Silver and gold nanoparticles: A toxicological aspect
Journal of Science & Technology 118 (2017) 020-025
Silver and Gold Nanoparticles: a Toxicological Aspect
Dang Minh Hieu
Hanoi University of Science and Technology – No. 1, Dai Co Viet Str., Hai Ba Trung, Ha Noi, Viet Nam
Received: September 06, 2016; accepted: June 9, 2017
Abstract
Silver and gold nanoparticles have been found in vast number of applications, especially in medicine. With
increasing and intensively uses of these nanoparticles, there is a growing concern, recently, on their
environmental impacts when they are released into environments. In this study, the size- and shape-
dependent cytotoxicity of silver and gold nanoparticles have been examined. Silver nanoparticles inhibited
the growth of the mold Aspergillus niger, and the one-dimension (Np1) and two-dimension (Np2)
nanoparticles indicated more effective than the round ones (Np0). On the other hand, gold nanoparticles of
the three types: nanostars (AuNS), polyethylene glycol coated nanostars (PEG-NS) and TAT peptide tagged
nanostars (TAT-NS), placed impact on the BT549 human breast cancer cells with reduction in the cell
viability. The PEG-NS showed more remarkable impact on the cells in compare to the others.
Keywords: Silver nanoparticles, Gold nanoparticles, Toxicity.
1. Introduction
biological systems. Several reports recently have
pointed out some toxic activities of AgNPs such as:
immunotoxicology and cytotoxicity [11, 12] and
genotoxicology by chromosomal alterations, nucleus
ablation, etc. [5, 12]. Abdelhalim and co-workers [13]
have observed various adverse effects of AuNPs on
tissue, cellular and subcellular levels of rat liver
include cloudy swelling, polymorphism, binucleation,
hyaline vacuolation, karyopyknosis, karyorrhexis,
karyolysis and necrosis, etc. Another study by Ma
and co-workers [14] also pointed out the role of
AuNPs on the accumulation of autophagosome and
the impairment of lysosome degradation capacity.
Since metal NPs, in general, are biopersistent and
biodurable, the fast increase in their applications over
the past decades has raised concerns on their effects
on health and environments [15]. Recent report has
pointed out that NPs, which tend to accumulate in the
sludge instead of in the effluents of wastewater
treatment plants, can have impact on microbial
community in agricultural soils as it showed
reduction in the fungal component when the sludge
was mixed with soils [16]. Another report has
suggested that AgNPs affect the growth and induce
modifications in nutritional content of radish [17].
Within the scope of this study, I would like to discuss
on the size- and shape-dependent cytotoxicity aspect
of silver and gold nanoparticles.
Nanoparticles * (NPs) and related technology
have gained great development over the last two
decades. Metal NPs now can be found easily
commercial available and in various applications
especially in medicine and biological sciences. From
laboratories to industry, NPs have received many
positive reviews from their users. However, there are
still very few people and researchers assessing the
downside of using NPs. Since various kinds of NPs
from different materials are currently in use, some
scientists still believe in acceptable impact of their
side effects. Nevertheless, more risk assessments of
NPs on the environments as well as plant and animal
health are necessary [1].
Among NPs, silver nanoparticles (AgNPs) and
gold nanoparticles (AuNPs) receive a lot of concerns
in all aspects from synthesis to their applications. Due
to their unique physiochemical, electrical,
mechanical, optical and thermal properties, the
AgNPs can be found in wide range of applications
including activities against bacterial and viral threats
[2, 3], incorporation in nano-scale sensors for fast
response and lower limit detection, diagnosis, drug
delivery, wound dressing, chemotherapeutic agent,
etc. [4-6]. On the other hands, the AuNPs owing to
their unique chemical and optical properties can be
found intensively in applications for drug delivery [7,
8] and biomedical imaging and diagnosis [4, 6, 9, 10].
2. Materials and Methods
2.1. Materials
As the use of metal NPs is continually
increasing, there is a demand for better understanding
the effects of the particles on ecological and
The mold strains using in this study was an
isolated strain Aspergillus niger D15 obtained from
the laboratory of Department of Microbiology –
* Corresponding author: Tel.: (+84) 4-3869-2764
Email: hieu.dangminh@hust.edu.vn
Biochemistry
Biotechnology and Food Technology, Hanoi
- Molecular Biology, School of
20
Journal of Science & Technology 118 (2017) 020-025
Fig.1. (a) SEM images of three different types of AuNPs. All AuNPs were prepared at concentration of 100
ppm. Scale bars: 100 nm. (b) Schematics for the making of TAT tagged gold nanostars.
University of Science and Technology (HUST). The
BT549 breast cancer cell line (ATCC® HTB-122™)
was a stable cell line provided by Vo-Dinh Lab. at
Department of Biomedical Engineering, Duke
University.
filtered by 0.22 μm nictrocellulose membrane. AuNS
(~60 nm diameter) were prepared using a seed
mediated method by quickly mixing AgNO3 (100 μl,
2-3 mM) and ascorbic acid (50 μl, 0.1 M) together
into 10 ml of HAuCl4 (0.25 mM) with 12 nm citrate
gold seeds (100 μl, OD520: 3.1) followed by
filtration using 0.22 μm nictrocellulose membrane.
PEG-NS were prepared by adding final 5 μM of
PEG-SH to freshly synthesized gold nanostars for 10
minutes followed by one centrifugal wash then
resuspending in pure ethanol. TAT-NS were prepared
by mixing final 100 μM of TAT peptide in 1 nM of
PEG-NS for 48 hours followed by two centrifugal
washes in ethanol. Fig. 1b shows schematics for the
three different types of gold nanostars. The
characteristics of AuNPs including zeta potential,
diameter and concentration were assessed by
nanoparticle tracking analyzer NanoSight NS500
(Malvern Instruments Ltd., UK).
Gold(III) chloride trihydrate (HAuCl4·3H2O), L
(+)-ascorbic acid (AA), trisodium citrate dehydrate, 1
N
hydrochloric acid solution (HCl), O-[2-(3-
Mercaptopropionylamino)ethyl]- O′-
methylpolyethylene glycol (mPEG-SH, MW 5000),
silver nitrate (AgNO3, 99.995%) were purchased
from Sigma-Aldrich (St. Louis, MO). Cystein-
terminated TAT peptide (residues 49–57, sequence
Arg-Lys-Lys-Arg-Arg-Arg-Gln-Arg-Cys-CONH2)
was purchased from SynBioSci (Livermore, CA).
AgNPs of Np0 (round shape), Np1 (1-dimension
shape) and Np2 (2-dimension shape) with longest
diameter of 30 to 40 nm, 10 to 100 nm and 10 to 100
nm, respectively (Fig. 1a) were obtained from
International Training Institute for Material Science
(ITIMS) at HUST, and all preserved in deionized
water at 100 ppm of concentration.
2.3. Growth inhibition test for A. niger
A. niger D15 was cultivated on PDA (Potato
dextrose agar) medium at 30oC. After 72 hours of
incubation, spores were harvested and preserved in
physiological
saline
medium
followed
by
determination of the spore density with
hemocytometer equipment. The spore solution was
then kept at 4oC in refrigerator until use.
2.2. Silver and gold nanoparticle synthesis
Three types of AuNPs: AuNS (AuNPs with star-
shape), PEG-NS (the AuNS coated with polyethylene
glycol) and TAT-PEG-NS (the PEG-NS tagged with
TAT peptide, a human immunodeficiency virus type
1 (HIV-1) encoded TAT peptide) were prepared
following methods described by Yuan et al. [18] and
Fales et al. [19]. Briefly, citrate gold seeds were
prepared by adding 15 ml of 1% trisodium citrate to
100 ml of boiling HAuCl4 (1 mM) under vigorous
stirring for 15 minutes. The solution was cooled and
The inhibition tests were conducted in glass
tubes. Each tube was prepared with 5ml of PDB
(Potato dextrose broth) medium containing AgNPs at
a certain concentration. The test concentrations for
NPs were 50, 25 and 12.5 ppm. Spore solution was
added to each test tube to the spore density of 104
spores / ml. The control tube contained spores
suspended in 5 ml of PDB without NPs. Tubes were
then incubated at 30oC with shaking for 24 hours. 100
21
Journal of Science & Technology 118 (2017) 020-025
cell lysis. Plates were then allowed to incubate at
room temperature for 10 minutes to stabilize
luminescent signal before recording with
FLOUstar® OMEGA multi-mode microplate reader
(BMG Labtech, Germany).
l of suspension from each tube was spread on plate
containing PDA medium follow with incubation at
30oC. The growth of A. niger can be justified on the
culturing plates at 24 and 48 hours of incubation.
a
2.4. Cell viability test for human breast cancer cells
3. Results and discussion
The BT549 cells were cultured in RPMI-1640
growth media (10% fetal bovine serum (FBS);
Invitrogen, Carlsbad, CA), in an incubator with a
humidified atmosphere (5% CO2) according to the
ATCC’s protocol. The viability of the cells was
measured using the CellTiter-Glo® luminescent Cell
Viability Assay (Promega, Wyoming, USA). In
principle, the amount of ATP molecules formed will
be proportional to the number of cells alive in
medium. By measuring the amount of ATP through
the luciferase reaction which catalyzes the transform
of beetle luciferin into oxyluciferin in the presence of
Mg2+, ATP and molecular oxygen and creates
luminescent light, the number of cells in culture can
be estimated. The detailed protocol for the Assay can
Briefly, BT549 cells in exponential growth phase
were prepared in 96-well plates, 100 l per well.
Blank wells contained only RPMI-1640 medium
without cells. AuNPs of three different types were
added to experimental wells to three different final
concentrations: 0.1, 0.2 and 0.3 nM, and incubated in
the incubator with a humidified atmosphere (5%
CO2). Control wells contained 100 l of cell
suspension without AuNPs. At certain time points,
plates were taken out and 100 l of CellTiter-Glo®
Reagent was added to each well. Contents were
mixed for 2 minutes on an orbital shaker to induce
3.1. Shape-dependent inhibitory effects of AgNPs
on the growth of A. niger
In these experiments, the inhibitory effects of
AgNPs on A. niger growth were tested with three
different kinds of NPs: round-shape, two- and one-
dimension as described above in the Materials.
Results indicated in Fig. 2 shows the growth of A.
niger at 24 hours of incubation on PDA plates. Spores
treated with different AgNPs at different
concentrations showed different growth capacity.
Data clearly indicated the inhibitory effects of NPs on
the spore growth, which showed increased with the
concentrations of NPs (Fig. 2a).
Comparing images taken at 48 hours of
incubation, at 50 ppm of concentration, one- and two-
dimensional AgNPs (Np1 and Np2) showed more
efficient at growth inhibition than the three-
dimensional NPs in term of retardation in spore
formation when comparing the color of colonies
formed. In case of Np0, from 24 hours to 48 hours of
incubation, all colonies developed into totally dark
colored colonies from the white ones indicated high
level of spore formation while in the case of the other
NPs, the white colonies developed into partly dark
colonies indicated uncompleted spore formation.
Several toxicity mechanisms for AgNPs have been
Fig. 2. Shape-dependent inhibitory effects of AgNPs on the growth of A. niger. (a) Mold growth at 24 hours of
incubation after treated with different AgNPs and at different concentrations. (b) Mold growth at 24 and 48
hours of incubation after treated with different AgNPs at 50 ppm of the particles’ concentration.
22
Journal of Science & Technology 118 (2017) 020-025
reported. The intensively discussed mechanism is that
were tested for their toxicity to the human breast
cancer cells, BT549. Results of the viability test, as
shown in Fig. 3, indicated that BT549 cells were
affected by all types of AuNPs. While the bare
particles (AuNS) and the TAT-NS showed little
impact on the cell viability with relatively unchanged
amount of ATP after 26 hours of incubation (Fig. 3a
and 3c), the PEG coated one showed remarkable
impact since it indicated reductions in the ATP
contents (Fig. 3b). In all cases, the impact of AuNPs
on the cells did not indicated correlation with the
concentrations of the nanoparticles. The control case
showed growing trend of the ATP content correspond
to increasing number of the cells which indicates
normal cell functions.
AgNPs can interact with cell membrane proteins,
disrupt the integrity of cell membrane, activate
signaling pathway, leading to inhibition in cell
proliferation [20]. Another mechanism which has also
been discussed involves the cellular uptake of AgNPs
by diffusion or endocytosis that cause mitochondrial
dysfunction, generation of Reactive Oxygen Species
(ROS), leading to damage of proteins and nucleic
acids inside the cells, and finally inhibition of cell
proliferation or causing cell death [20-23]. A previous
review has discussed the toxic effect of AgNPs to a
broad spectrum of common fungi and a possible toxic
mechanism of disruption of cell membrane, inhibition
of normal building process [5]. The real underlying
mechanism of action of AgNPs against fungi,
however, so far has not been unveiled
Due to their physical and chemical properties,
AuNPs recently have become attractive for biological
and biomedical applications, especially as delivery
vehicles for drugs, diagnostic tools and optical
nanomaterials [24]. In general, the toxicity of
nanomaterials can occur in several different
mechanisms in the body, which are the induction of
oxidative stress by free radical formation, interact
with cellular components, disrupt or alter cell
3.2. Shape-dependent toxicity of gold nanoparticles
on human breast cancer cells
Three types of AuNPs, which are nanostars
(AuNS), PEG coated nanostars (PEG-NS) and the
PEG-NS tagged with TAT peptide, the HIV-1
encoded peptide that were well studied for its
function as facilitating the cell penetration, (TAT-NS)
Fig. 3. Shape-dependent toxicity of (a) gold nanostars (AuNS), (b) PEG coated nanostars (PEG-NS), and
(c) TAT tagged nanostars (TAT-NS) on BT549 human breast cancer cells. Lines represents the trend-lines.
23
Journal of Science & Technology 118 (2017) 020-025
functions, and cell/tissue accumulation, etc. [25].
Aknowledgements
The author thanks to Dr. Ho Phu Ha, School of
Due to their unique properties, the toxicity of
nanomaterials can be unique from xenobiotics and
may be driven by the size, shape, chemical
composition and surface characteristics. These could
affect the mode of endocytosis, cellular uptake,
efficiency of particle processing in the endocytic
pathway, distribution and accumulation, interaction
with other molecules or cell components, formation
of free radical or decide short- or long-term toxicity.
In this study, there are three types of AuNPs have
been used in which the AuNS (around 60 nm
diameter) could be the smallest in size and the other
two could be similar in size. PEG was used for
coating the AuNS to improve its stability, and thus
increase the size of the particles. PEG, a common
pharmaceutical excipient, is used extensively in
commercial quantum dots (QDs) for stabilizing QDs
in acidic environment inside the cell after
endocytosis. Previous study on PEG-QDs found no
significant toxicity on cells, but differences in
accumulation and clearance [26]. However, another
study by Zhang and co-workers [27] has observed
size-dependent in vivo toxicity of PEG coated AuNPs
on mice at a dose of 4000 µg/kg body-weight.
Although the study could not conclude the smaller
particles have greater toxicity, the authors suggested
the further metabolism of the particles should be
considered as an important issue. On the other hand,
TAT peptide is a well-studied member of the cell-
penetrating peptides (CPPs) family, which facilitates
the transfer of the nanoparticles across cell boundary
[24]. Although there is no direct evidence, the
toxicity of AuNPs in this study could come from the
accumulation and/or interaction of the particles with
cell components, and that the toxicity of PEG-NS was
highest in compare to the others might correlate to the
ease of the transportation across cell membrane
which may decide how the integrity of the membrane
could remain.
Biotechnilogy and Food Technology and the
International Training Institute for Material Science,
HUST for generous providing silver nanoparticles.
The author also thanks to Prof. Vo Dinh Tuan,
Fitzpatrick Institute for Photonics, Duke University,
USA for providing chance to visit and practice the
making of gold nanoparticles. The author
acknowledges kind support from the members of Vo-
Dinh Lab during the visit stay.
References
[1] Ai J. et al. Nanotoxicology and nanoparticle safety in
biomedical designs. Int J Nanomed. 6 (2011): 1117-
1127.
[2] Lara H. H. et al. Silver nanoparticles are broad-
spectrum bactericidal and virucidal compounds. J
Nanobiotechnology 9 (2011):30.
[3] Taylor E. and Webster T. J. Reducing infection
through nanotechnology and NPs. Int J Nanomed. 6
(2011): 1463-1473.
[4] Austin L. A. et al. The optical, photothermal, and
facile surface chemical properties of gold and silver
NPs in biodiagnostics, therapy and drug delivery.
Arch Toxicol. 88 (2014): 1391-1417.
[5] Ge L. et al. Nanosilver particles in medical
applications: synthesis, performance and toxicity. Int
J Nanomed. 9 (2014): 2399-2407.
[6] Srisam M. et al. Single Nanoparticles Plasmonic
Sensors. Sensors 15 (2015):22774-22792.
[7] Calixto G. M. F. et al. Nanotechnology-Based Drug
Delivery Systems for Photodynamic Therapy of
Cancer: A Review. Molecules 21 (2016):342.
[8] Mocan L. et al. Advances in cancer research using
gold nanoparticles mediated photothermal ablation.
Clujul Medical 89(2) (2016):199-202.
[9] Vo-Dinh T. et al. Plasmonic Nanoprobes: From
Chemical Sensing to Medical Diagnostics and
Therapy. Nanoscales 5(21) (2013):10127-10140.
4. Conclusions
In conclusion, the study has assessed the health
and environmental risks of silver and gold
nanoparticles. It pointed out that AgNPs can inhibit
the growth of the mold A. niger and the one- and two-
dimension particles shows more effective than the
round (none-dimension) ones. Three types of AuNPs
shows affected the viability of the BT549 human
breast cancer cells. However, the PEG-NS indicates
highest toxicity to the cells in compare to the two
other AuNPs. Future study should focus on the
interaction, accumulation, distribution of the
nanoparticles in order to unveil the mechanism of
their toxicity.
[10] Bucharskaya A. et al. Towards Effective
Photothermal/Photodynamic
Treatments
Using
Plasmonic Gold Nanoparticles. Int. J. Mol. Sci. 17
(2016):1295.
[11] Fröhlich E. Value of phagocyte function screening for
immunotoxicity of NPs in vivo. Int J Nanomed. 10
(2015): 3761-3778.
[12] Katsnelson B. A. et al. Some inferences from in vivo
experiments with metal and metal oxide NPs: the
pulmonary phagocytosis response, subchronic
systemic toxicity, genotoxicity, regulatory proposals,
searching for bioprotectors (a self-overview). Int J
Nanomed. 10 (2015): 3013-3029.
24
Journal of Science & Technology 118 (2017) 020-025
[13] Abdelhalim M. A. K. et al. Gold nanoparticles Enhanced Raman Scattering. J. Phys. Chem. 118
induced cloudy swelling to hydropic degeneration,
cytoplasmic hyaline vacuolation, polymorphism,
binucleation, karyopyknosis, karyolysis, karyorrhexis
and necrosis in the liver. Lipids in Health and
Disease 10 (2011):166.
(2014): 3708-3715.
[20] McShan D. et al. Molecular Toxicity Mechanism of
Nanosilver. J Food Drug Anal. 22(1) (2014): 116-
127.
[21] Rinna A. et al. Effect of silver nanoparticles on
mitogen-activated protein kinases activation: role of
reactive oxygen species and implication in DNA
damage. Mutagenesis 30 (2015): 59-66.
[14] Ma X. et al. Gold Nanoparticles Induce
Autophagosome Accumulation through Size-
Dependent Nanoparticle Uptake and Lysosome
Impairment. ACS Nano 5(11) (2011):8629-8639.
[22] Kaba S. I. and Egorova E. M. In vitro studies of the
toxic effects of silver nanoparticles on Hela and U937
cells. Nanotechnology, Science and Applications 8
(2015): 19-29.
[15] Utembe W. et al. Dissolution and biodurability:
Important parameters needed for risk assessment of
nanomaterials. Particle and Fibre Toxicology 12
(2015): 11.
[23] Bao H. et al. New Toxicity Mechanism of Silver
Nanoparticles: Promoting Apoptosis and Inhibiting
Proliferation. Plos ONE 10(3) (2015): e0122535.
[16] Durenkamp M. et al. NPs within WWTP sludges
have minimal impact on leachate quality and soil
microbial community structure and function. Environ.
Pollut. 211 (2016): 399-405.
[24] Lévy R. et al. Gold nanoparticles delivery in
mammalian live cells: a critical review. Nano
Reviews 1 (2010): 4889.
[17] Zuverza-Mena N. et al. Effect of silver NPs on radish
sprouts: Root growth reduction and modifications in
the nutritional value. Front. Plant Sci. 7 (2016): 90.
[25] Aillon K. L. et al. Effects of nanomaterial
physicochemical properties on in vivo toxicity. Adv
Drug Deliv Rev 61(6) (2009): 457-466.
[18] Yuan H. et al. TAT Peptide-Functionalized Gold
Nanostars: Enhanced Intracellular Delivery and
Efficient NIR Photothermal Therapy Using Ultralow
Irradiance. J. Am. Chem. Soc. 134 (2012): 11358-
11361.
[26] Ballou B. et al. Noninvasive imaging of quantum dots
in mice. Bioconjug. Chem 15 (2004):79–86.
[27] Zhang X-D. et al. Size-dependent in vivo toxicity of
PEG-coated gold nanoparticles. Int J Nanomed. 6
(2011):2071-2081.
[19] Fales A. M. et al. Development of Hybrid Silver-
Coated Gold Nanostars for Nonaggregated Surface-
25
6 trang
18/04/2022
920
Bạn đang xem tài liệu "Silver and gold nanoparticles: A toxicological aspect", để tải tài liệu gốc về máy hãy click vào nút Download ở trên
File đính kèm:
- silver_and_gold_nanoparticles_a_toxicological_aspect.pdf
