Professional Documents
Culture Documents
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
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.
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
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
121
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).
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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.
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).
Collagen and Fibronectin were analyzed in supernatants of cultured CF-31 cells by Procollagen
Type I C-Peptide EIA Kit (Takara, MK101) and
A.
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
120
100
80
60
40
20
0
35
50nm
B.
40
45
50
55
AuNPs diameter, nm
60
D.
140
Nanoparticles number
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.
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T. Mironava et al.
very small, probably resulting from the gold thin lm
coating used for conductivity (Figure 2g).
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
K Ti
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.
95
13 nm (2 days)
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.
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T. Mironava et al.
A.
B.
I.
2 microns
D.
J.
1.8
1.2
0.9
0.6
0.3
0.0
E.
1 micron
F.
2 microns
45 nm AuNPs
13 nm AuNPs
1.5
K.
250
200
3 days
6 days
45 nm AuNPs
13 nm AuNPs
150
100
50
0
3 days
L.
2 microns
G.
1 micron
H.
C.
1 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.
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
A.
B.
1 micron
200 nm
1 micron
13 nm
1.0
0.8
0.6
0.4
0.2
0.0
D.
200 nm
120
110
100
90
80
70
60
50
40
30
20
10
0
13 nm
F.
1 micron
H.
K.
200 nm
37C
4C
45 nm
1.0
0.8
0.6
0.4
0.2
0.0
I.
L.
40
45 nm
35
AuNPs ng/cell, 104
C.
E.
T. Mironava et al.
30
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, %
20
1.5
40
20
1.5
1.0
1.0
0.5
0.5
0.0
0.0
0
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).
T. Mironava et al.
A. 40000
Number of cells
30000
25000
40000
95 g/ml
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
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.
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.
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.
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3 days exposure
A.
Control
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.
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.
Control
Control
13 nm AuNPs
13 nm AuNPs
45 nm AuNPs
45 nm AuNPs
100
100
80
80
% collagen / cell
% of fibronectin / cell
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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