Reprotoxicity of Gold, Silver, and Gold-Silver Alloy Nanoparticles On Mammalian Gametes

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Reprotoxicity of gold, silver, and gold-silver alloy nanoparticles on

mammalian gametes
Article in The Analyst · October 2013

DOI: 10.1039/c3an01463k · Source: PubMed
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Reprotoxicity of gold, silver, and gold-silver alloy nanoparticles on mammalian gametes

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DOI: 10.1039/C3AN01463K
Daniela Tiedemann1#, Ulrike Taylor1#, Christoph Rehbock2, Jurij Jakobi2, Sabine Klein1,
Wilfried A. Kues1, Stephan Barcikowski2*, Detlef Rath1*
1
Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Federal Research Institute of
Published on 07 October 2013. Downloaded by Universitaet Duisburg Essen on 17/10/2013 07:01:10.
Animal Health, Mariensee, Germany

2
Technical Chemistry I and Center for Nanointegration Duisburg-Essen (CENIDE),
University of Duisburg-Essen, Essen, Germany
Analyst Accepted Manuscript

# Both authors contributed equally
* Corresponding authors
Concerning biological matters:
Prof. Dr. Detlef Rath
Institute of Farm Animal Genetics (FLI)
Höltystraße 10
31535 Neustadt
Germany
Phone: +49 5034 871 144
Fax: +49 5034 871 101
rath@tzv.fal.de
Concerning nanoparticles and laser technology:
Prof. Dr. Stephan Barcikowski
Technical Chemistry I and Center for Nanointegration Duisburg-Essen (CENIDE)

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University of Duisburg-Essen
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DOI: 10.1039/C3AN01463K
Universitätsstraße 7
45141 Essen
Germany
Phone: +49 201 183 3150
Fax: +49 201 183 3049

stephan.barcikowski@uni-due.de
Keywords: oocytes, maturation, spermatozoa, toxicity, pig model

Abstract
Metal and alloy nanoparticles are increasingly developed for biomedical applications, while a
firm understanding of their biocompatibility is still missing. Various properties have been
reported to influence the toxic potential of nanoparticles. This study aimed to assess the
impact of nanoparticle size, surface ligands and chemical composition of gold, silver or gold-
silver alloy nanoparticles on mammalian gametes. An in vitro assay for porcine gametes was
developed, since these are delicate primary cells, for which well-established culture systems
exist and functional parameters are defined. During coincubation with oocytes for 46h neither
any of the tested gold nanoparticles nor the gold-silver alloy particles with a silver molar
fraction of up to 50% showed any impact on oocyte maturation. Alloy nanoparticles with 80%
silver molar fraction and pure silver nanoparticles inhibited cumulus-oocyte maturation.
Confocal microscopy revealed a selective uptake of gold nanoparticles by oocytes, while
silver and alloy particles mainly accumulated in the cumulus cell layer surrounding the
oocyte. Interestingly sperm vitality parameters (motility, membrane integrity and
morphology) were not affected by any of the tested nanoparticles. Only sporadic association
of nanoparticles to the sperm plasma membrane were found by transmission electron

microscopy. In conclusion, mammalian oocytes reacted sensitive to silver containing

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nanoparticles. Likely, the delicate process of completing meiosis in maternal gametes features
high vulnerability towards nanomaterial derived toxicity. The results imply that released Ag+-
ions are responsible for the observed toxicity, but the incorporation into an alloy seemed to
alleviate the toxic effects to a certain extent.

Introduction
Nanotechnology is considered to be one of the key technologies with high future impact on
technical as well as biological and medical applications 1. Due to their small size and relative

large surface area nanoparticles (NP) have unique physiochemical properties. These attributes
may cause adverse effects when introduced into biological systems 1. Therefore, it is
2
important to thoroughly understand the risks exerted by nanomaterial exposure to provide
reliable information to governments and regulating authorities deciding about nanomaterial
safety guidelines 3.
4-15
With respect to potential repro-toxicological effects mainly piscine embryos and to a
16-21 22-28
lesser extend avian embryos and mammalian embryos have been investigated,
showing a varying degree of sensitivity related to nanoparticle composition, production
method, and exposure route.
Gamete quality plays a vital role for ontogenesis 29. The influence of nanoparticles on a single
gamete may already cause tremendous developmental differences. Impairment of gametes

30
may affect reproductive functions or have pathological influence on the next generation .
Yet, despite their importance, studies concerning the sensitivity of gametes towards
nanoparticle exposure are comparatively scarce. Regarding spermatozoa, no toxicity was

31, 32
found for PVA and PVP-coated iron and europium hydroxide NPs . Moderate effects
were detected for titan dioxide, gold, silver and zinc oxide NP 33-37, while one study indicated
severe effects for europium trioxide NP 32. Oocytes have only been examined after exposure
to TiO2NP 38 and CdSe-core quantum dots (QD) 39. Both treatments had a negative influence
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on oocyte quality, which in case of the QDs could be obliterated after coating with ZnS.
We developed an in vitro assay for assessing the repro-toxic potential of NP on mammalian
gametes. The approach allows investigating well-defined primary cells with clearly stated
functions using internationally standardized protocols of in vitro fertilization (IVF). Oocytes
and spermatozoa possess very different features with regard to metabolic activity, membrane
composition, and compartmentalization, which facilitates to investigate in how far such
properties influence sensitivity towards potentially toxic substances. The cumulus-oocyte-
complexes and spermatozoa used in the study were of porcine origin. The pig as reference

model is frequently used in embryo-toxicologal studies 40-43. Porcine physiology, metabolism,
44
and pathology reflect the human situation much better than small animal models . A
previous phylogenetic study suggested that the pig genome is threefold closer to the human
45
than the mouse , and recently the newest genome assembly (10.2) of the porcine genome
was released (www.ensembl.org/sus_scrofa/Info/Index).
A currently emerging field for the synthesis of colloidal metal and alloy nanoparticles is
46 47 48 49
pulsed laser ablation in liquid (PLAL) . One advantage of this method is the high
purity of the obtained colloids as no organic stabilizers are required during synthesis. This is
particularly useful in toxicological assays in order to rule out cross contaminations by organic
ligands. Excellent colloidal stability in these particles is caused by partial oxidation of the
nanoparticle surface during the ablation process, which e.g. in the case of gold contains 3.3 –
6.6 % of Au+/Au3+ species 50. These oxidized metal surfaces attract oxygen from the aqueous
medium yielding a pH-dependent equilibrium between Au-OH and Au-O- groups on the
51
surface and causing a negative zeta potential . Another advantage of PLAL is its high
52, 53
flexibility as to the used solvent and particularly concerning the used educt material
54 55 56
giving access to metal and semiconductor nanoparticles . Additionally, this synthesis
57 58 59 60 61 62, 63
route has been successfully used for the fabrication of alloy nanoparticles ,
which are often difficult to produce by chemical reduction methods due to the unavailability
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of suitable precursor species.
Besides playing an important role in biomedical applications, the in this project tested types
of nanoparticles are addressed as model particles with "tuneable" properties. The interactions
at the nano-bio-interface depend on nanoparticle size, material composition, shape, surface
area, surface properties, solubility, and charge 3, 64, 65. As alloy formation is known to alter the
properties of raw materials, this may further change their biological impact. This complex
matrix necessitates systematic approaches in order to predict the impact of the different
properties on the toxic potential of the nanoparticles. The obtained findings will increase our

understanding of mechanisms concerning nanoparticle biocompatibility.
Results and discussion
Development of an in vitro reprotoxicity-assay employing porcine gametes
Figure 1 schematically depicts the experimental design to assess reprotoxicity of different NP.
Prior to ovulation oocytes mature and become competent for fertilization in the ovarian
follicle. The important timing of these events is controlled by the oocytes surrounding
granulosa cells (cumulus cell layers). They prevent the resumption of the meiotic processes
until the oocyte starts to undergo the final development before ovulation. These processes are
mimicked under in vitro conditions and serve as a sensitive functional test. Cumulus-oocyte-
complexes were isolated from porcine ovaries, collected at a slaughterhouse. The in vitro
maturation process of 46h to gain fertilization-competent oocytes is a standard procedure
when performing IVF. Timing and completeness of oocyte maturation is viewed as sensitive
toxicological parameter, since it is already impaired by substance concentrations far below

66, 67
concentrations that influence the viability of the matured oocyte . As functional
parameters of oocyte maturation the formation of metaphase plate of the second meiosis and
cumulus cell expansion were assessed.

Porcine spermatozoa derived from fresh ejaculates were co-incubated with nanoparticles for
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two hours, and the following functional parameters were determined: Sperm motility, sperm
membrane integrity, and morphological abnormalities.
For oocyte maturation trials pure gold nanoparticles were employed in various sizes (6 nm
and 20 nm) and concentrations (10 µg/ ml and 30 µg/ml) carrying different surface ligands
(BSA and BSA/Citrate) to examine whether differences in those properties influence the toxic
potential. In spermatozoa only one pure gold nanoparticle variant was tested (20 nm, 10
µg/ml, BSA coating). Furthermore, by using several variants of gold-silver alloys as well as
pure silver nanoparticles, the effect of an increasing electro-chemical potential (respective ion

release rate) and hazardous potential of the related metal ions on cumulus-oocyte-complexes
and spermatozoa were tested. Stabilisers and reducing agents, which are difficult to exclude in
precursor-based, chemically produced gold and silver nanoparticles, may have effects on their
own. In the present study particles were of maximum purity realized by synthesis of colloids
employing laser ablation of a bulk solid target in water 48, 68, 69.
When nanoparticles are transferred into biological settings they are usually exposed to a
70
multitude of proteins, which adsorb to the nanoparticle surface forming a protein corona .
65, 71
Such coronas may considerably influence the biological identity of the particle . To
standardize the conditions and at the same time mimic the in vivo settings, all nanoparticle
variants were ex situ coated with bovine serum albumin (BSA).
Characterisation of the produced gold, silver and gold-silver alloy nanoparticles
The synthesised gold nanoparticles exhibited a distinct surface plasmon resonance based on
an extinction peak around 520 nm, while a peak at 424 nm was observed for silver
nanoparticles. The extinction peak for alloy particles ranged between 497 nm for gold-silver
80:20 alloys, 451 nm for the gold-silver 50:50 alloys and 415 nm for the gold-silver 20:80
alloys. Figure S1 displays the UV-Vis spectra of all employed nanoparticles in detail. Figure
2A shows exemplary AuAg alloys with different molar fractions. The appearance of only one
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peak in the UV-Vis spectrum is a clear indicator of alloy formation. The wavelength of the
SPR-peak linearly scales with the alloy composition (Figure 2B), as it has been previously
described in literature58. An EDX line scan of a high – angular annular dark field micrograph
confirmed the formation of homogeneous AuAg nanoparticles (Figure 2C). An exemplarily
TEM micrograph of well-distributed laser-generated Au50Ag50 alloy nanoparticles after

stabilization with BSA is provided in figure 2D.
The primary particle diameter consisted of 6 and 20 nm for AuNP when tested at 10µg/ml and
8 nm for the tests at 30 µg/ml. Gold-silver alloy nanoparticles measured 6 nm (20% and 50%

silver molar fraction) and 7 nm (80% silver molar fraction), respectively. Silver nanoparticles
had a size of 11 nm. All values including additional characteristics and number dosages are
listed in table 1. Frequency distributions of the nanoparticle diameters as measured in an
analytical disc centrifuge are depicted in figure S2. A more detailed characterization of these
nanoparticles at average diameters of 6 nm and 20 nm has recently been published 72. In this
paper size control of laser-fabricated nanoparticles was performed by the addition of low
salinity electrolytes like NaCl, present during the laser ablation synthesis. Significant
differences in the average diameters of these particles were verified using analytical disk
centrifugation, SEM and TEM. Reproducibility of this process was demonstrated by
comparing 3 samples fabricated under equivalent conditions. It should be noted that for
smaller nanoparticles good reproducibility (6±1.5 nm) and narrow size distributions
(PDI=0.1) were found. Larger nanoparticles showed higher error margins (20±7 nm) and
wider size distributions (PDI=0.3). In order to further compare the size distributions of the
two samples used in this work and to quantify overlaps, the total number of particles found in
the fractions >10 nm and < 10 nm were calculated. In samples with 6 nm diameter about 89 %
of the particles are < 10 nm and 11% are > 10 nm, while in 20 nm samples an opposite trend
is found (15% < 10 nm; 85% > 10 nm).

Exemplarily the zeta potential of the 6 and 20 nm sized pure gold nanoparticles was measured
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before BSA coating as well as 24h after medium transfer to display the relevant surface
charge for interactions at the nano-bio-interface. As expected due to the BSA and the post-
transfer adsorption of various medium derived proteins the zeta potential of the 6 nm gold
particles increased from -44 ±0.9 mV to -2.87 ± 0.73 mV, and of 20 nm gold particles from -
42 ±0.7 mV to -7.1 ±0.5 mV. However, results obtained from the zeta potential measurements
must not be overinterpreted when discussing the total surface charge of nanoparticles with
regard to colloidal stability. The main limitation in this case is the comparison between
particles dispersed in simple aqueous media and those measured in the presence of proteins

and other medium components, while in both cases simple Smoluchowski fittings were used.
As it is well known from literature, interpretation of zeta potential measurements is hindered
by the presence of complex ligands like proteins containing various charged organic residues.
These may significantly interfere with the retardation force during the measurement of the
electrophoretic mobility73. Even though in this case zeta potential measurements at a sum >
30 mV are found for particles dispersed in medium, which is generally considered as an
indicator for impaired colloidal stability, excellent long-term stability could be observed in all
the used samples. Most probably steric stabilization by the BSA protein corona prevents
particle aggregation. Long term stability of the used particles in Androhep semen extender
over a time period of 28 days has been verified in previous studies conducted by the authors72.
As indicated by time-resolved UV-Vis measurements, a constant scattering intensity at 800
nm, and hence no significant aggregation occurred during long-term exposition to the cell
culture medium.
Effect of gold, silver and gold-silver alloy nanoparticles on cumulus-oocyte complexes
Exposure of cumulus-oocyte-complexes to pure gold nanoparticles during in vitro maturation
resulted in a considerable accumulation of particles inside the oocyte as determined by laser

scanning confocal microscopy (LSCM; Figure 3B). By LSCM only particles and particle
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74 DOI: 10.1039/C3AN01463K
aggregates with a diameter >60 nm can be visualised . Therefore, the observed signals were
probably derived from particle aggregates, since primary particle size did not exceed 20 nm.
Cumulus cells surrounding the oocyte did not contain any detectable gold aggregates. Yet,
despite the high content of intracellular particles none of the tested gold nanoparticle variants
showed an effect on oocyte maturation, regardless of nanoparticle size (6 nm versus 20 nm),

coating (BSA versus sodium citrate) or concentration (10 µg/ml [equal to a number dose of
1.94x1010 NP/oocyte] versus 30 µg/ml [equal to 8.65x1010 NP/Oocyte]) (Figure 4).
Interestingly, NP containing silver reacted quite differently. In contrast to pure gold

nanoparticles, even the particles with least silver molar fraction (20%) were preferentially
found in the cumulus cell layers (Figure 3C, D). While nanoparticle uptake behavior was
concentration independent, a clear concentration dependency was noted for toxic aspects.
After coincubation with nanoparticles containing 20% and 50% silver the maturation rate did
not differ from the controls. If the silver molar fraction increased to 80% and above, oocyte
maturation decreased significantly (Figure 4). Coincubation of oocytes with Ag+ ions (added
as AgNO3 [12,5 µg/ml]), in a concentration based on the calculated Ag+ ion content of the
alloy particles consisting of 80% of silver, led to a complete arrest of maturation.
Why pure gold nanoparticles and silver containing nanoparticles differed so substantially in
their uptake behavior can only be speculated about. The particles surface net charge, which is
one of key features for interactions at the nano-bio-interface 64 , did not differ among particle
types. All particles were BSA coated before transfer into the maturation medium. However,
after medium transfer particles were in contact with a new set of biomolecules. Adsorbing
rapidly to the particles' surface, they fit a new biological identity to the nanoparticle, which is
75
the one that is recognized and ultimately acted upon by the cellular machinery .
Biomolecule adsorption kinetics differ in dependence of nanoparticle properties 76. Therefore,
one possible hypothesis for the differential particle uptake, i.e. uptake of pure gold particles
preferentially into oocytes and uptake of silver containing particles preferentially into
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cumulus cells, might be that the presence of silver in a nanoparticle led to the adsorption of a
biomolecule mixture, which facilitates the preferential accumulation at the cumulus cell layer.
With regard to toxicity, of the various parameters tested (size, surface modification,
composition) the particle composition had the largest impact. The observation that the
reprotoxic potential of gold nanoparticles is low and that a concentration dependant toxic
effect of silver containing nanoparticles exists is in good agreement with literature 4, 5, 7-9, 11, 16,
23, 77
. While being the first report testing gold nanoparticles on mammalian cumulus-oocyte-
4, 5
complexes, it confirms the finding of other repro-toxicological studies using piscine and

avian embryos 16. In the present work a size effect could not be confirmed. However, a study
examining gold clusters (<2 nm) found a detrimental effect after application of a 10000 fold
higher dose (1014 NP/embryo, 6). Nevertheless, in our opinion, the high biocompatibility
recommends use of gold nanoparticles in bio-applications such as novel biomarkers, cancer
imaging, and therapy as well as drug delivery 78, 79.
Silver nanoparticles displayed considerable repro-toxicity in several trials performed with

4, 7-9, 11, 77 23
embryos of piscine and murine origin . Interestingly, no effect was found in
18-20
studies testing the toxicity of silver nanoparticles on chicken embryos . In this study we
varied silver concentration by using gold-silver alloy particles with different molar fractions
of silver observing an concentration dependent detrimental effect on maturation rate. As the
silver containing particles were primarily found in cumulus cells, it has to be discussed
whether this was the consequence of an inhibited cumulus expansion allowing continious
transport of the germinal vesical break down inhibitor into the germinal vesicle. A possible
cause for the toxic effect might be Ag+-ions dissolving of the nanoparticles. The effect of ion
release of silver nanoparticles is an object of current investigations and is widely discussed in

80, 81 82 93
the literature . It has been shown that gold-silver nanoparticles have different
influence on human mesenchymal stem cells (hMSC) compared to pure silver and gold
83
nanoparticles . It has to be emphasised that laser-generated silver-gold nanoparticles
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compared to chemically prepared nanoparticles have a homogeneous ultrastructure. Resulting
from the ablation process, element distribution is homogeneous (Figure 2C), whereas a
chemical reduction process leads to slight element gradient due to the different reduction
potentials83. Nevertheless, alloying of the silver nanoparticles with gold leads to different
dissolution kinetics of silver ions. It could be expected that silver ion release of alloy
nanoparticles depends only on the silver content and is controlled by the oxidation resistance
84
of alloys with increasing gold fraction due to the change of the electrochemical potential .
85
However, some anomalies for gold-silver systems were shown by Alissawi et al. . The

authors found that the entropy of mixing of gold-silver nanoalloy in polytetrafluoroethylene
nanocomposite had big influence on silver ion release. For a short time (40 min) after
exposure of alloys to water, beside the pure silver, the highest ion release was found for
Au50Ag50, with a minimum for Au70Ag30 and a slow increase to Au90Ag10. After one day of
exposure, the ion release turned and increased relative to the amount of silver in the alloy 85.
Considering these findings, the toxicity of gold-silver alloy nanoparticles may also depend on
exposure time to the biological system. Since nanoparticle coincubation with cumulus-oocyte-
complexes took place over 46h silver ion release had probably already become proportional to
the amount of silver in the alloy. As the positive controls with Ag+-ions showed similar
detrimental effects as the higher doses silver nanoparticles, it seems likely that the dissolution
of silver ions from the nanoparticle surface is the mode of action 86. It is noteworthy that the
alloy particles containing 50% silver had no statistically significant effect. In opposite
approximately the same silver concentration (3.3 µg/ml) and less has repeatedly been reported
7-9, 77
to have reprotoxic effects . This suggests that incorporation of silver into an alloy
structure may be used to control the toxicity of silver ions and might therefore allow broader
biomedical applications of such alloy nanoparticles.

Effect of gold, silver, and gold-silver alloy nanoparticles on spermatozoa

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Porcine spermatozoa derived from fresh ejaculates were co-incubated with nanoparticles for
two hours, and essential fertility parameters such as motility, membrane integrity, and
morphological abnormalities were determined.
Co-incubation of spermatozoa with BSA coated gold, silver and gold-silver alloy
nanoparticles did not result in nanoparticle uptake by the spermatozoa. Even adsorption to
intact membrane could only be witnessed occasionally (Figure 5). With regard to gold
nanoparticles this stands in contrast to other studies investigating gold nanoparticle-sperm-

34 36
interactions, reporting internalisation of citrate stabilised and PVP coated gold

nanoparticles. However, Taylor et al. 87 reported membrane association but not internalisation
of ligand-free and oligonucleotide conjugated gold nanoparticles. Concerning silver

36
nanoparticles our results are in agreement with Moretti et al. , who examined PVP coated
silver nanoparticles and also mentioned a lack of nanoparticle adsorption and internalisation.
Again it is presumably the final biomolecular corona, which determines how nanoparticle-
sperm interactions proceed. A comparison of the mentioned studies does not permit the
34 36
derivation of a clear trend though, since neither Wiwanitkit et al. , nor Moretti et al.
provide information regarding the ingredients of the dilution medium, thus no conclusion can
87
be drawn how the final corona might be composed. Taylor et al. used ligand-free and
oligonucleotide conjugated gold nanoparticles in a medium containing anorganic salts and
fructose, but no protein source. It seems plausible that those particles bind to the sperm
surface since ligand-free AuNP display a positive surface charge making them attractive to
88
the net negatively charged sperm surface membrane , while oligonucleotide conjugated
nanoparticles could have bound directly to nucleic acid specific binding sites possessed by
spermatozoa 89. In the current trials, BSA coated nanoparticles were suspended in a medium
containing BSA, which surprisingly seems to inhibit nanoparticle adsorption to the sperm
90
membrane, since albumins have been described to adsorb to the sperm membrane .
However, the adsorption of albumins to the surface of gold nanoparticles has been described
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to induce a conformational change, resulting in diminished α-helix content and a higher
91
content of β-sheets and random or expanded structures , which might interfere with the
adsorption of the BSA-gold nanoparticle complexes to the sperm membrane.
Equally surprising was the complete lack of toxicity, which was observed after coincubation
with the various nanoparticle types by measuring post-incubation sperm motility, membrane
integrity, and morphology (Figure 6). All previously published studies reported a negative
34, 36, 87 36
effect of gold as well as silver nanoparticles on sperm viability and functionality
parameters. However, at least with respect to gold all studies described close membrane

87 34, 36
association of the nanoparticles or even nanoparticle penetration while in the current
work such contact was not observed at all. This suggests that detrimental effects of gold
nanoparticles require direct interactions with the exposed spermatozoa. With regard to silver
36
nanoparticles, Morreti et al. did not observe any sperm-nanoparticle contact while still
observing toxicity with nanoparticle doses approximately equal to the ones used in this study.
Therefore, silver nanoparticles do not seem to require direct contact to cause an effect. This is
not surprising, since it is generally presumed that Ag+-ions dissolving from the silver
nanoparticles are the active agent of silver nanoparticle toxicity 86. However, it has also been
described that Ag+-ions are adsorbed to BSA 92

and that the presence of BSA can reduce the
93
cytotoxic effect of silver nanoparticles . Therefore we hypothesize that BSA coating of the
nanoparticle as well as additional BSA in the sperm dilution medium captured the majority of
free Ag+-ions and thus alleviated silver nanoparticle toxicity on sperm. The fact that the co-
incubation of spermatozoa with free Ag+-ions in form of AgNO3 diluted in sperm extension
medium equally had no significant effect, underlines the proposed hypothesis (Figure 6).
It is noteworthy that in opposite to the effects on oocytes and cumulus-oocyte-complexes,
albumin seems to protect spermatozoa from the Ag+-ion derived toxicity of silver
nanoparticles. In their case the Ag+-ions, which remained unbound to albumin due to the
chemical equilibrium sufficed to inflict detrimental damage. Apparently, the delicate

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processes, which cumulus-oocyte-complexes undergo when completing the second meiotic
divisions, make them considerably more vulnerable compared to spermatozoa whose
chromatin is completely inert. This emphasizes the proposition made by Beker van
66 67
Woudenberg et al. and Lazzari et al. that oocyte maturation is one of the most sensitive
parameters for the assessment of toxicity. Sperm functions seem to be better protected against
BSA coated nanoparticles as long as their membrane system is intact. However, sperm
development may be impaired during spermatogenesis as data from Antonini 94 indicates after
exposure of men to welding dust. Unfortunately, other than the final steps of oogenesis,

equivalent phases of spermatogenesis are not established as in vitro methods.
Experimental
Oocyte maturation in vitro
Pig ovaries were collected at a local abattoir and transported to the laboratory in an isolated
bucket within two hours. Upon arrival the ovaries had a temperature of 30-32°C. Ovaries
were washed with saline and cumulus-oocyte-complexes were aspirated from follicles with a
size of 3-4 mm and washed in Dulbecco´s phosphate buffered saline (AppliChem, Darmstadt,
Germany) supplemented with 1% new born calf serum. Only cumulus-oocyte-complexes with
at least three complete layers of cumulus cells and evenly granulated cytoplasm were selected
for the experiments. Groups of 50 cumulus-oocyte-complexes were placed into wells of 4-
well culture dishes (Nunc A/S, Kamstrupvej, Denmark) containing 500µl maturation medium
supplemented with 125µL nanoparticle solution (Table 2). After 46h at 38.5°C and 5.5% CO2
the degree of cumulus cell expansion was rated on a scale from 0-3 95 (0 = no expansion, 1 =
slightly expanded, still close association to zona pellucida, 2 = expanded, still some cells with
contact to each other, 3 = highly expanded, no more cell to cell contact) before removing
them from the maturation medium. Cumulus cells were removed by vigorous pipetting and
oocytes were fixed in ethanol and acetic acid (3:1) on glass slides. Lacmoid stain was applied
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after 24h to visualize the DNA of nucleus and polar body. Maturation rate was determined by
number of oocytes having reached the metaphase of the second meiosis and the according
polar body extrusion. The experiments were repeated seven times, which equals 350 oocytes
per treatment group.

Viability of spermatozoa
For sperm viability tests fresh ejaculated spermatozoa were used from three large-white boars
of proven fertility. The sperm rich phase of each ejaculate was collected and the initial

concentration was determined with the NucleoCounter® SP-100™ (ChemoMetec A/S,
Allerød, Denmark). Spermatozoa were washed twice in Androhep™ (Minitüb GmbH,
Tiefenbach, Germany) (Table 3) and diluted to a final concentration of 100x106
spermatozoa/ml with Androhep™, to which the NP had previously been added. One millilitre
samples were incubated for 2h at 37°C. Motility was evaluated using computer assisted sperm
analysis (CASA, HTM-IVOS 12, Hamilton Thorne Biosciences, Beverly, USA) ) and under a
phase contrast microscope (200x). Membrane integrity was tested with propidium iodide (PI,
Life Technologies, Darmstadt, Germany). Samples were incubated for 10 minutes and
analysed flow cytometrically using a FACScan (Becton Dickinson Biosciences, Heidelberg,
Germany). Morphology of spermatozoa was evaluated using phase contrast microscopy
(1000x under oil; 200 cells). Seven repeats of each treatment group were performed.
Particle synthesis
Size controlled gold nanoparticles of 6 nm and 20 nm were synthesized by pulsed laser
ablation of a gold wire in liquid-flow and in the presence of highly diluted electrolytes (3 µM;
100 µM) 72. The particles were stabilized ex situ with a final BSA concentration of 2.5 g/l and
further NaCl and water was added in order to adjust the ionic strength in all samples to a
constant level of 50 µM. Additional sodium citrate at a final concentration of 1 mM was

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DOI: 10.1039/C3AN01463K
added to some of the samples synthesized in the presence of 100 µM NaCl.
Gold nanoparticles at high concentrations (120 µg/ml) were synthesized using ns-pulsed laser
ablation (Nd-YAG (Rofin Power Line E); λ=1064 nm, repetition rate =20 kHz, power =5 W)
of a gold foil (99.99 % purity, Allgemeine-Gold, thickness = 0.5 mm) in 200 µM NaCl
applying a flow-through chamber. The chamber was fabricated out of acryl glass with a total
volume of 620 µl and tubings with an inner diameter of 0.3 cm. The flow was propelled by a
syringe pump (ProSense NE 300) and kept at a constant rate of 5 ml/min. Focusing was done
with a f-theta lens (f=100 mm) and the focused beam was moved on the target using a scanner

system (Scanlab, SCANcube10) ablating a rectangular pattern with a diameter of 2 mm and a
length of 5 mm on the target. Ablation was carried out for 5 min yielding a total sample
volume of 25 ml. After synthesis BSA with a final concentration of 0.5 g/l was added for
stabilization.
Silver and gold-silver alloy nanoparticles were synthesized by pulsed ps-laser ablation (Expla
Atlantic 1064 at λ= 1064 nm, repetition rate = 100 kHz, power = 15 W) of Ag (Goodfellow
GmbH) and alloy foils ( Ag20Au80, Ag50Au50 , Ag80Au20, custom made by the “Institute for
noble metal and metal chemistry Schwäbisch-Gmünd”) with a thickness of 0.5 mm in

96
deionised water using an aluminium batch chamber (Figure 2E) with a total volume of 30
ml. Focusing was done with a f-theta lens (f=100 mm) and the focused beam was moved on
the target using a scanner system (Scanlab, SCANcube10) ablating a helical pattern with a
diameter of 6 mm. After synthesis the samples were diluted to a total mass concentration of
150 µg/ml. Stabilization was done with BSA at a final concentration of 0.66 g/l for alloy
nanoparticles and with 2.5 g/l for silver nanoparticles.
Particle characterization
The mass concentrations in the silver and alloy targets were gravimetrically determined by
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DOI: 10.1039/C3AN01463K
weighing the ablated targets prior to ablation and afterwards, using a microbalance (Precisa
XR205 SM-DR). For gold samples, mass concentrations were determined by UV-Vis
spectroscopy acquiring spectra in a range from λ= 200 nm – λ=900 nm in a cuvette with 10
mm path length and 1.5 ml volume using a Thermo Scientific Evolution 201 photometer
(Thermo Fisher Scientific, Dreieich, Germany). Concentrations were calculated based on a

calibration correlating the gold interband absorption at λ=380 nm to the gold mass
concentration 72. Furthermore, UV-Vis spectroscopy of alloy nanoparticles were performed in
order to monitor the position of the surface plasmon resonance (SPR) and hence to verify

alloy formation during the laser synthesis process.
Size determinations were conducted with an analytical disk centrifuge (ADC) (DC 24000;
CPS-instruments) at 24000 rpm against a saccharose gradient in a sample volume of 0.1 ml.
Measurements were carried out in a size range between 1000 – 4 nm and dependent on
particle densities, deduced from the compositions of the equivalent targets. Measurements
took 10 to 30 min. The main peaks of the number and mass distributions were fitted by log-
normal functions, while the corresponding particle diameters were deduced from the mean
value. Errors originated from the standard deviations of these curves. The total particle
concentrations in the samples were determined based on the number distributions and the total
mass concentrations. The particle diameters were determined from the d50 values of the
cumulative frequency distributions as depicted in figure S2.
Zeta-potential measurements were carried out using a Malvern Zetasizer Nano ZS in
disposable capillary cells with a volume of 750 µl. The samples were equilibrated for two
minutes at 25°C and measurements of each sample were repeated three times. The zeta-
potential distributions were calculated applying standard Smoluchowski fitting. Interpretation
was performed by determining the mean values of the resulting peak. In samples containing
multiple peaks, average mean values, weighted by the peak integrals were calculated. Errors
in all samples were derived from standard deviations of the triplicate measurements. For
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DOI: 10.1039/C3AN01463K
particle specifications see table 1.
Laser scanning confocal microscopy (LSCM)
Confocal localization of metal nanoparticles had been established previously 74. In the current
experiments with cumulus-oocyte-complexes the settings for the LSM510 with an Axioplan
200 (Carl Zeiss Micro Imaging GmbH, Jena, Germany) were applied as listed in table 4 in
multi-tracking mode. The detection parameters were adjusted to avoid auto reflection in the
controls without nanoparticles.

A total of 25 medial optical sections of the oocytes at a distance between successive sections
of 400 nm were documented. Thus maximum stacks of 143x143 µm x 10 µm were
investigated with a 63x Plan Apochromat objective (NA 1.4) covering at least one quarter of
the total cumulus-oocyte-complex of about 100 µm in diameter for the oocyte alone and with
additional 50 to 100 µm for the cumulus layers. Pixel size was 400x400x700 nm and pixel
times 1.6 µsec.
Detailed analyses were performed on 70x70x1 µm volumes.
Statistical analysis
Statistical analysis was performed with SigmaStat Version 2.03 (StatCon, Witzenhausen,
Germany). Oocyte maturation passed the test for normal distribution and effects were
evaluated by a One Way ANOVA (Tukey test). For sperm cell viability parameters a One
Way Anova on Ranks (Kruskal-Wallis) was performed as data were not normally distributed.
Conclusion
In this systematic study, AuNP showed no detrimental effects on cumulus-oocyte-complexes
or spermatozoa, regardless of size, surface ligands or concentration. This further underlines

the suitability of gold nanoparticles to be developed for biomedical applications. Silver alloy
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DOI: 10.1039/C3AN01463K
nanoparticles displayed a silver ratio-dependant toxicity on cumulus-oocyte-complexes. A
silver molar ratio higher than 50% caused negative effects on porcine gametes. We
hypothesize that dissolved Ag+-ions are responsible for the observed effects. However the
incorporation of silver into an alloy seems to reduce the toxicity as compared to to pure silver
nanoparticles. The different sensitivity of sperm and oocytes to silver nanoparticle toxicity is
probably due differences regarding the activity of the cell nucleus and the nanoparticle uptake
pattern. It is important to point out that silver containing nanoparticles are able to inhibit
oocyte maturation which may lead to fertility problems in vivo.

Acknowledgements
We gratefully acknowledge the skilfull assistance of Birgit Sieg, Antje Frenzel, Ina Ott, Jörg
Kramer, Meike Stünkel, Lisa Gamrad, Vivian Merk, Valeria Terehina and Rolf Poppenga.
This work was financially supported by the the Priority Program 1313 of the German
Research Foundation.
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Tables
Table 1: Specifications of the tested nanoparticles.
Chemical Particle size (nm) Particle corona Mass concentration Particles/mL Particles/oocyte Particles/sperm
composition (µg/mL) (maturation) (coincubation)

Au 6 BSA 10 1.94x1012 1.94x1010
Au 6 citrate+BSA 10 1.93x1012 1.93x1010
Au 20 BSA 10 1.24x1011 1.24x109 1.24x103
Au 8 BSA 30 8.65x1012 8.65x1010
AuAg 8:2 6 BSA 10 5.04x1012 5.04x1010 5.04x104
AuAg 5:5 6 BSA 10 5.93x1012 5.93x1010 5.93x104
AuAg 2:8 7 BSA 10 7.20x1012 7.20x1010 7.20x104
Ag 11 BSA 10 1.29x1010 1.29x108 1.29x102

Table 2: Maturation medium for porcine oocytes. View Article Online

DOI: 10.1039/C3AN01463K
DMEM/Ham´s F-12 (PAA, Pasching, Austria) Concentration

supplemented with
Penicillin 0.06 mg/mL
Streptomycin 0.05 mg/mL
Glutamine 2.5 mM
Fetal calf serum (FCS) 10%

PMSG 10 IU/mL
HCG 10 IU/mL
EGF 50 ng/mL

IGF1 100 ng/mL
β G 5 ng/mL
Table 3: Semen extender (mod.Androhep™)
Substance Concentration
Trisodium-2-hydrate 8 g/L
Titriplex III EDTA 2.4 g/L
BSA fraction 5 2.5 g/L
Sodium hydrogencarbonate 1.2 g/L
Hepes acid 9 g/L
D(+) glucose monohydrate 26 g/L
Dihydrostreptomycinsulfate 0.812 g/L
Penicillin-G-sodium 0.303 g/L
destilled water Dissolve in and fill up to 1L

Published on 07 October 2013. Downloaded by Universitaet Duisburg Essen on 17/10/20
Table 4: Instrument settings of the laser scanning confocal microscope.
Excitation Detected Laser power Main beam Detection band Detection Detection gain Detection offset
wavelength (nm) signal (mW) splitter pass (nm) channel
514 Scattered SPR 0.54 80/20 505 – 530 2 <270 -0.07

488 Scattered SPR 0.54 80/20 475 – 525 2 <270 -0.07
543 Scattered SPR 0.1 80/20 520 – 550 2 <270 -0.07
633 difference 0.1 488/543/633 None transmission 250 - 300 -0.5

interference
contrast (DIC)
Figures View Article Online

DOI: 10.1039/C3AN01463K

Figure 1: Overview of the in vitro coincubation of nanoparticles with porcine cumulus-
oocyte-complexes and spermatozoa. Coloured circles depict nanoparticles: red = AuNP,
shades of orange = AuAgNP, yellow = AgNP. Blue coating symbolises BSA. Green coating
symbolises citrate. Numbers indicate nanoparticles size (nm). Arrows show which particles
are used in the experimental parts.

View Article Online

DOI: 10.1039/C3AN01463K
Figure 2: (A) Exemplary AuAg colloids with different molar fractions. (B) Correlation of
silver molar fraction to maximum surface plasmon resonance extinction peak (C) TEM-EDX
line scan with insert showing high – angular annular dark field micrograph. (D) TEM
micrograph of a Ag50Au50 nanoparticle dipersion after stabilisation with BSA. (E) Aluminium
batch chamber for the synthesis of silver and gold-silver alloy nanoparticles.
View Article Online

DOI: 10.1039/C3AN01463K

Figure 3: Representative laser scanning microscope images of porcine cumulus-oocyte
complexes after 46h coincubation during in vitro maturation. (A) Negative control; (B) gold
nanoparticles; (C) gold-silver alloy nanoparticles; (D) silver nanoparticles; Bars = 10
micrometer.
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DOI: 10.1039/C3AN01463K
Figure 4: Oocyte maturation after 46h of in vitro maturation in the presence of various
nanoparticle types: (A) Percentage of cumulus-oocyte-complexes displaying a metaphase
plate and polar body [values are mean±SD; * a, b p< 0.05]. (B) Mean value of cumulus
expansion, every cumulus-oocyte-complex test group was assessed and the cumulus
expansion rated on a scale of 0 - 3 [values are mean±SD; * a, b p< 0.05]

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DOI: 10.1039/C3AN01463K
Figure 5: Representative TEM-micrograph of a sperm head after coincubation for 2 h at 37°C
with gold nanoparticles, 20 nm, BSA coated (10 µg/ml): (A) Overview. Black rectangle
pointing out area magnified below. (B) Region showing nanoparticles adhesion in higher
magnification. Arrows depicting gold nanoparticles.

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DOI: 10.1039/C3AN01463K
Figure 6: Sperm vitality parameters after co-incubation for 2 h at 37°C with various
nanoparticle types: (A) Sperm cell motility, (B) Sperm membrane integrity and (C) Sperm
morphology. Shown is percentage of spermatozoa, which differs compared to the control
[values are mean±SD; * a, b p< 0.05]

View Article Online
DOI: 10.1039/C3AN01463K
The influence of metal and alloy nanoparticles on functionality and viability of farm animal
derived oocytes and sperm was examined to increase our understanding of nanoparticle
reprotoxicity.
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Reprotoxicity of Gold, Silver, and Gold-Silver Alloy Nanoparticles On Mammalian Gametes

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Reprotoxicity of Gold, Silver, and Gold-Silver Alloy Nanoparticles On Mammalian Gametes

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Reprotoxicity of gold, silver, and gold-silver alloy nanoparticles on

Article in The Analyst · October 2013

Christoph Rehbock Jurij Jakobi

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Reprotoxicity of gold, silver, and gold-silver alloy nanoparticles on mammalian gametes

Wilfried A. Kues1, Stephan Barcikowski2*, Detlef Rath1*

Animal Health, Mariensee, Germany

University of Duisburg-Essen, Essen, Germany

Analyst Accepted Manuscript

Concerning biological matters:

Prof. Dr. Detlef Rath

Institute of Farm Animal Genetics (FLI)

Phone: +49 5034 871 144

Fax: +49 5034 871 101

Concerning nanoparticles and laser technology:

Prof. Dr. Stephan Barcikowski

Technical Chemistry I and Center for Nanointegration Duisburg-Essen (CENIDE)

Phone: +49 201 183 3150

Fax: +49 201 183 3049

Keywords: oocytes, maturation, spermatozoa, toxicity, pig model

Analyst Accepted Manuscript

Confocal microscopy revealed a selective uptake of gold nanoparticles by oocytes, while

oocyte. Interestingly sperm vitality parameters (motility, membrane integrity and

of nanoparticles to the sperm plasma membrane were found by transmission electron

microscopy. In conclusion, mammalian oocytes reacted sensitive to silver containing

alleviate the toxic effects to a certain extent.

Analyst Accepted Manuscript

reliable information to governments and regulating authorities deciding about nanomaterial

showing a varying degree of sensitivity related to nanoparticle composition, production

method, and exposure route.

gamete may already cause tremendous developmental differences. Impairment of gametes

nanoparticle exposure are comparatively scarce. Regarding spermatozoa, no toxicity was

We developed an in vitro assay for assessing the repro-toxic potential of NP on mammalian

functions using internationally standardized protocols of in vitro fertilization (IVF). Oocytes

composition, and compartmentalization, which facilitates to investigate in how far such

properties influence sensitivity towards potentially toxic substances. The cumulus-oocyte-

Analyst Accepted Manuscript

was released (www.ensembl.org/sus_scrofa/Info/Index).

at the nano-bio-interface depend on nanoparticle size, material composition, shape, surface

Analyst Accepted Manuscript

Results and discussion

Development of an in vitro reprotoxicity-assay employing porcine gametes

maturation process of 46h to gain fertilization-competent oocytes is a standard procedure

toxicological parameter, since it is already impaired by substance concentrations far below

cumulus cell expansion were assessed.

membrane integrity, and morphological abnormalities.

Analyst Accepted Manuscript

variants were ex situ coated with bovine serum albumin (BSA).

Characterisation of the produced gold, silver and gold-silver alloy nanoparticles

confirmed the formation of homogeneous AuAg nanoparticles (Figure 2C). An exemplarily

TEM micrograph of well-distributed laser-generated Au50Ag50 alloy nanoparticles after

stabilization with BSA is provided in figure 2D.

Analyst Accepted Manuscript

listed in table 1. Frequency distributions of the nanoparticle diameters as measured in an

centrifugation, SEM and TEM. Reproducibility of this process was demonstrated by

is found (15% < 10 nm; 85% > 10 nm).

Analyst Accepted Manuscript

As it is well known from literature, interpretation of zeta potential measurements is hindered

Wilfried A. Kues1, Stephan Barcikowski2, Detlef Rath1