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Anovelone-potgreensynthesisofselenium
nanoparticlesandevaluationofitstoxicityin
zebrafishembryos
ARTICLE·OCTOBER2014
DOI:10.3109/21691401.2014.962744·Source:PubMed
CITATION
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4AUTHORS,INCLUDING:
KrishnanSundar
MuthuKumaran
KalasalingamUniversity
KalasalingamUniversity
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Artificial Cells, Nanomedicine, and Biotechnology, 2014; Early Online: 1–7
Copyright © 2014 Informa Healthcare USA, Inc.
ISSN: 2169-1401 print / 2169-141X online
DOI: 10.3109/21691401.2014.962744
A novel one-pot green synthesis of selenium nanoparticles
and evaluation of its toxicity in zebrafish embryos
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Kalimuthu Kalishwaralal, Subhaschandrabose Jeyabharathi, Krishnan Sundar & Azhaguchamy Muthukumaran
Department of Biotechnology, Kalasalingam University, Krishnankoil, Tamilnadu, India
the developments in nanotechnology, much effort has been
made to produce selenium at the nanoscale level and to
evaluate its potential use in various medicinal applications
such as cancer therapy (Huang et al. 2013). Selenium nanoparticles have been found to exhibit strong anti-oxidative
(Li et al. 2013), anti-leishmanial (Soflaei et al. 2014) and
anti-bacterial effects (Tran and Webster 2011).
Recent developments in nanotechnology mainly focus on
the synthesis and development of metal nanoparticles. The
methods of nanoparticle synthesis still face the challenge
of optimizing the size and monodispersity (Mónica et al.
2013). The physical and chemical methods of nanoparticle
synthesis are more popular. They are expensive processes
involving the use of toxic chemicals and the possibilities of
toxic chemicals absorbed on the surface of the nanoparticles
is high (Schrade et al. 2013). These short comings can be
overcome by the use of eco-friendly synthesis of nanoparticles (Sriram et al. 2010). Recently, several reports are
available on the use of bacteria, yeast, fungi and plant
sources that all play an important role in biosynthesis of
nanoparticles (Kalishwaralal et al. 2010, Syed et al. 2013).
Bacteria such as Zooglea ramigera (Srivastava and Mukhopadhyay 2013), Bacillus selenitireducens (Switzer et al. 1998),
Selenihalanaerobacter shriftii (Blum et al. 2001), Bacillus
cereus (Dhanjal and Cameotra 2010) and Pseudomonas
alcaliphila (Zhang et al. 2011), can survive and grow at high
selenium concentrations and they have applications in the
adsorption of these metal ions. The toxicity of selenium ions
is reduced or eliminated by changing the redox state of the
selenium ions and in the process leading to the formation
of well-defined nanoscale particles. However, synthesis of
selenium nanoparticles by bacteria also has some drawbacks. The major problem encountered is the isolation and
purification of the nanoparticle from the selenium resistant bacteria. Most of these techniques involve capital and
energy intensive downstream processing steps including
sonication and ultracentrifugation (Kalishwaralal et al. 2010,
Kalimuthu et al. 2008). As an alternative to this, a low cost
and eco-friendly method for the biosynthesis of selenium
ions to selenium nanoparticles (SeNPs) is described here;
Abstract
Over the last 50 years, compelling evidence has accumulated
on the beneficial role of selenium in human health. In the
present study, different proteins were evaluated as reducing
agents for the eco-friendly synthesis of selenium nanoparticles
from an aqueous solution of sodium selenite. This method
is a simple, low cost green synthesis alternative to chemical
synthesis. The high conversion of selenium ions to selenium
nanoparticles (SeNPs) was achieved by a reaction mixture
of 0.1 g bovine serum albumin and 0.1 g sodium selenite at
a reaction temperature of 121°C for 20 min duration. The
selenium nanoparticles were characterized by fourier transform
infrared (FTIR), scanning electron microscopy (SEM) and
energy-dispersive X-ray spectroscopy. The FTIR spectral
bands were sharp with strong absorption peaks at 1649 and
1551 cmⴚ 1. SEM analysis of the synthesized selenium
nanoparticles clearly showed the spherical shape with an average
size ranging from 500 to 600 nm. The toxicity of SeNPs was
evaluated using zebrafish embryos as a model system. SeNPs
induced malformations in zebrafish embryos in a concentrationdependent manner. Selenium nanoparticles at 15–25 μg/ml
concentration caused pericardial edema, tail malformation
and decrease in heart rate in zebrafish embryos. Treatments
with lower concentrations did not alter the heart rate or display
any heart abnormalities. This study underlines the importance
of identifying optimal SeNP concentration that could have
potential therapeutic applications.
Keywords: bovine serum albumin, heart rate, nanoselenium
embryo, pericardial edema, scanning electron microscopy,
toxicity, zebrafish
Introduction
Selenium is an essential trace element identified in the
early 1950s as vital for the survival of several organisms,
including mammals; selenium is important for various
aspects of human health, including cardiovascular health
(Klayman and Gunter 1973, Suadicani et al. 1992). Due to
Correspondence: Dr. A. Muthukumaran, Department of Biotechnology, Kalasalingam University, Krishnankoil – 626126, Tamilnadu, India. Tel: ⫹ 91 4563
289042. Fax: ⫹ 91 4563 289322. E-mail: [email protected]
(Received 11 July 2014; revised 2 September 2014; accepted 3 September 2014)
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K. Kalishwaralal et al.
the method involves one-pot synthesis of selenium nanoparticles using bovine serum albumin as the reducing and
stabilizing agent.
Selenium-deficiency leads to endemic cardiomyopathy
and Keshan disease in humans (Tiekink 2012). Both the
diseases were first identified in parts of China where the soil
is low in selenium content. An inverse association between
the risk of heart disease and serum selenium concentration below 79 μg/l was also reported from a Danish study
(Kardinaal et al. 1997). Cardiopulmonary bypass surgery,
in patients with a low level of selenium in the blood, leads
to organ failure (Stoppe et al. 2013). Treatment with SeNPs
could be used as a therapeutic agent in cardio-related disorders; but pre-evaluation of toxicity of SeNPs is necessary
before considering the therapeutic options. To address this
issue, zebrafish (Danio rerio) embryos were selected as an
experimental model for evaluating cardiotoxicity. To the
best of our knowledge, there is no report available till date
on the biocompatibility and chronotropic effects (heart rate)
of selenium nanoparticles in zebrafish embryos.
Materials and methods
Biosynthesis of selenium nanoparticles
Three different proteins namely bovine serum albumin
(BSA), lipase and protease purchased from HiMedia
Laboratories, India were used. Aliquots (0.1 g) of each of
these proteins were mixed with 50 ml volume of sodium
selenite (0.1 g) (HiMedia, India). The solution was kept in
an autoclave under conventional sterilization conditions
(121°C, 15–20 psi for 20 min).
Purification of nanoparticles
The nanoparticles carrying protein suspension were
centrifuged at 12,000 rpm (1725 ⫻ g) for 10 min and then
the supernatant was discarded. Protein containing the
Figure 1. Schematic diagram- sodium selenite to selenium nanoparticles using BSA as a reducing agent.
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Selenium nanoparticles and evaluation of its toxicity in zebrafish embryos
3
Figure 2. SEM analysis of the selenium nanoparticles. A. SEM image of the selenium nanoparticles produced by BSA (5,000 ⫻ magnification)
B. Particle size analysis at 9,500 ⫻ magnification C. Particle analysis at high resolution (20,000 ⫻ magnification) D. EDX analysis of selenium
nanoparticles.
nanoparticles was washed and resuspended in deionized
water and again centrifugation was done at the same speed
mentioned above. The washing step was repeated three
times at room temperature.
Transformer - Infrared Spectrophotometer (Shimadzu –
8400, Japan) by KBr pellet method as described by Narmato
(1997).
Size determination with SEM
Treatment of zebrafish embryos with selenium
nanoparticles
The size and shape of nanoparticles were measured by Scanning Electron Microscopic analysis using a Philips JSM 6390
model (USA) electron microscope. Essentially for analysis,
one drop of sample suspension was taken for scanning electron microscopy (SEM). The selected areas within SEM sections were subjected to elemental composition analysis using
an energy-dispersive X-ray spectroscopy (EDX) microanalysis system coupled to the scanning electron microscope.
Briefly, the zebrafish embryos at the early blastula stage
were transferred to a petri plate at 10 embryo/petri plate.
The embryos were treated with different concentrations of
selenium nanoparticles (5, 10, 15, 20, 25 μg/ml) for 24 h.
The experiment was performed in triplicate and 10 embryos
were used as a control. The development of the zebrafish
embryos until hatching of the larvae was observed with an
optical microscope (Primo Star Carl Zeiss, Germany).
FTIR spectral analysis
Statistical methods
The synthesized selenium nanoparticles were further
analyzed for their chemical structure using Fourier
The data of results are reported as the mean ⫾ standard
deviation from at least three separate experiments.
Figure 3. FTIR analyses of synthesized selenium nanoparticles using BSA.
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K. Kalishwaralal et al.
Figure 4. Zebrafish embryos exposed to different concentrations of (5–25 μg/ml) selenium nanoparticles taken at 24 h. (A- control; B-5 μg/ml;
C- 10 μg/ml; D-15 μg/ml; E- 20 μg/ml; F- 25 μg/ml).
Results and discussion
Bovine serum albumin-mediated synthesis of selenium
nanoparticles
BSA is a most abundant plasma protein which consists of
three homologous domains (domains I, II, and III) in their
tertiary structure, and its cysteine residues form 17 disulfide
bonds to produce a double-loop bridging pattern (Carter
and Ho 1994, He and Carter 1992). When the protein is
exposed to heat at 121°C, the structure of BSA will facilitate
the breaking of the disulfide bonds and further promote
the unfolding of the protein, exposing more -SH groups
(Figure 1). These -SH and hydroxyl groups could be used for
the reduction of Se (IV) to Se (0) by BSA, resulting in change
of color of the reactant solution from clear white to clear
red (data not shown). The color formation in the reaction
mixture is due to the excitations of surface plasmon resonance of the selenium nanoparticles formed in the reaction
mixture (Charles et al. 2011). However, it is interesting to note
that when the same reaction was carried out in the presence
of other proteins tested like lipase and protease, they could
not produce any color change in the solution. This may be
due to the lack of free and exposed -SH groups in lipase
and protease enzymes which could not have contributed
for the production of the selenium nanoparticles (Au et al.
2010).
Analysis of selenium nanoparticles using SEM
Figure 5. (A) Viability of zebrafish embryos exposed to various
concentrations of selenium nanoparticles after 96 hpf. (B) Percentage
of malformation (tail, pericardial edema) induced by different
concentrations of selenium nanoparticles. (C) Effects of selenium
nanoparticles on heart rates of zebrafish embryos at 96 hpf.
Particle size analysis of selenium nanoparticles was
done using SEM and the result is presented in Figure 2A.
The photomicrograph of SeNPs showed a wide distribution
of size in the sample; the observed SeNPs were spherical in
shape (Figure 2B). The SEM photomicrograph of selenium
nanoparticles showed a size of 500–600 nm which is very
narrow in size distribution (Figure 2C). This observation is in
contrast to the previous reports; selenite-resistant bacteria
such as Sulfurospirillum barnesii, Bacillus selenitireducens,
and Selenihalanaerobacter shriftii produce selenium nanoparticles as unstructured, amorphous aggregates that range
Selenium nanoparticles and evaluation of its toxicity in zebrafish embryos
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in diameter from 200 to 800 nm as analyzed by SEM (Oremland et al. 2004). However, Charles et al. (2011) demonstrated
the synthesis of Se nanospheres (approximately 300 nm)
using both E. coli expressing recombinant Se factor A and
purified recombinant protein. The EDX profile shows a
strong selenium signal along with weak sulfur group peaks.
The result indicated that 92.76% (wt.) of the sample had the
presence of selenium nano particles. The detection of the
presence of sulfur 7.24% (wt.) in the EDX spectra, confirms
the presence of sulfur containing protein/peptide molecules
bound to the surface of the nanoparticles (Syed et al. 2013)
(Figure 1D).
Fourier transformer – infrared spectrophotometer studies
FTIR analysis was performed to characterize the surface
chemistry of selenium nanoparticles produced by BSA and
analysis of FTIR indicated protein mediated synthesis of
selenium nanoparticles; the strong absorption bands at
1649 and at 1551 cm⫺ 1 are characteristic of amide I and C-H
vibrations of CH2 groups of protein moiety respectively, with
albumin as the stabilizing and capping agent surrounding
the selenium nanoparticles (Figure 3).
Effect of selenium nanoparticles on zebrafish embryo
Zebrafish embryos have been used as an in vivo assay
model and could potentially be used for preclinical drug
development for human diseases. Zebrafish and humans
share 85% homology at the genome level (Barbazuk et al.
2000). In order to evaluate the toxicity of SeNPs, zebrafish
embryos were exposed to 500 nm SeNPs at different concentrations (5–25 μg/ml). Zebra fish eggs contain a layer of
5
chorionic membrane as an outside barrier with numerous
distributed pore canals (0.5–0.7 μm in diameter). As illustrated in Figure 4, large sized selenium nanoparticles were
adsorbed on the outer surface of the embryonic chorion in
a concentration dependent manner. This is in corroboration
with the observation of Bai et al. (2010), who observed direct
penetration of SeNPs into the zebrafish embryo. The effect of
SeNPs on the viability of zebrafish embryo was also observed
(Figure 5A). Though there was no mortality noted in lower
concentrations (5–10 μg/mL), exposing these embryos to
higher SeNP concentrations (20 and 25 μg/mL) resulted
in a significant percentage of mortality compared to that
of control group at 96 h post fertilization (hpf ). Zebrafish
embryos exposed to 15–25 μg/ml of SeNPs induced various
developmental abnormalities which were observed and documented at 96 hpf. The lower concentrations (5–10 μg/mL)
of SeNP did not show any significant malformation (such as
pericardial edema and tail malformation) throughout the
tested time points (Figure 6A–I) whereas both pericardial
edema and tail malformation were observed at 20–25 μg/ml
SeNPs-treated embryos (Figure 5B). These observations corroborate with that of Duan et al. (2013), who also reported
malformations of pericardial edema and tail malformation
in embryos treated with silica nanoparticles.
Effect of selenium nanoparticles on cardiovascular
functions of zebrafish embryo
Several studies have shown that selenium may have a
protective effect against cardiovascular disease (Neve
1996). The exact link between selenium nanoparticles and
cardiac dysfunction is not well known. A 2–3 fold risk of
Figure 6. Effects of different concentration of selenium nanoparticles on zebrafish development. Larva control shown at 96 hpf (A–C), 5 μg/ml
SeNP (D–F), 10 μg/ml SeNP (G–I), 15 μg/ml SeNP (J–L), 20 μg/ml SeNP (M–O), 25 μg/ml SeNP (P–R). Larvae shown are representative of at least
three replicative experiments and approximately 30 treated embryos.
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K. Kalishwaralal et al.
Declaration of interest
The authors report no declarations of interest. The authors
alone are responsible for the content and writing of the
paper.
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References
Figure 7. Malformations (e.g., pericardial edema) induced by
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Acknowledgments
The authors thank Kalasalingam University for the facilities
provided. Mr. A. Raja of Karunya University, Coimbatore is
acknowledged for his help with SEM & EDX analysis.
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