Challenge in understanding size and shape dependent toxicity of gold nanomaterials in human skin keratinocytes

Article abstract of DOI:10.1016/j.cplett.2008.08.039

As the nanotechnology field continues to develop, assessing nanoparticle toxicity is very important for advancing nanoparticles for biomedical application. Here we report cytotoxicity of gold nanomaterial of different size and shape using MTT test, absorp

Full text of DOI:10.1016/j.cplett.2008.08.039

Chemical Physics Letters 463 (2008) 145–149
Contents lists available at ScienceDirect
Chemical Physics Letters
journal homepage: www.elsevier.com/locate/cplett
Challenge in understanding size and shape dependent toxicity of gold nanomaterials
in human skin keratinocytes
*
Shuguang Wang, Wentong Lu, Oleg Tovmachenko, Uma Shanker Rai, Hongtao Yu, Paresh Chandra Ray
Department of Chemistry, Jackson State University, 1400 J.R. Lynch Street, Jackson, MS 39217, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
As the nanotechnology field continues to develop, assessing nanoparticle toxicity is very important for
advancing nanoparticles for biomedical application. Here we report cytotoxicity of gold nanomaterial
of different size and shape using MTT test, absorption spectroscopy and TEM. Spherical gold nanoparti-
cles of different sizes are not inherently toxic to human skin cells, but gold nanorods are highly toxic due
to the presence of CTAB as coating material. Due to toxicity of CTAB, and aggregation of gold nanomate-
rials in the presence of cell media, we have demonstrated that it is difficult to understand the cytotoxicity
of gold nanomaterials individually.
Received 26 June 2008
In final form 11 August 2008
Available online 15 August 2008
Ó 2008 Elsevier B.V. All rights reserved.
1. Introduction
port the systematic study of cytotoxicity of gold nanomaterial of
different size and shape.
The nanoscience revolution that sprouted throughout the 1990s
is having promises to benefit society [1–24]. Since their size scale is
similar to that of biological molecules (e.g., proteins, DNA) and
structures (e.g., viruses and bacteria), enormous interest is growing
to exploit nanoparticles for various biomedical applications [1–24].
Due to valuable size and shape dependent properties, gold nano-
particles have been exploited as tools in biology for the last fifteen
years [1–24]. Their high electron density has made them popular
labels in electron microscopy, and because their surface properties
enable a great variety of functionalization procedures. Having ex-
tremely small size with highest possible surface area enables them
to access a variety of biological environments. These advantages,
along with the universal biocompatibility, gold nanoparticles are
promising to be used as vehicles for drug and gene delivery. Fur-
thermore, gold nanoparticles have recently been demonstrated in
cell imaging, targeted drug delivery, and cancer diagnostics and
therapeutic applications [4–24]. As nanotechnology field continues
to develop, studies on the cytotoxicity of nanoparticles, with re-
spect to their size and shape, are required in order to advance
nanotechnology for biomedical applications. This will be important
for assessing nanoparticle toxicity, for advancing nanoparticles for
imaging, drug delivery, and therapeutic applications and for
designing multifunctional nanoparticles. Though nanoparticle pro-
duction has been estimated to increase from 2300 tons produced
today to 58000 tons by 2020, it is surprising that knowledge on
the toxicity effect of nanoparticle exposure is in infancy and also
reported results are confusing [25–30]. Driven by the need, we re-
2. Experimental
Gold nanoparticles of different particle sizes were synthesized
using the citrate reduction method as reported recently [9–16].
Transmission electron microscope (TEM) images and UV–Vis
absorption spectra were used to characterize the nanoparticles,
as we reported recently [9–16]. The particle concentration was
measured by UV–Vis spectroscopy using molar extinction coeffi-
cients at the wavelength of maximum absorption of each gold
colloid as reported recentlyꢁ1
[
,
e(15) 518 nm = 3.6 ꢀ 108 cmꢁ1 Mꢁ1
,
,
e
e
and
(30) 530 nm = 3.0 ꢀ 109 cmꢁ1 M ꢁ1
e
(40) 533 nm = 6.7 ꢀ 109 cmꢁ1 Mꢁ1
(50) 535 nm = 1.5 ꢀ 1010 cmꢁ1
M
,
e
(60) 540 nm = 2.9 ꢀ 1010 cmꢁ1 M-1,
e
(80) 550 nm = 6.9 ꢀ 1010 cmꢁ1 Mꢁ1] [9–16].
Gold nanorods were synthesized using a seed-mediated, surfac-
tant-assisted growth method in
a
two-step procedure
[10,14,16,23]. Colloidal gold seeds (ꢂ1.5 nm diameter) were first
prepared by mixing aqueous solutions of hexadecylcetyltrimethy-
lammonium bromide (CTAB, 0.1 M, 4.75 mL) and HAuCl4 (0.01 M,
0.2 mL) followed by addition of an aqueous solution of NaBH4
(0.01 M, 0.6 mL). The colloidal gold seeds were then injected into
an aqueous growth solution of CTAB (0.1 M, 4.75 mL), silver nitrate
(0.01 M, varying amounts of silver nitrate between 20 and 120 mL
were used to achieve the desired nanorod aspect ratio), HAuCl4
(0.01 M, 0.2 mL), and ascorbic acid (0.1 M, 0.032 mL). Nanorods
were purified by several cycles of suspension in ultrapure water,
followed by centrifugation. Nanorods were isolated in the precipi-
tate, and excess CTAB was removed in the supernatant. Nanorods
were characterized by TEM and absorption spectroscopy as shown
in Fig. 1.
* Corresponding author. Fax: +1 601 979 3674.
E-mail address: paresh.c.ray@jsums.edu (P.C. Ray).
0009-2614/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.cplett.2008.08.039

146
S. Wang et al. / Chemical Physics Letters 463 (2008) 145–149
Fig. 1. Absorption spectra, photograph and TEM images of gold nanorods of different aspect ratios.
To explore the cytotoxicity of gold nonmaterials, we have used
lar uptake of different sizes and shapes of colloidal gold nanoparti-
cles. Their results show that gold nanoparticles and nanorods are
capable of entering the cells. The cellular uptake increases with
incubation time until it is saturated at 8 h.
human skin cell line, HaCaT keratinocytes, a transformed human
epidermal cell line obtained from Dr. Norbert Fusening of the ger-
many cancer research center, Heidelberg, Germany. The HaCaT
cells were grown in Dulbecco’s Modified Eagle’s Medicum (DMEM)
with 10% FBS in 25 cm2 culture flasks cultured in a humidified incu-
bator with 5% CO2 at 37 °C. After cells grew to the desired concen-
tration, they were centrifuged at 2000 rpm for 5 min. The cells were
washed twice with 1 ꢀ PBS and resuspended in DMEM to reach a
final cell concentration of 1 ꢀ 105 cells/mL for further treatment.
After treating the cells with gold nanomaterials at different time
intervals, the cell viability was determined using the MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.
Viable cells are capable of metabolizing MTT while dead cells are
To make sure that cellular uptake reaches maximum, all our re-
sults are for 24 h incubation. As shown in Fig. 2A, no difference was
found in cell viability between cell with and without gold nanopar-
ticles of different sizes. This indicates that spherical gold nanopar-
ticles of different sizes are not inherently toxic to human cells at
our test concentration. To further confirm this, we also continu-
ously exposed the cell for 48 h and no toxicity was observed. Con-
nor et al. [28] and Takahasi et al. [30] have shown that
nanoparticles or nanorods are usually clustered inside the cell.
They concluded that cell death might be the result of a harmful
influence by the aggregates. To understand whether gold nanoma-
terials are only aggregated inside the cell or may be aggregated
outside the cell and then entered as aggregate, we did a time
dependence study of gold nanoparticle’s surface plasmon reso-
nance band (SPB) between 510–570 nm. The SPB origin is attrib-
uted to the collective oscillation of the free conduction electrons
induced by an interacting electromagnetic field. These resonances
are also denoted as surface plasmon’s. Our experiments indicate
that the shift in plasmon band energy to longer wavelengths by
about 150–400 nm (Fig. 2B) in the presence of DMEM, which indi-
cated strong aggregation of gold nanoparticles (TEM image,
Fig. 2C). This aggregation is due to the presence of high concentra-
tion of sodium salt in DMEM. To understand whether other compo-
not. 10 lL of nanomaterial were added in 90 lL cell media and
incubated for 24 h at 37 °C with 5% CO2. After treatment, the med-
ium was removed and the culture wells were washed in PBS buffer.
Then we added 50
60 min. The cells were then centrifuged and the suspension was
discarded. 200 L DMSO was added to each well and incubated
lL of 5 mg/mL MTT solution and incubated for
l
for 10 min. The absorbance at 540 nm was recorded using Multis-
kan Ascent Plate Reader with the ASCENT software (Labsystems).
3. Results and discussion
The cell viabilities for gold nanoparticles of different sizes are
shown in Fig. 2. Chithrani et. al. [29] have measured the intracellu-

S. Wang et al. / Chemical Physics Letters 463 (2008) 145–149
147
A
140
120
100
80
60
40
20
0
B
C
2.5
2.0
1.5
1.0
0.5
0.0
Only DMEM
DMEM with GNP after 1 minute
DMEM with GNP after 10 minutes
DMEM with GNP after 15 minute
Only GNP
400 500 600 700 800 900 1000
Wavelength (nm)
Fig. 2. (A) Viability of cells incubated with gold nanoparticle of different sizes. (B) Time dependent absorption change in the presence of DMEM of 30 nm gold nanoparticles.
(C) TEM image for gold nanoparticle in the presence of cell media.
nents of DMEM media are also responsible for aggregation, we per-
formed aggregation experiment with simulated media without so-
dium salt. We have not observed any aggregation even after 24 h.
Even experiments with different sodium salt concentration indi-
cate that for simulated media with sodium salt concentrations less
than 0.1 M, there was no aggregation. Our experiments show that
the plasmon band shifted to longer wavelengths with time, indi-
cating bigger cluster formation. Therefore, our results show that
nanoparticles aggregated outside the cell and may enter the cell
as aggregated form. It is known that unique properties of nanopar-
ticles change as it forms bigger clusters and, as a result, our exper-
iments indicate that it is really challenging to study the gold
nanoparticle toxicity individually in cell media.
be removed by centrifugation. Our data indicate that cell viability
increases with centrifugation. As we discussed before, we started
with 0.1 M, 4.75 ml, CTAB for gold nanorod synthesis. After nano-
rod formed, the total volume was 120 ml (CTAB diluted 25 times).
To remove the excess CTAB from the solution, we centrifuged sev-
eral times. For each centrifugation, we have diluted nanorod solu-
tion 10 times. So after three centrifugations, we have diluted CTAB
by 25000 times. Since we do not know how much CTAB has been
used for nanorod formation, we estimated that maximum CTAB in
solution is <0.1 lM. CTAB-coated nanorods are positively charged.
Their mutual repulsion prevents aggregation so that gold nanorods
are stable in solution even for several months.
As shown in Fig. 3A, 1 lM CTAB is not toxic. Therefore, we do
Fig. 3A shows the cell viabilities for gold nanorods of aspect ra-
tio 3. Our experiment indicates that gold nanorod is highly toxic. It
was surprising that gold nanoparticles are not toxic, whereas gold
nanorods exhibit very high toxicity, though gold nanorods are syn-
thesized from gold nanoparticles of small size using a seed-medi-
not expect toxicity after three centrifugations. Our experimental
data indicate there is 40% cell death even after three centrifuga-
tions (Fig. 3A). We believe this is due to the fact that gold nano-
rod-bound CTAB layer will eventually enter solution in the
presence of cell media due to aggregation of nanorods as shown
in Fig. 3B and C. This is due to the screening effect of the sodium
salts present in DMEM, leading to more linked particles and hence
larger damping of the surface plasmon absorption of Au nanorod
surfaces. The aggregated gold nanorods in cell media make it diffi-
cult to monitor their toxicity. We tried to remove further CTAB
from the nanorod-bound CTAB layer by performing 4–5 times cen-
trifugation, and our experimental results shows that nanorods are
not stable if CTAB is removed further by centrifuging it more than 3
times. We believe that this is due to the lack of repulsive interac-
tion among individual nanorods and, as a result, nanorods break
to nanoparticles and some nanorods undergo aggregation.
ated, surfactant-assisted growth method. CTAB is
a unique
surfactant used for the synthesis of nanorods and is widely used.
CTAB selectively forms a tightly packed bilayer on the side faces
of nanorods, which leads the ends of the rods to be more exposed
to facilitate anisotropic growth along the longitudinal axis. Since
the extra chemical present in gold nanorod that is not in gold
nanoparticle is CTAB, we studied the toxicity of CTAB (Fig. 3A).
Our results indicate that CTAB alone shows similar toxicity at
the concentration of 100
l
M. Concentration dependent study
M concentration (data not
shows that CTAB is toxic even at 10
l
shown in Fig. 3A). In gold nanorods, there are nanorod-bound CTAB
as well as excess free CTAB in solution, which have not been used
during nanorod formation. The excess CTAB left in the solution can
To protect from CTAB which is bound with gold nanorod, we
have exposed the nanoparticles to poly(styrenesulfonate) (PSS)

148
S. Wang et al. / Chemical Physics Letters 463 (2008) 145–149
A
160
140
120
100
80
60
40
20
0
1.0
0.8
0.6
0.4
0.2
0.0
B
C
only GNR
GNR + DMEM after 1 minute
GNR + DMEM after 10 minutes
500
600
700
800
900
1000
Wavelength/nm
Fig. 3. (A) Viability of cells incubated with gold nanorod (GNR, aspect ratio 3.0) with CTAB, only CTAB and PSS coated GNR. (B) Time dependent absorption change in the
presence of DMEM for gold nanorod with aspect ratio 3.0. (C) TEM data for gold nanorod in presence of cell media.
polyelectrolyte solution. The extra PSS in solution was separated
by centrifugation of the nanorod solution at 8000 rpm. The pellet
was redispersed in N-(2-hydroxyethyl)piperazine-N0-2-ethanesul-
fonic acid (HEPES) solution. The nanorods prepared by seed-medi-
ated surfactant (CTAB) methods are positively charged. Our zeta
potential measurement using Zetasizer NanoZS shows that posi-
tively charged surface (Zeta potential 58.7 mV) of the nanorods
changed to a negatively charged surface (Zeta potential ꢁ69.8
mV) when exposed to PSS. Absorption spectra measurement indi-
cate that kmax remains about same after PSS coating, which indicate
there are no aggregation during coating process, which is also con-
firmed by TEM experiment. Our TEM data indicated that the PSS
coated outside the CTAB bilayer (as shown in Fig. 4A).
Fig. 4 shows the cell viability for PSS-coated gold nanorods with
different aspect ratios. Our experiment indicates that PSS coated
gold nanorod is not toxic. Therefore, the toxicity of gold nanorod
160
A
B
140
120
100
80
60
40
20
50 nm
0
Fig. 4. (A) TEM data for PSS-coated gold nanorod. (B) Viability of cells incubated with PSS-coated gold nanorod (3.2 aspect ration) of different aspect ratios.

S. Wang et al. / Chemical Physics Letters 463 (2008) 145–149
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Acknowledgements
We wish to thank NSF-PREM grant # DMR-0611539, NSF-MRI
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