Narayanan and El-Sayed
2007; Huang et al. 2007a; Corma and Garcia 2008;
Nanotech Full Report 2008). Due to the increasing
demand, numerous commercial suppliers of AuNPs
exist where these particles are manufactured in large
quantities where there is signicant exposure to workers. Yet, recent reports (Hoet et al. 2004; Nanotech
Full Report 2008) indicate that AuNP penetration
into tissue and their effects on cells is still unclear. It is
also extremely important since AuNPs are easily
functionalized which allows the production of new
drugs with chemical groups that target cancer cells,

Correspondence: Dr Tatsiana Mironava, Department of Materials Science and Engineering, State University of New York at Stony Brook, Stony Brook,
NY 11794-2275, USA. Fax: +1 631 632 8052. E-mail: tania.mironova@gmail.com
ISSN 1743-5390 print/ISSN 1743-5404 online
2010 Informa UK Ltd.
DOI: 10.3109/17435390903471463

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Gold nanoparticles toxicity

and due to the high electron density of Au, allow for
enhanced imaging (Eghtedari et al. 2004; Huang et al.
2006, 2007b; Durr et al. 2007; Rayavarapu et al.
2007). Other specic biomedical applications of these
particles involve chemotherapy (via drug delivery) and
directed thermal irradiation of tumors, with both
approaches representing a promise for cancer treatment (Mukherjee et al. 2005; El-Sayed et al. 2006;
Harjanto 2006; Paciotti et al. 2006; Dixit et al. 2007;
Huang et al. 2007c, 2007d, 2007e; Oyelere et al.
2007; Gannon et al. 2008). Further, the majority of
reports dealing with the effects of AuNPs on normal
cells have focused on the short term effects (212 h) of
particle exposure instead of more long term consequences that can affect overall cellular function. Collectively, results from these studies are thoroughly
discussed in detail in recent reviews (Lewinsky
et al. 2008; Murphy et al. 2008). The detrimental
effects cannot be detected simply by general proliferation or apoptosis assays, since the presence of the
AuNPs inside the cells may be interfering with the
proper execution of other specic functions. Furthermore, the short experimental times (less than 24 h)
frequently reported in the literature (Goodman et al.
2004; Connor et al. 2005; Khan et al. 2007; Hauck
et al. 2008; Ponti et al. 2008) are not sufcient to
observe many of these effects, which were showed to
appear after 72 h, and several rounds of cell division
(Pernodet et al. 2006). Even after impaired function is
detected, none of the previous studies has addressed
the essential question of possible recovery once
AuNPs are removed. Lastly, our laboratory has previously shown that human dermal broblasts are
signicantly affected by the presence of 13 nm size
AuNPs, with the most dramatic effect being a large
decrease in cell area which correlated with decreased
cell proliferation and migration, as well as ECM
organization (Pernodet et al. 2006).
Since the skin is the primary source of contact for
nanoparticles, and since the interaction with nanoparticles is cell specic (Goodman et al. 2004; Connor
et al. 2005; Chithrani et al. 2006; Cornell
2006; De la Fuente et al. 2006; Krpetic et al. 2006;
Takahashi et al. 2006; Patra et al. 2007; Ryan et al.
2007), we chose to continue our studies using human
dermal broblasts and explore: (a) Whether the
decreased cell area was associated with a concomitant
change in cytoskeletal protein expression; (b) whether
a critical exposure concentration or time exists for
which recovery was possible once AuNPs were
removed; (c) the mechanism for particle penetration
into the cells and (d) whether signicant differences
could be discerned in these phenomena between
AuNPs of two different sizes (13 nm and 45 nm),
synthesized with identical surface chemistry.

121

Materials and methods

Synthesis of Au/citrate nanoparticles
13 nm Au/Citrate nanoparticles were synthesized
according to Turkevich (1985a, 1985b) protocol.
HAuCl4 (0.1% ml, solution in HCl 30% [Aldrich])
in 95 ml MilliQ water in a tree-necked ask with
condenser and thermometer was heated to boiling
point while stirring, after that sodium citrate (200 mg,
dehydrated, 99%, [Aldrich]) in 5 ml of water was
added to the solution. Over a couple minutes, the
color of solution changed from yellow to grey and
nally to purple. The solution was gently boiled for
4050 min and then cooled down at room temperature. For 45 nm Au/citrate nanoparticles synthesis
was carried out in a three-necked ask under condenser. 25 mg of KAuCl4 was boiled in 98 ml MilliQ
water following by addition of 2.5 ml of 1.5% sodium
citrate under vigorous stirring, after changing the
color from greyish to purple nanoparticles solution
was cooled down at room temperature. In both cases
the resulting particles were coated with negatively
charged citrate and, therefore, were uniformly
suspended.

Cell culture
Primary human dermal broblasts (CF-31, Caucasian female, 31 years old, National Institute on Aging
(NIA) Bank, passage 714 only) were plated at cell
density 2,500 cells per well and cultured in 24-well
dishes. Dulbeccos Modied Eagles Medium
(DMEM) was used with 1% of penicillin-streptomycin (PS) and 10% of fetal bovine serum (FBS) (all
purchased from Sigma). 1 ml of medium containing
AuNPs (with concentrations in the range of 0189 mg/ml
in case of 13 nm and 026 mg/ml in case of 45 nm)
was added to each well 24 h after plating. The wells
were incubated with AuNPs for the chosen time
points (up to 6 days) and then counted or xed,
stained and imaged. All incubations were performed
at 37 C and 5% CO2. Each experiment had a
control (cells grown in medium without AuNPs),
and was performed in triplicate and repeated at least
three times.

Cell counting
To determine the cell number during the growth
curve experiments cells were plated at an initial density of 2,500 cells per well and counted using hemocytometer at the specic time point (up to eight days).

122

T. Mironava et al.

Each grid square of the hemocytometer slide represents a volume of 10-7 m3, and cells were counted in
10 squares in 1 ml of the cell suspension. Each condition had triplicates and all experiments were conducted three times. Cell suspensions were mixed for
uniform distribution and were diluted enough so that
the cells did not aggregate.

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Cell staining for confocal microscopy

Cell area and overall morphology as a function of time
and concentration was monitored using a Leica confocal microscope. For these experiments, cells were
xed with 3.7% formaldehyde for 15 min following
exposure to AuNPs for three and six days. Alexa Fluor
488-Phalloidin was used for actin ber staining and
Propidium Iodide for nuclei staining. In addition, a
set of images was obtained using an Hg lamp with the
excitation lter of 450490 nm and the emission lter
of long pass (LP) at 515 nm.

step ethanol dehydration. Samples were coated with a

thin layer of atomic Au for conductivity and imaged
by SFEG SEM LEO 1550.

Apoptosis induction and quantication

Induction of apoptosis was induced by the use of
Recombinant Human Tumor Necrosis Factor-alpha
(TNF-a, Biosource). An anti-ACTIVE Caspase-3
pAb (Promega) and Cy3 conjugated secondary antibody was used to determine whether cells underwent
apoptosis after exposure to AuNPs for three and six
days at different concentrations (13 nm AuNPs: 95,
142 and 190 mg/ml: 45 nm AuNPs: 13, 20 and 26 mg/ml).
Cells were also stained with DAPI to reveal the
nuclei (Invitrogen). The percentage of apoptotic
cells per sample was determined using the images
captured during uorescent microscopy. For this
experiment, 15 random images were captured for
each sample (samples were in triplicate and the
experiment was repeated twice).

TEM
Western blotting
TEM analysis was used to assess the size distribution
of the AuNPs as well as the fate of internalized
particles. One drop of the original AuNPs solution
(95 mg/ml 13 nm and 13 mg/ml 45 nm particles) was
placed on 300 mesh copper grip, which was coated
with formvar lm. The sample was then dried out at
room temperature. Gaussian distributions of diameters were calculated from the samples with more
than 170 nanoparticles. After exposure to AuNPs for
three and six days at 142 mg/ml (13 nm) and 20 mg/ml
(45 nm), the cells were xed in a solution of 2%
paraformaldehyde and 2.5% glutaraldehyde in 0.1 M
Phosphate Buffered Saline (PBS), stained in 2%
uranyl acetate, dehydrated with ethanol, then embedded in Propylene oxide. The specimen was cut into
ultrathin sections (90 nm) with Reichart UltracutE
ultramicrotome and stained on the grid with uranyl
acetate and lead citrate. The samples were imaged
using a FEI Tecnai12 BioTwinG2 transmission electron microscope. Digital images were acquired with
an AMT XR-60 CCD Digital Camera System and
compiled using Adobe Photoshop program.

SEM
SEM analysis was used to assess the uptake of particles by exposing cells to 20 mg/ml 45 nm AuNPs and
to 142 mg/ml 13 nm AuNPs for three days, following
by xing (3.7% formaldehyde in PBS) and then multi

Proteins were extracted with RIPA Lysis and Extraction Buffer (25 mM TrisHCl, pH 7.6, 150 mM NaCl,
1% NP-40, 1% sodium deoxycholate, 0.1% SDS) and
were separated by SDS-PAGE (0.9 mg of protein was
applied per lane) and blotted onto nitrocellulose
membrane (Millipore, Beverly, MA, USA). The
membranes were blocked with 5% non-fat milk and
probed with diluted monoclonal antibodies (Antiactin Clone AC-40, obtained from Sigma and Antib-tubulin [E7]) developed by Klymkowsky and
obtained from the Developmental Studies Hybridoma Bank (Department of Biological Sciences, The
University of Iowa, Iowa City, IA, USA) at 4 C for
1 h. After washing, bound antibodies were detected
with a goat anti-rabbit IgG or anti-mouse IgG coupled
to HRP (1:10,000) at room temperature for 1 h. The
signal was visualized by using ECL (Amersham Pharmacia Biosciences, Piscataway, NJ, USA).

Clathrin-mediated endocytosis inhibition

Cells were cultured in 24-well dish for 24 h, after that
PAO (Gibson et al. 1989) solution in 1% DMSO was
added to maintain a nal concentration 20 mM. After
20 min of pre-treatment with PAO, the AuNPs were
added to medium and the cells were cultured for
another 2 h, rinsed with PBS, and xed for TEM
as described above.

Gold nanoparticles toxicity

Endocytosis inhibition
Cells were plated in tissue culture asks (75 cm2)
under normal conditions (37 C, DMEM, 5% CO2)
for 24 h. The cells were then exposed to AuNPs for
24 h either at low temperature (4 C, DMEM, 5%
CO2) or at 37 C as control. The cells were then
counted and sent to Columbia Analytical Inc. for
gold content analysis by atomic absorption
spectroscopy.

Collagen and Fibronectin were analyzed in supernatants of cultured CF-31 cells by Procollagen
Type I C-Peptide EIA Kit (Takara, MK101) and

A.

human Fibronectin EIA Kit (Takara, MK115) as

described in instructions provided by the manufacturer. Samples with high concentration of collagen
and bronectin were appropriately prediluted with
Sample Diluent.

Results
Particle characteristics
Two different sizes of citrate AuNPs were synthesized
and were characterized by TEM (Figure 1a, 1b).
Quantitative measurements shown by histograms
(Figure 1c, 1d) reveal that the average particles sizes
were 13 1.1 nm and 45 3.2 nm.

C.
140

Nanoparticles number

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80
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35

50nm

B.

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45
50
55
AuNPs diameter, nm

60

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140
Nanoparticles number

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Collagen and Fibronectin expression

123

120
100
80
60
40
20
0

50nm

10

11
12
13
14
AuNPs diameter, nm

15

Figure 1. AuNPs imaged by TEM and their Gaussian size distribution histograms. (a) and (c) 45 5.1 nm particles, (b) and (d) 13 1.8 nm
particles.

124

T. Mironava et al.
very small, probably resulting from the gold thin lm
coating used for conductivity (Figure 2g).

Size and time-dependent uptake of AuNPs

Since gold is more electron dense than the surrounding organic material it was clearly visible under SEM
(Figure 2). The images shown in Figure 2 clearly
reveal the outline of the cell and the brighter regions,
indicative of the presence of gold, especially around
the nuclei (Figure 2ad). This observation was conrmed by EDS/X-ray Microanalysis, which shows the
Au Ka peak at 2.3 keV, indicating the presence of
AuNPs clusters inside the cells (Figure 2e). In the
control cells (not exposed to AuNPs), the Au peak is

The impact of AuNPs on cell proliferation

Results in Figure 3 show that for both particle sizes,
the cell doubling time is increasing with higher
AuNPs concentration and longer exposure. If we
compare the concentrations for which the cell doubling time increases from 3545 h, we nd that the
concentration of the 13 nm particles, on average, is

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A.

B.

10 m

10 m

C.

D.

200 nm

E.
1.2

F.
1.1

Au

G.
Au

Si

0.7
KCnt

2.0

Si

0.9

0.9

200 nm

1.6

0.6
KCnt
0.4

0.5

Si

1.2
KCnt
0.8
O

0.2

O
C
Zn

Zn

Ca

0.2 C
Au
O Zn

0.0

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Ca

Zn

0.0
0

5 6
keV

9 10

5 6
keV

Au

9 10

0.4

C Zn Au K

Ti

0.0
0

5 6
keV

Zn Au
7

9 10

Figure 2. (a) and (c). SEM images of cells exposed to 45 nm AuNPs (20 mg/ml); (b) and (d) SEM image of cells exposed to 13 nm AuNPs (142
mg/ml); (e), (f) and (g) EDAX spectrum of cells with 13 nm, 45 nm and control, respectively.

Doubling time, hours

Gold nanoparticles toxicity

AuNPs:

95

13 nm (2 days)

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Doubling time, hours

90

13 nm (4 days)

85

13 nm (6 days)

80

45 nm (2 days)

75

45 nm (4 days)

70

45 nm (6 days)

125

80
45 nm

70
60

13 nm

50
2

4
6
Day of exposure

65
60
55
50
45
40
Control

35
0

20

40

60

80

100

120

140

160

Figure 3. Doubling time for cells exposed to different concentrations of AuNPs 13 nm and 45 nm for 2, 4 and 6 days; inset: Doubling time
versus time of exposure for samples exposed to 20 mg/ml 45 nm and 142 mg/ml 13 nm AuNPs.

seven times higher than that of the 45 nm particles (at

day 2 and 4) and increases to nearly a factor of 10 after
six days of incubation. The inset graphs show that the
doubling time increases as a function of time for both
particles sizes suggesting that saturation is not occurring, and for a specic concentration of AuNPs
(20 mg/ml 45 nm and 142 mg/ml 13 nm), the effect
is almost equivalent at day 4 and 6.

Intracellular fate of internalized AuNPs

The data clearly shows that for both particle sizes the
AuNPs are sequestered inside large vacuoles and do
not penetrate either the nucleus or the mitochondria
(Figure 4). The vacuoles are distributed uniformly
across the cytoplasm (Figure 4ah), consistent with
the particle distribution observed in the dry samples
imaged with SEM (Figure 2). Closer examination also
shows that even after six days, no particles were
detected inside the nucleus or mitochondria
(Figure 4c, 4g). We also present higher magnication
images revealing that neither the 13 nm nor the 45 nm
particles are uniformly distributed within vacuoles.
Rather, we nd that both types of particles are
attracted to the vacuole membrane, leaving the interior almost empty (Figure 4l, 4m). In the case of the
13 nm particles, their density around the membrane is
greater and we can see sections of the membrane
protruding inside the vacuole with particles still

attached to it (Figure 4l). In contrast, with the

45 nm particles, only a monolayer string of particles
covers the membrane surfaces and no particles were
seen attached to the thinner tendrils.
Furthermore, with increasing incubation time additional AuNPs (both sizes) enter the cell, causing the
formation of greater number of vacuoles as well as
increasing the diameter of the existing vacuoles
(Figure 4i, 4j). Specically, we nd that the number
and diameter of vacuoles per cell increases at the same
rate for both types of AuNPs (average of seven cells
for each conditions were examined). The major difference between samples exposed to the two AuNP
sizes is the number of particle clusters per vacuole,
which is roughly ve times greater with the 13 nm
particles (Figure 4k).
Finally, close examination of TEM cross sections
obtained after three and six days of incubation with
both types of AuNPs indicates that in all cases the
vacuoles clustered inside the cytoplasm. Magnied
regions near the cell membrane show that even
though occasional particle clusters are observed, no
vacuoles are present. Hence, we have no direct evidence of exocytosis occurring to prevent accumulation of particles within the cells. Rather, the number
of particles within the cells nearly doubles for both
types of particles, when the incubation time is
increased from 36 days.
In order to determine whether the mode of penetration of the AuNPs in the cells is a function of

126

T. Mironava et al.
A.

B.

I.

2 microns

D.

J.

1.8

1.2
0.9
0.6
0.3
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E.

1 micron
F.

Vacuole number per cell

2 microns

45 nm AuNPs
13 nm AuNPs

1.5

K.
250
200

3 days

6 days

45 nm AuNPs
13 nm AuNPs

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100
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L.

2 microns
G.

1 micron
H.

Particles number per vacuole

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Vacuole size, micron

2 microns

200

6 days

45 nm AuNPs
13 nm AuNPs

150
100
50
0
3 days

M.

2 microns

1 micron
100 nm

6 days
N.

100 nm

Figure 4. TEM section of cells exposed to AuNPs; (a) and (b) 142 mg/ml 13 nm particles three days exposure; (c) and (d) 142 mg/ml 13 nm
nanoparticles six days exposure, (e) and (f) 20 mg/ml 45 nm AuNPs three days exposure; (h) and (g) 20 mg/ml 45 nm gold six days exposure;
(i) control; (j) vacuole size distribution; (k) number of vacuoles per cell; (l) number of particles/clusters per vacuole, (m) and (n) high
magnication of cell vacuole led with to 13 and 45 AuNPs, respectively.

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Gold nanoparticles toxicity

particle size, i.e., do some particles penetrate by
passive diffusion through the cell membrane
(Geiser et al. 2005) or do they enter via endocytosis
(Connor et al. 2005), clathrin mediated endocytosis
was inhibited using PAO (Gibson et al. 1989). Following treatment of cells with PAO prior to AuNP
exposure, the cells were xed, sectioned, and examined with TEM (Figure 5). The results were quantied by measuring the ratio of AuNPs adjacent to the
cell membrane versus the number in the cytoplasm.
Figure 5 shows that the number of AuNPs clustered
in the vicinity of the cell membrane, and the number
that penetrated the cells in the absence of PAO is
nearly the same for both particle sizes. With exposure
to PAO the ratio is nearly unchanged for the 13 nm
particles (Figure 5e), indicating that clathrinmediated endocytosis is not the predominant particle
penetration pathway. In Figure 5fi we show crosssections for cells treated with and without PAO and
exposed to 45 nm AuNPs, revealing that almost no
particles are present inside the cytoplasm of the treated cells, while a signicant number is present in the
untreated control cells. In fact, a strong accumulation
of AuNPs is clearly seen outside of the membrane in
the treated cells (Figure 5i) and as a result, the ratio is
reduced to nearly zero for the 45 nm particles, as
compared to that of the control cells (Figure 5k),
indicating that clathrin-mediated endocytosis is the
predominant mode of entry.
To further investigate if the major pathway for
13 nm AuNPs penetration is diffusion through membrane driven concentration gradients or non-receptor
mediated endocytosis (phagocytosis), we utilized an
approach involving temperature. Specically, when
we decreased the incubation temperature to 4 C,
almost 85% of 13 nm AuNPs penetration rate into
the cells was inhibited (Figure 5j). In contrast, only
73% of penetration was inhibited in case of 45 nm
AuNPs (Figure 5l), indicating that phagocytosis
appears to be the prime mode of 13 nm AuNPs
penetration, but also is partly responsible for the
cell penetration of the 45 nm AuNPs.

Cell recovery
Based on these results, an interesting question arises:
Do cells have a mechanism for eliminating AuNPs
and thereby recover from their adverse effects? In the
simplest scenario the cells can pass the nanoparticles
to daughter cells, therefore in the absence of particles
in the media, the AuNPs concentration inside the cell
becomes increasingly diluted. Hence, if the nanoparticles are removed from the environment, can
the cells eventually recover from AuNP exposure?

127

Thus, we tried to address these questions by specifically investigating whether cells containing AuNPs
are still capable of dividing and passing the particles to
the daughter cells.
In Figure 6 images of cells immediately before the
removal of the particles, and ve days later show that
after a three-day incubation AuNPs uptake is greater
for the 13 nm particles (Figure 6b) than for 45 nm
(Figure 6e). After ve days of incubation without
AuNPs, a drastic reduction in the average amount
of particles per cell is observed in cells exposed to both
particle sizes (Figure 6c, 6f). Thus, the major mechanism for AuNPs reduction appears to be cell division
(Figure 6d), where the particles are seen to be divided
between the two daughter cells.

Particle-mediated apoptosis
To investigate whether exposure to AuNPs leads to
detrimental effects for the cells, we examined the rate
of apoptosis. Figure 7 shows the percentage of cells
undergoing apoptosis exposed to different AuNPs
concentrations and incubated for three and six
days. Specically, we nd that in the presence of
the 45 nm AuNPs there is a higher rate of apoptosis
with either longer exposure or higher particle concentrations in comparison to that with the 13 nm
AuNPs. Whereas the apoptotic rate for cells exposed
to 13 nm AuNPs at day 3 starts at 23% and increases
to 42% at the highest concentration, with the 45 nm
AuNPs, it ranges from 4475%. Similarly, after six
days, apoptosis further increases to 61% with the
lowest and increases to 97% with the highest concentration of 13 nm, whereas with the 45 nm AuNPs the
rate is 91100% at the different concentrations
(Figure 7). It is clear that for the 45 nm AuNPs
that apoptosis: (1) Occurs at nearly one tenth of
the concentration of that of the 13 nm particles, (2)
increases steeply with higher concentration, and (3) is
nearly 100% for all concentrations after six days of
incubation. Hence, even though the damage may
appear to be equivalent, i.e., approximately 40% for
142 mg/ml of 13 nm particles, versus 13 mg/ml for the
45 nm particles, the rate of increase with concentration and incubation time is much faster with the larger
AuNPs.
Next we tested to see if removal of AuNPs can
rescue the cells from apoptosis. Figure 8 shows that
even after removal of the AuNPs, the rate of cell
growth in the exposed cells is slower than that of
the control unexposed cells, with the slowest being
in cells exposed to the 45 nm particles. In the inset we
plotted the percentage recovery after a total of
an eight-day incubation (three days with AuNPs

128
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AuNPs ng/cell, 104

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T. Mironava et al.

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25
20
15
10
5

1 micron

200 nm

0
37C

4C

Figure 5. (ab) TEM sections of the cells exposed to 142 mg/ml 13 nm gold; (cd) cells treated with PAO (20 mM) and exposed to 142 mg/ml
13 nm AuNPs; (e) 13 nm AuNPs ratio inside and outside of the cell with PAO and without inhibition (p = 0.9461); (fh) cells exposed to
20 mg/ml 45 nm nanoparticles; (gi) cells treated with PAO and exposed to the same concentration of 45 nm gold; (j) amount of gold per cell for
samples exposed to 13 nm AuNPs at 37 C and 4 C (p < 0.001); (k) 45 nm AuNPs ratio inside/outside cell (p < 0.0001); (l) amount of gold per
cell for samples exposed to 45 nm AuNPs at 37 C and 4 C (p = 0.0123).

A.

B.

C.

D.

E.

F.

129

Figure 6. (a) Control; (b) Cells exposed to 142 mg/ml of 13 nm AuNPs for three days; (c) Cells for the same concentration of 13 nm AuNPs
after ve days recovery following three days exposure; (d) Cell transmitting nanoparticles to daughter cell upon dividing; (e) Cells exposed to
20 mg/ml of 45 nm AuNPs for three days; (f) Cells for the same concentration of 45 nm AuNPs after ve days recovery.

A. 100

3 days exposure
6 days exposure

80

B. 100
80

60

3 days exposure
6 days exposure

60

40

Apoptosis, %

Apoptosis, %

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Gold nanoparticles toxicity

20

1.5

40
20
1.5

1.0

1.0

0.5

0.5
0.0

0.0
0

25 50 75 100 125 150 175 200 225

13 nm AuNP concentration, mg/ml

5
10
15
20
25
30
45 nm AuNP concentration, mg/ml

Figure 7. Apoptosis rate of cells exposed to (a) 13 nm for three and six days at different concentrations (p = 0.0527 and 0.0143, respectively)
and (b) 45 nm AuNPs for three and six days at different concentrations (p = 0.0315 and 0.0083, respectively).

incubation, ve days of recovery), which is calculated

by dening control as undergoing 100% recovery.
With the 13 nm AuNPs the rate of cell proliferation
increases immediately after removal of the particles
and recovery reaches 61%, 48% and 37% for increasing concentration (Figure 8a inset). With the 45 nm

AuNPs the rates of recovery are much slower with

respect to increasing particle concentrations (42%,
22% and 11%).
Since no division occurs during apoptosis, recovery
is also not possible, and thus the number of AuNPs in
the cells is not reduced. Therefore, we replotted the

T. Mironava et al.

A. 40000

Number of cells

30000
25000

40000
95 g/ml

75 100 125 150 175 200

AuNPs, g/ml

20000

142 g/ml

Recovery

Exposure

15000
10000

45 nm AuNPs

60

45000

Recovery, %

Recovery, %

35000

B. 50000

Control

13 nm AuNPs
60
50
40
30
20
10
0

Number of cells

130

190 g/ml

35000

50

Control

40
30
20
10
0
10

30000

15

20

25

AuNPs, g/ml

25000

Exposure

13 g/ml

Recovery

20000
20 g/ml

15000
10000

5000

5000

26 g/ml

0
1

Day

4
5
Day

Figure 8. Cell recovery. (a) and (b) dermal broblast cells CF-31 exposed to different AuNPs concentrations for three days and then allowed to
recover for ve days. Time where nanoparticles were removed is outlined with dashed line; control is equal to 100% recovery.

100000

100000

Control
13 g/ml AuNPs 45 nm
20 g/ml AuNPs 45 nm
26 g/ml AuNPs 45 nm
Cell number

Control
95 g/ml AuNPs 13 nm
142 g/ml AuNPs 13 nm
190 g/ml AuNPs 13 nm
Cell number

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10000

10000

Days

Days

Figure 9. Cell recovery. Growth curves for the recovery after AuNPs exposure for three days (cell number was corrected by apoptotic cell
subtraction on day 3). Dermal broblast cells CF-31 were exposed to different concentrations of 13 nm (a) and 45 nm (b) AuNPs, respectively.

data by subtracting the fraction of cells that have

undergone apoptosis after three day incubation,
and thus no longer divide. The data shows that the
cell number (as a function of incubation time after
correcting for the fraction of non-dividing apoptotic
cells on day 3) for both types of particles is nearly
identical to that of the control cells indicating that the
doubling time of the exposed cells, even if they still
contain nanoparticles is similar.

Particle-mediated microlament disruption

In Figure 10ah we show confocal images of cells after
exposure to different concentrations of both 13 nm

and 45 nm AuNPs for three and ve days and

following recovery (Figure 10ip). Figure 10e
and Figure 10m shows the average cell aspect ratio
corresponding indicating that it is bigger in the
exposed cells than control. Furthermore, we can
clearly see that there are fewer cells for the samples
exposed to 45 nm AuNPs (Figure 10fh), which are
also more elongated and appear ready to lift off the
substratum, as compared to cells exposed to the 13
nm AuNPs (Figure 10bd). Removing the AuNPs
and allowing the cells to recover for another ve days,
has a dramatic effect; the 13 nm AuNPs exposed cells
have the same appearance as the unexposed control
cells even at the highest concentration. In contrast, for
the larger particles, the recovery is much slower, and

Gold nanoparticles toxicity

131

3 days exposure
A.

B.

C.

D.

F.

G.

H.

170.6 m
E.
Cell aspect ratio

12
8

Exposure
Recovery

6
4
2
0
0 50 100 150 200
13 nm AuNPs, mg/ml
5 days following recovery

I.

J.

K.

L.

M.
12

N.

O.

P.

Cell aspect ratio

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10

10

Exposure
Recovery

8
6
4
2
0

0 5 10 15 20 25 30
45 nm AuNPs, mg/ml

Figure 10. Human dermal broblasts CF-31 imaged with confocal microscopy after three days for (a) control and cells exposed to 13 nm
AuNPs at the following concentrations, (b) 95 mg/ml, (c) 142 mg/ml, and (d) 190 mg/ml and to 45 nm AuNPs at (f) 13 mg/ml, (g) 20 mg/ml and
(h) 26 mg/ml. The cells were also monitored following recovery after ve days of AuNPs removal (jp), compared to control. (i) Cell aspect ratio
after exposure and recovery (e) 13 nm AuNPs, (m) 45 nm AuNPs.

132

T. Mironava et al.
3 days exposure
A.

Control

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D.

5 days recovery

14 days recovery

B.

C.

E.

F.

H.

I.

11.24 m

13 nm AuNPs
G.

45 nm AuNPs
Figure 11. CF-31 imaged with confocal microscopy after three days of exposure and 5 and 14 days following recovery. (a), (b), and (c) control;
(d), (e) and (f) cells exposed to 142 mg/ml of 13 nm nanoparticles; (g), (h), and (i) cells exposed to 20 mg/ml of 45 nm.

even after ve days many cells still appear elongated

and their density remains low (Figure 10np).
We further show a magnied view of cells, that
shows how the actin bers are well extended across
the cell cytoplasm in the control conditions
(Figure 11ac). On the other hand, after three days
of AuNPs exposure to either 13 or 45 nm AuNPs, the
actin laments are broken or appearing disrupted
and thinner, appearing as an aspiration of dots
(Figure 11d, 11g). After ve days of recovery the
disruption of the actin laments are less obvious in
the case of 13 nm AuNPs (Figure 11e) as compared
to those still present with the 45 nm particles
(Figure 11h). Stretched actin laments were observed
again with the 13 nm AuNPs indicating that the cells

are in the process of recovery while for the larger

particles, the damaged actin was still visible, even
though some stretched laments were also observed.
We also nd, that following 14 days of recovery, the
cytoskeleton of the cells exposed to both size particles
completely recover (Figure 11f, 11i) and resemble
that of the control cells (Figure 11c).
In order to examine whether these effects on the
cytoskeleton were a direct result of less production of
actin or tubulin, we examined the expression of these
proteins from the particle exposed and control
cells. Figure 12 shows no difference between the
control and any of the exposed samples, indicating
that the amount of actin and beta tubulin is
unaffected.

Gold nanoparticles toxicity

a

Discussion and conclusion

Beta-tubulin 55 kDa
actin

45 kDa

Figure 12. Western blot. (a) control, cells cultured for three days
with AuNPs; (b) 142 mg/ml 13 nm; (c) 20 mg/ml 45 nm.

Another possible cause for the observed reduction

in cell area may be a decrease in the expression of
ECM proteins. Therefore, we measured the amount
of collagen (type I) and bronectin expressed by the
cells after incubation for three days with 20 and 142
mg/ml of 13 nm and 45 nm AuNPs, respectively, and
then after recovery for ve and 14 days. The magnitude of the reduction after three days of exposure for
both proteins is within one standard deviation for the
two particle sizes and appears to be mostly a function
of the exposure conditions (Figure 13). It is also
important to notice, that collagen is reduced at a
greater level than bronectin and indicating that
ECM proteins produced after exposure to AuNPs
is altered to a low collagen and higher bronectin
composition (a ratio contains 20% less collagen than
bronectin when compared with control composition
for cells exposed to 13 nm AuNPs and 35% less
collagen in case of cells exposed to 45 nm particles).
Furthermore, the data shows that the production of
bronectin partially recovers after ve days and fully
recovers after 14 days, whereas the production of
collagen only slightly recovers after ve days and is
still not fully recovered after 14 days. These results are
consistent with those of the actin bers, shown
in Figure 11.

In this manuscript we attempted to elucidate some of

the fundamental aspects regarding the effects of
AuNPs on primary dermal broblasts. In a typical
tissue or organ, cells are organized in different layers,
interact with each other, and damage in one layer can
have an impact on the other cells in an unpredictable
fashion. We chose to study dermal broblasts since
their interactions within skin are well known (i.e., in
terms of gene expression, protein deposition etc.).
Therefore, our approach is to rst understand the
effects of AuNPs on the function of cells in vitro, and
then to try to model the effects at the tissue level.
While the most common approach in studying the
effects of nanoparticles reported in numerous publications (Goodman et al. 2004; Connor et al.
2005; Khan et al. 2007; Hauck et al. 2008; Lewinsky
et al. 2008; Ponti et al. 2008) is to cell proliferation,
our results allow a deeper understanding of additional
effects on cells, in the context of how living cells
tolerate the particles once they penetrate the cell.
In attempt to directly compare the effects of the
13 nm and 45 nm size AuNPs, we chose exposure
concentrations based on their effect on doubling time
(Figure 2). We observed that for a 26 day time
interval, the concentration difference for two nanoparticle sizes (for the same doubling time increase)
varies by 6- to 13fold. This signicant variation
occurs because of the concentration-doubling time
non-linearity over time. The average concentration
that causes the same increase in cell doubling time for
cells exposed to 13 nm particles is larger than the
concentration of the 45 nm particles by roughly a
factor of eight. Therefore, a set of three

Control

Control

13 nm AuNPs

13 nm AuNPs

45 nm AuNPs

45 nm AuNPs

100

100

80

80

% collagen / cell

% of fibronectin / cell

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133

60
40
20

60
40
20

0
3

8
Days

17

8
Days

17

Figure 13. Fibronectin (a) and collagen (b) expression, cells treated with 142 mg/ml 13 nm and 20 mg/ml 45 nm AuNPs for three days,
recovered for ve and 14 days after exposure.

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134

T. Mironava et al.

concentrations: 13, 20 and 26 mg/ml was chosen for

45 nm AuNPs, and a set of 95, 142 and 190 mg/ml for
13 nm AuNPs.
In this study we found that the nanoparticle uptake
by cells was dependent on exposure time, particle size
and concentration. We also found that the 45 nm
particles, at much lower concentrations result in a
more signicant increase of the doubling time than
the 13 nm particles. These results were surprising in
view of the ndings of Pan et al (2007) who reported
that maximum toxicity occurs for a particle diameter
of 1.4 nm (Pan et al. 2007). In order to understand
the basis of the observed toxic effects between the
different particles, we performed several experiments
where the distribution of AuNPs in the cell could be
measured and have shown that they penetrate the cell
membrane and accumulate in large vacuoles.
Even though the mechanism of sequestration for
both particles sizes appears to be similar, the method
of penetration into the cytoplasm was determined to
be different. In previous studies investigating nanoparticle penetration, gold particles of different shapes
or sizes were coated with transferrin (Yang et al.
2005; Chithrani et al. 2006; Chithrani and
Chan 2007) for which known membrane recognition
pathways exist. In our case, the particles were stabilized with citrate for which no specic recognition site
(s) on the plasma membrane is known. Also, to rule
out any independent effects of citrate on cells, we
exposed cells to citrate and found no differences in
cell growth when compared to control cells. Further,
most of the previous work was also performed with
cancer cells or cell lines whose characteristics, to an
extent, are known to differ from primary cells.
The data described herein shows that we have
identied the AuNPs cell penetration pathways in
dermal broblasts; for 45 nm nanoparticles clathrin
mediated endocytosis is the major pathway, while for
the 13 nm it is phagocytosis. Another possibility may
be that the particle size affects the amount of protein
adsorbed, which in turn can affect the recognition
pathway triggered when the particles attach to the cell
membrane. Since the radius of gyration of albumin is
approximately 3.5 nm, more molecules are likely to be
adsorbed on the larger particles, with radii of 22 nm
and surface area of ~ 1200 nm than on the smaller
particles with radii of 6 nm and surface area ~ 120 nm.
Also, the dependence of the uptake on temperature
may help to clarify whether non clathrin mediated
endocytosis is responsible for particle penetration.
Sudhakaran et al. (2007) have shown that edocytosis
in human dermal broblasts decreases to approximately 15% with decreasing the temperature to
16 C; a similar decrease was observed by Mamdouh
et al. (1996) in epithelial cells, who also found that at

4 C endocytosis in epithelial cells is reduced by nearly

95%. Our results show that both small and large
AuNPs uptake was inhibited by 85% and 73%,
respectively. Although the amount of inhibition is
signicant, it is still smaller that that reported for
endocytosis, indicating that other pathways may
also play a role in overall particle uptake. Further
work is in progress, which will hopefully clarify the
issue, of the involvement of both the amount and
nature of the adsorbed proteins as a function of
particles size. Lastly, the ratio of particles inside
and outside the cell, in the untreated control cells
is an indication of the efciency of the particular
penetration mechanism used by the cells to bring
the AuNPs across the cell membrane to the interior
of the cell (PAO data). From this data it appears that
the penetration process used to bring in the smaller
AuNPs is more efcient than that used for entry of the
larger particles.
Regardless, with both particles, once inside the cell,
they are sequestered in vacuoles, where they localize
to the membrane. These vacuoles are considered full
when almost all available membrane surfaces are
covered with particles. As a result, the concentration
of AuNPs that they can hold is inversely proportional
to the cross sectional area of the particles. Comparing
cross-sections obtained from cells incubated for three
and six days, we nd that the mean diameter of the
vacuoles increases by approximately a factor of 2 while
the number of particles per vacuole increases by a
factor of 45. Hence the number of particles within a
vacuole increases in a manner proportional to the
available increased area which the particles could
subtend on the membrane surface. On the other
hand, the size of the vacuoles is similar for the two
types of particles after equivalent incubation times,
and the number of vacuoles is nearly the same after six
days of incubation. Thus, it is the number of vacuoles,
rather than the absolute concentration of particles
within the cell, that plays the largest role in disrupting
normal cellular function. However, we do observe
loose particles that are visible in the cytoplasm of
cells exposed to 45 nm AuNPs for six days. This
observation might be the result of vacuole structural
instability at certain local particle concentrations. In
essence, it is more difcult for the inner surface of a
vacuole to equilibrate larger 45 nm AuNPs, leading to
vacuole collapse and nanoparticle release in the cytoplasm. Therefore, our observations that toxicity correlates with vacuole number must be qualied by the
fact that some vacuoles ruptured when exposed to 45
nm AuNPs, and thus were not counted. With the
same intact vacuole number, the higher toxicity of
cells exposed to the 45 nm AuNPs probably arises
from additional vacuoles that collapsed and release

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Gold nanoparticles toxicity

the AuNPs in the cytoplasm leading to interference
with normal cellular function and ultimately
damaging the cell.
Furthermore, we also determined the apoptotic
rates for cells exposed to different AuNPs sizes and
concentrations and showed that the previously
reported decrease in cell proliferation was erroneously
interpreted as an increase in doubling time. We measured the fraction of cells undergoing apoptosis by
assaying the production of caspase 3, which is a
protein produced in the early stage of apoptosis. Cells
producing this protein are adherent, but no longer
divide, therefore for obtaining the precise doubling
time, the number of cells have to be corrected by
subtraction of apoptotic cells. AuNPs were previously
reported to have no toxic effects (Patra et al. 2007),
but others found that they induce apoptosis in certain
cell types (Cornell 2006; Pan et al. 2007; Patra et al.
2007), clearly indicating cell type dependence of
AuNPs cytotoxicity. However, even though Patra
et al. (2007) and Pan et al. (2007) reported apoptosis
in melanoma, broblast and macrophage cell lines,
they did not test it with apoptosis specic assays.
Furthermore, we found that for the 13 nm particles,
the fraction of cells undergoing apoptosis increases
linearly with concentration at both incubation at three
and six days, however, for 45 nm AuNPs it increases
in exponential fashion with higher concentration for
three days and almost 100% for all concentrations
tested for six-day exposure.
Even though numerous papers have explored the
effects of AuNPs exposure in different cell culture
systems (Connor et al. 2005; Chithrani et al.
2006; Cornell 2006; Krpetic et al. 2006; Pernodet
et al. 2006; Takahashi et al. 2006; Patra et al.
2007; Ryan et al. 2007), no data has been reported
on the ability of any cell type to recover once the
particles have been removed from the media. Our data
for cell proliferation where we found a decrease in
growth rate with increasing AuNPs concentration are
in agreement with other studies (Goodman et al.
2004; Cornell 2006; Krpetic et al. 2006;
Pernodet et al. 2006) regardless of nanoparticle coating. The second observation we made is cell area
reduction that was previously also detected
by Pernodet et al. (2006). In order to try to provide
a possible explanation for this reduction in cell area
we looked at the integrity of the cytoskeleton. Actin
bers are responsible for distributing internal forces
by attaching one end the integrins receptors expressed
on the cell membrane, which adhere to proteins
secreted by the cell in the extra cellular matrix.
We show that the actin bers are well extended
across the cell cytoplasm in the control cells, whereas
in those exposed to both 13 nm and 45 nm particles,

135

actin laments were disrupted and appear as small

dots. After ve days of recovery, the cells exposed to the
13 nm particles appear to generate large and strong
actin bers, whereas in the cells exposed to the 45 nm
particles, a signicant amount of disrupted actin laments even after ve days are present. By day 14 all
cells had recovered with an intact actin cytoskeleton.
This type of ber disruption due to the presence of
AuNPs can occur either due to down regulation of
cytoskeletal proteins or triggering a signaling pathway
which somehow interferes with the self assembly of
the actin, possibly due to physical interference by the
large amounts of vacuoles. Interestingly, Western blot
analyses reveals that the amount of actin and betatubulin formed by the cells is not affected by the
presence of the nanoparticles, indicating that it is
probably the latter that is responsible for the observed
cytoskeletal disruption. We reasoned that the large
number of vacuoles as well as loose particles found
in samples exposed to 45 nm AuNPs interferes with
the ability of actin bers to effectively form linkages
with the ECM via integrins. As a result the cell area is
decreased and disrupted bers are observed in the
cells. In order to carry proper tension, the actin bers
must also be anchored to integrins that are bound to
Arg-Gly-Asp (RGD) domains found on the secreted
ECM proteins. Therefore, the lack of tension in the
bers may also be caused by a disruption in the
expression of the ECM proteins, which for broblasts
are primarily collagen and bronectin. When we
investigated this possibility, we found that signicantly less collagen and bronectin were expressed
by the cells exposed to nanoparticles, which may
explain the observed drastically reduced cell area.
Furthermore, since collagen reduction is higher
than bronectin, the composition of the ECM is
altered. Loss of collagen is generally associated with
increased ECM rigidity and aging. Hence, this shift in
composition may explain the increased modulus of
the bers previously reported by Pernodet et al.
(2006) while the decreased overall quantity is consistent with the smaller and thinner bers for cells
cultured with AuNPs.
Lastly, we also found that as the cells divide the
concentration of particles is decreased, which in turn
allows the cells to reduce the number of vacuoles and
form normal actin bers and increase their production
of ECM proteins. Hence, after 14 days near full
recovery occurs for cells expose to both types of
AuNPs. Taken together, these data indicate that
AuNPs are toxic for human dermal broblasts and
the toxicity rate depends on the concentration and
size of nanoparticles, as well as the time of exposure.
Even though the internalized fate of 13 nm and 45 nm
AuNPs is similar, the major penetration pathways are

136

T. Mironava et al.

different. In addition, our results indicate that overall

toxicity of different sized AuNPs does not depend on
total gold concentration in the cell. Rather, the vacuole number and stability play a signicant role in the
induction of and the disruption of the cytoskeleton.

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