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Reprotoxicity of gold, silver, and gold–silver alloy nanoparticles on mammalian gametes† Cite this: DOI: 10.1039/c3an01463k
Daniela Tiedemann,‡a Ulrike Taylor,‡a Christoph Rehbock,b Jurij Jakobi,b Sabine Klein,a Wilfried A. Kues,a Stephan Barcikowski*b and Detlef Rath*a 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 46 h 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 with the sperm plasma membrane was found by transmission electron microscopy. In conclusion, mammalian oocytes
Received 1st August 2013 Accepted 2nd October 2013
were sensitive to silver containing nanoparticles. Likely, the delicate process of completing meiosis in
DOI: 10.1039/c3an01463k
maternal gametes features high vulnerability towards nanomaterial derived toxicity. The results imply that released Ag+-ions are responsible for the observed toxicity, but the compounding into an alloy
www.rsc.org/analyst
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 relatively large surface area, nanoparticles (NPs) have unique physiochemical properties. These attributes may have adverse effects when introduced into biological systems.1 Therefore, it is important to thoroughly understand the risks exerted by nanomaterial exposure2 to provide reliable information to governments and regulating authorities deciding about nanomaterial safety guidelines.3 With respect to potential reprotoxicological effects mainly piscine embryos4–15 and to a lesser extent avian embryos16–21 and a
Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Federal Research Institute of Animal Health, H¨oltystraße 10, 31535, Neustadt, Mariensee, Germany. E-mail: [email protected]; Fax: +49 5034 871 101; Tel: +49 5034 871 144
b
Technical Chemistry I and Center for Nanointegration Duisburg-Essen (CENIDE), University of Duisburg-Essen, Universit¨ atsstraße 7, 45141, Essen, Germany. E-mail: [email protected]; Fax: +49 201 183 3049; Tel: +49 201 183 3150 † Electronic supplementary 10.1039/c3an01463k
information
(ESI)
available.
‡ Both authors contributed equally.
This journal is ª The Royal Society of Chemistry 2013
See
DOI:
mammalian embryos22–28 have been investigated, showing a varying degree of sensitivity related to the nanoparticle composition, production method, and exposure route. Gamete quality plays a vital role in ontogenesis.29 The inuence of nanoparticles on a single gamete may already cause tremendous developmental differences. Impairment of gametes may affect reproductive functions or have pathological inuence on the next generation.30 Yet, despite their importance, studies concerning the sensitivity of gametes towards nanoparticle exposure are comparatively scarce. Regarding spermatozoa, no toxicity was found for PVA and PVP-coated iron and europium hydroxide NPs.31,32 Moderate effects were detected for titanium dioxide, gold, silver and zinc oxide NPs,33–37 while one study indicated severe effects for europium trioxide NPs.32 Oocytes have only been examined aer exposure to TiO2NPs38 and CdSe–core quantum dots (QDs).39 Both treatments had a negative inuence on oocyte quality, which in the case of the QDs could be obliterated aer coating with ZnS. We developed an in vitro assay for assessing the reprotoxic potential of NPs on mammalian gametes. The approach allows investigation of well-dened primary cells with clearly stated functions using internationally standardized protocols of in vitro fertilization (IVF). Oocytes and spermatozoa possess very
Analyst
View Article Online
Published on 07 October 2013. Downloaded by Laurentian University on 01/11/2013 07:07:55.
Analyst different features with regard to metabolic activity, membrane composition, and compartmentalization, which facilitates to investigate in how far such properties inuence the sensitivity towards potentially toxic substances. The cumulus–oocytecomplexes and spermatozoa used in the study were of porcine origin. The pig as a reference model is frequently used in embryo-toxicological studies.40–43 Porcine physiology, metabolism, and pathology reect the human situation much better than small animal models.44 A previous phylogenetic study suggested that the pig genome is threefold closer to that of humans than that of mice,45 and recently the newest genome assembly (10.2) of the porcine genome was released (http// :www.ensembl.org/sus_scrofa/Info/Index). A currently emerging eld for the synthesis of colloidal metal and alloy nanoparticles is pulsed laser ablation in liquid (PLAL).46–49 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 for 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 pHdependent equilibrium between Au–OH and Au–O groups on the surface and causing a negative zeta potential.51 Another advantage of PLAL is its high exibility as to the used solvent52,53 and particularly concerning the used raw material giving access to metal54,55 and semiconductor nanoparticles.56 Additionally, this synthesis route has been successfully used for the fabrication of alloy nanoparticles,57–63 which are oen difficult to produce by chemical reduction methods due to the unavailability of suitable precursor species. The types of nanoparticles tested in this project have not only been chosen due to their important role for biomedical applications, but also 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 ndings will increase our understanding of mechanisms concerning nanoparticle biocompatibility.
Results and discussion Development of an in vitro reprotoxicity-assay employing porcine gametes Fig. 1 schematically depicts the experimental design to assess reprotoxicity of different NPs. Prior to ovulation oocytes mature and become competent for fertilization in the ovarian follicles. 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 nal development before ovulation. These Analyst
Paper
Fig. 1 Overview of the in vitro coincubation trials of nanoparticles with porcine cumulus–oocyte-complexes and spermatozoa. Coloured circles depict nanoparticles: red ¼ AuNPs, shades of orange ¼ AuAgNPs, yellow ¼ AgNPs. Blue coating symbolises BSA. Green coating symbolises citrate. Numbers indicate nanoparticle size (nm). Arrows show which particles are used in the experimental parts.
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 46 h to gain fertilization-competent oocytes is a standard procedure when performing IVF. Timing and completeness of oocyte maturation are viewed as sensitive toxicological parameters, since it is already impaired by substance concentrations far below concentrations that inuence the viability of the matured oocyte.66,67 As functional parameters of oocyte maturation the formation of a metaphase plate of the second meiosis and cumulus cell expansion were assessed. Porcine spermatozoa derived from fresh ejaculates were coincubated with nanoparticles for 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 mg mL1 and 30 mg mL1) carrying different surface ligands (BSA and BSA/citrate) to examine whether differences in those properties inuence the toxic potential. In spermatozoa only one pure gold nanoparticle variant was tested (20 nm, 10 mg mL1, 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 was tested. Stabilisers and reducing agents, which are difficult to be excluded 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 multitude of proteins, which
This journal is ª The Royal Society of Chemistry 2013
View Article Online
Paper adsorb to the nanoparticle surface forming a protein corona.70 Such coronas may considerably inuence the biological identity of the particle.65,71 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).
Published on 07 October 2013. Downloaded by Laurentian University on 01/11/2013 07:07:55.
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 the gold–silver 80 : 20 alloys, 451 nm for the gold– silver 50 : 50 alloys and 415 nm for the gold–silver 20 : 80 alloys. Fig. S1† displays the UV-vis spectra of all employed nanoparticles in detail. Fig. 2A shows exemplary AuAg alloys with different molar fractions. The appearance of only one 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 (Fig. 2B), as it has been previously described in the literature.58 An EDX line scan of a high-angular annular dark eld micrograph conrmed the formation of homogeneous AuAg nanoparticles (Fig. 2C). An exemplarily TEM micrograph
Fig. 2 (A) Exemplary AuAg colloids with different molar fractions. (B) Correlation of gold molar fraction with a maximum surface plasmon resonance extinction peak. (C) TEM-EDX line scan with inset showing high-angular annular dark field micrograph. (D) TEM micrograph of a Ag50Au50 nanoparticle dispersion after stabilisation with BSA. (E) Aluminium batch chamber for the synthesis of silver and gold–silver alloy nanoparticles.
This journal is ª The Royal Society of Chemistry 2013
Analyst of well-distributed laser-generated Au50Ag50 alloy nanoparticles aer stabilization with BSA is provided in Fig. 2D. The primary particle diameter consisted of 6 and 20 nm for AuNPs when tested at 10 mg mL1 and 8 nm for the tests at 30 mg mL1. Gold–silver alloy nanoparticles measured to be 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 Fig. 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, the size control of laser-fabricated nanoparticles was performed by the addition of low salinity electrolytes like NaCl, present during the laser ablation synthesis. Signicant differences in the average diameters of these particles were veried 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 ¼ 0.1) were found. Larger nanoparticles showed higher error margins (20 7 nm) and wider size distributions (PDI ¼ 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 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. The long-term stability of the used
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View Article Online
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Published on 07 October 2013. Downloaded by Laurentian University on 01/11/2013 07:07:55.
Table 1
Paper
Specifications of the tested nanoparticles
Chemical composition
Particle size (nm)
Particle corona
Mass concentration (mg mL1)
Particles/mL
Particles/oocyte (maturation)
Au Au Au Au AuAg 8 : 2 AuAg 5 : 5 AuAg 2 : 8 Ag
6 6 20 8 6 6 7 11
BSA Citrate + BSA BSA BSA BSA BSA BSA BSA
10 10 10 30 10 10 10 10
1.94 1012 1.93 1012 1.24 1011 8.65 1012 5.04 1012 5.93 1012 7.20 1012 1.29 1010
1.94 1010 1.93 1010 1.24 109 8.65 1010 5.04 1010 5.93 1010 7.20 1010 1.29 108
particles in Androhep semen extender over a time period of 28 days has been veried in previous studies conducted by the authors.72 As indicated by time-resolved UV-vis measurements, a constant scattering intensity at 800 nm, and hence no signicant 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; Fig. 3B). By LSCM only particles and particle aggregates with a diameter >60 nm can be visualised.74 Therefore, the observed signals were probably derived from particle aggregates, since the primary particle
Fig. 3 Representative laser scanning microscope images of porcine cumulus– oocyte complexes after 46 h coincubation during in vitro maturation. (A) Negative control; (B) gold nanoparticles; (C) gold–silver alloy nanoparticles; (D) silver nanoparticles; bars ¼ 10 micrometer.
Analyst
Particles/sperm (coincubation)
1.24 103 5.04 104 5.93 104 7.20 104 1.29 102
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 mg mL1 [equal to a number dose of 1.94 1010 NP per oocyte] versus 30 mg mL1 [equal to 8.65 1010 NP per oocyte]) (Fig. 4). Interestingly, NPs 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 (Fig. 3C and D). While nanoparticle uptake behavior was concentration independent, a clear concentration dependency was noted for toxic aspects. Aer 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 signicantly (Fig. 4). Coincubation of oocytes with Ag+ ions (added as AgNO3 [12,5 mg mL1]), 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 particle surface net charge, which is one of the key features for interactions at the nano–biointerface,64 did not differ among particle types. All particles were BSA coated before transferring into the maturation medium. However, aer transferring into the medium, particles were in contact with a new set of biomolecules. Adsorbing rapidly to the particles' surface, they t a new biological identity to the nanoparticle, which is the one that is recognized and ultimately acted upon by the cellular machinery.75 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 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 modication, composition), the particle composition had the largest impact. The observation that the reprotoxic potential of gold nanoparticles is low and that a concentration
This journal is ª The Royal Society of Chemistry 2013
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Fig. 4 Oocyte maturation after 46 h 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].
dependent toxic effect of silver containing nanoparticles exists is in good agreement with the literature.4,5,7–9,11,16,23,77 While being the rst report testing gold nanoparticles on mammalian cumulus–oocyte-complexes, it conrms the nding of other reprotoxicological studies using piscine4,5 and avian embryos.16 In the present work a size effect could not be conrmed. However, a study examining gold clusters (
Published on 07 October 2013. Downloaded by Laurentian University on 01/11/2013 07:07:55.
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Reprotoxicity of gold, silver, and gold–silver alloy nanoparticles on mammalian gametes† Cite this: DOI: 10.1039/c3an01463k
Daniela Tiedemann,‡a Ulrike Taylor,‡a Christoph Rehbock,b Jurij Jakobi,b Sabine Klein,a Wilfried A. Kues,a Stephan Barcikowski*b and Detlef Rath*a 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 46 h 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 with the sperm plasma membrane was found by transmission electron microscopy. In conclusion, mammalian oocytes
Received 1st August 2013 Accepted 2nd October 2013
were sensitive to silver containing nanoparticles. Likely, the delicate process of completing meiosis in
DOI: 10.1039/c3an01463k
maternal gametes features high vulnerability towards nanomaterial derived toxicity. The results imply that released Ag+-ions are responsible for the observed toxicity, but the compounding into an alloy
www.rsc.org/analyst
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 relatively large surface area, nanoparticles (NPs) have unique physiochemical properties. These attributes may have adverse effects when introduced into biological systems.1 Therefore, it is important to thoroughly understand the risks exerted by nanomaterial exposure2 to provide reliable information to governments and regulating authorities deciding about nanomaterial safety guidelines.3 With respect to potential reprotoxicological effects mainly piscine embryos4–15 and to a lesser extent avian embryos16–21 and a
Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Federal Research Institute of Animal Health, H¨oltystraße 10, 31535, Neustadt, Mariensee, Germany. E-mail: [email protected]; Fax: +49 5034 871 101; Tel: +49 5034 871 144
b
Technical Chemistry I and Center for Nanointegration Duisburg-Essen (CENIDE), University of Duisburg-Essen, Universit¨ atsstraße 7, 45141, Essen, Germany. E-mail: [email protected]; Fax: +49 201 183 3049; Tel: +49 201 183 3150 † Electronic supplementary 10.1039/c3an01463k
information
(ESI)
available.
‡ Both authors contributed equally.
This journal is ª The Royal Society of Chemistry 2013
See
DOI:
mammalian embryos22–28 have been investigated, showing a varying degree of sensitivity related to the nanoparticle composition, production method, and exposure route. Gamete quality plays a vital role in ontogenesis.29 The inuence of nanoparticles on a single gamete may already cause tremendous developmental differences. Impairment of gametes may affect reproductive functions or have pathological inuence on the next generation.30 Yet, despite their importance, studies concerning the sensitivity of gametes towards nanoparticle exposure are comparatively scarce. Regarding spermatozoa, no toxicity was found for PVA and PVP-coated iron and europium hydroxide NPs.31,32 Moderate effects were detected for titanium dioxide, gold, silver and zinc oxide NPs,33–37 while one study indicated severe effects for europium trioxide NPs.32 Oocytes have only been examined aer exposure to TiO2NPs38 and CdSe–core quantum dots (QDs).39 Both treatments had a negative inuence on oocyte quality, which in the case of the QDs could be obliterated aer coating with ZnS. We developed an in vitro assay for assessing the reprotoxic potential of NPs on mammalian gametes. The approach allows investigation of well-dened primary cells with clearly stated functions using internationally standardized protocols of in vitro fertilization (IVF). Oocytes and spermatozoa possess very
Analyst
View Article Online
Published on 07 October 2013. Downloaded by Laurentian University on 01/11/2013 07:07:55.
Analyst different features with regard to metabolic activity, membrane composition, and compartmentalization, which facilitates to investigate in how far such properties inuence the sensitivity towards potentially toxic substances. The cumulus–oocytecomplexes and spermatozoa used in the study were of porcine origin. The pig as a reference model is frequently used in embryo-toxicological studies.40–43 Porcine physiology, metabolism, and pathology reect the human situation much better than small animal models.44 A previous phylogenetic study suggested that the pig genome is threefold closer to that of humans than that of mice,45 and recently the newest genome assembly (10.2) of the porcine genome was released (http// :www.ensembl.org/sus_scrofa/Info/Index). A currently emerging eld for the synthesis of colloidal metal and alloy nanoparticles is pulsed laser ablation in liquid (PLAL).46–49 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 for 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 pHdependent equilibrium between Au–OH and Au–O groups on the surface and causing a negative zeta potential.51 Another advantage of PLAL is its high exibility as to the used solvent52,53 and particularly concerning the used raw material giving access to metal54,55 and semiconductor nanoparticles.56 Additionally, this synthesis route has been successfully used for the fabrication of alloy nanoparticles,57–63 which are oen difficult to produce by chemical reduction methods due to the unavailability of suitable precursor species. The types of nanoparticles tested in this project have not only been chosen due to their important role for biomedical applications, but also 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 ndings will increase our understanding of mechanisms concerning nanoparticle biocompatibility.
Results and discussion Development of an in vitro reprotoxicity-assay employing porcine gametes Fig. 1 schematically depicts the experimental design to assess reprotoxicity of different NPs. Prior to ovulation oocytes mature and become competent for fertilization in the ovarian follicles. 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 nal development before ovulation. These Analyst
Paper
Fig. 1 Overview of the in vitro coincubation trials of nanoparticles with porcine cumulus–oocyte-complexes and spermatozoa. Coloured circles depict nanoparticles: red ¼ AuNPs, shades of orange ¼ AuAgNPs, yellow ¼ AgNPs. Blue coating symbolises BSA. Green coating symbolises citrate. Numbers indicate nanoparticle size (nm). Arrows show which particles are used in the experimental parts.
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 46 h to gain fertilization-competent oocytes is a standard procedure when performing IVF. Timing and completeness of oocyte maturation are viewed as sensitive toxicological parameters, since it is already impaired by substance concentrations far below concentrations that inuence the viability of the matured oocyte.66,67 As functional parameters of oocyte maturation the formation of a metaphase plate of the second meiosis and cumulus cell expansion were assessed. Porcine spermatozoa derived from fresh ejaculates were coincubated with nanoparticles for 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 mg mL1 and 30 mg mL1) carrying different surface ligands (BSA and BSA/citrate) to examine whether differences in those properties inuence the toxic potential. In spermatozoa only one pure gold nanoparticle variant was tested (20 nm, 10 mg mL1, 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 was tested. Stabilisers and reducing agents, which are difficult to be excluded 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 multitude of proteins, which
This journal is ª The Royal Society of Chemistry 2013
View Article Online
Paper adsorb to the nanoparticle surface forming a protein corona.70 Such coronas may considerably inuence the biological identity of the particle.65,71 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).
Published on 07 October 2013. Downloaded by Laurentian University on 01/11/2013 07:07:55.
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 the gold–silver 80 : 20 alloys, 451 nm for the gold– silver 50 : 50 alloys and 415 nm for the gold–silver 20 : 80 alloys. Fig. S1† displays the UV-vis spectra of all employed nanoparticles in detail. Fig. 2A shows exemplary AuAg alloys with different molar fractions. The appearance of only one 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 (Fig. 2B), as it has been previously described in the literature.58 An EDX line scan of a high-angular annular dark eld micrograph conrmed the formation of homogeneous AuAg nanoparticles (Fig. 2C). An exemplarily TEM micrograph
Fig. 2 (A) Exemplary AuAg colloids with different molar fractions. (B) Correlation of gold molar fraction with a maximum surface plasmon resonance extinction peak. (C) TEM-EDX line scan with inset showing high-angular annular dark field micrograph. (D) TEM micrograph of a Ag50Au50 nanoparticle dispersion after stabilisation with BSA. (E) Aluminium batch chamber for the synthesis of silver and gold–silver alloy nanoparticles.
This journal is ª The Royal Society of Chemistry 2013
Analyst of well-distributed laser-generated Au50Ag50 alloy nanoparticles aer stabilization with BSA is provided in Fig. 2D. The primary particle diameter consisted of 6 and 20 nm for AuNPs when tested at 10 mg mL1 and 8 nm for the tests at 30 mg mL1. Gold–silver alloy nanoparticles measured to be 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 Fig. 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, the size control of laser-fabricated nanoparticles was performed by the addition of low salinity electrolytes like NaCl, present during the laser ablation synthesis. Signicant differences in the average diameters of these particles were veried 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 ¼ 0.1) were found. Larger nanoparticles showed higher error margins (20 7 nm) and wider size distributions (PDI ¼ 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 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. The long-term stability of the used
Analyst
View Article Online
Analyst
Published on 07 October 2013. Downloaded by Laurentian University on 01/11/2013 07:07:55.
Table 1
Paper
Specifications of the tested nanoparticles
Chemical composition
Particle size (nm)
Particle corona
Mass concentration (mg mL1)
Particles/mL
Particles/oocyte (maturation)
Au Au Au Au AuAg 8 : 2 AuAg 5 : 5 AuAg 2 : 8 Ag
6 6 20 8 6 6 7 11
BSA Citrate + BSA BSA BSA BSA BSA BSA BSA
10 10 10 30 10 10 10 10
1.94 1012 1.93 1012 1.24 1011 8.65 1012 5.04 1012 5.93 1012 7.20 1012 1.29 1010
1.94 1010 1.93 1010 1.24 109 8.65 1010 5.04 1010 5.93 1010 7.20 1010 1.29 108
particles in Androhep semen extender over a time period of 28 days has been veried in previous studies conducted by the authors.72 As indicated by time-resolved UV-vis measurements, a constant scattering intensity at 800 nm, and hence no signicant 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; Fig. 3B). By LSCM only particles and particle aggregates with a diameter >60 nm can be visualised.74 Therefore, the observed signals were probably derived from particle aggregates, since the primary particle
Fig. 3 Representative laser scanning microscope images of porcine cumulus– oocyte complexes after 46 h coincubation during in vitro maturation. (A) Negative control; (B) gold nanoparticles; (C) gold–silver alloy nanoparticles; (D) silver nanoparticles; bars ¼ 10 micrometer.
Analyst
Particles/sperm (coincubation)
1.24 103 5.04 104 5.93 104 7.20 104 1.29 102
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 mg mL1 [equal to a number dose of 1.94 1010 NP per oocyte] versus 30 mg mL1 [equal to 8.65 1010 NP per oocyte]) (Fig. 4). Interestingly, NPs 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 (Fig. 3C and D). While nanoparticle uptake behavior was concentration independent, a clear concentration dependency was noted for toxic aspects. Aer 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 signicantly (Fig. 4). Coincubation of oocytes with Ag+ ions (added as AgNO3 [12,5 mg mL1]), 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 particle surface net charge, which is one of the key features for interactions at the nano–biointerface,64 did not differ among particle types. All particles were BSA coated before transferring into the maturation medium. However, aer transferring into the medium, particles were in contact with a new set of biomolecules. Adsorbing rapidly to the particles' surface, they t a new biological identity to the nanoparticle, which is the one that is recognized and ultimately acted upon by the cellular machinery.75 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 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 modication, composition), the particle composition had the largest impact. The observation that the reprotoxic potential of gold nanoparticles is low and that a concentration
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Fig. 4 Oocyte maturation after 46 h 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].
dependent toxic effect of silver containing nanoparticles exists is in good agreement with the literature.4,5,7–9,11,16,23,77 While being the rst report testing gold nanoparticles on mammalian cumulus–oocyte-complexes, it conrms the nding of other reprotoxicological studies using piscine4,5 and avian embryos.16 In the present work a size effect could not be conrmed. However, a study examining gold clusters (
