Research J. Pharm. and Tech.

10(7): July 2017

ISSN 0974-3618 (Print) www.rjptonline.org

0974-360X (Online)

RESEARCH ARTICLE
An Investigation of Polymer Coated Metallic Nanoparticles Mediated
Influence on Gut Microbiota of Zebra Fish (Danio rerio)
S. Karthick Raja Namasivayam, V. Poornima
Department of Biotechnology, Sathyabama University, Chennai 119, Tamil Nadu, India
*Corresponding Author E-mail: biologiask@gmail.com

ABSTRACT:
Nanotechnology is currently gained increased attention in various fields of science and technology like imaging,
sensing, targeted drug delivery, gene delivery systems and artificial implants which is increasing the commercial
usage of products including drugs in the various parts of the world. Because of their extensive consumption, it is
necessary to study the harmful effects of nanomaterials particularly their ecotoxic effect and cytoxicity of
nontarget organism (NTOs). In the present study, an attempt has been made to study toxic effect of silver and
gold nanoparticles coated with chitosan on the gut microbiota of adult zebra fish adopting culture dependent
methods. Respective metallic nanoparticles were synthesized by chemical reduction and stabilization of
precursor salt and the synthesized polymer coated nanoparticles were characterized by electron microscopy
studies and fourier transform spectroscopic studies. Adults of zebra fishes were administrated with lethal dose 50
(LD50) of polymer coated respective nanoparticles and their stress on gut microbiota studied by analysis of gut
tissue homogenate of tested fishes adopting culture dependent methods. Chitosan coated silver nanoparticles
brought about maximum reduction of microbial count which reveals drastic reduction of microbial count as
increasing time periods. Less toxic effect on microbial count has been inferred from chitosan coated gold
nanoparticles.

KEYWORDS: Gut microbiota, zebra fish, metallic nanoparticles, chitosan, polymer.

INTRODUCTION:
Due to the physical and ethical issues associated with
performing experiments on humans, biomedical research
utilizes primarily animal models to study biologic
processes conserved between humans and lower
vertebrates such as rats and mice. Although these models
have significant advantages, they are also expensive to
maintain, difficult to manipulate embryonically, and
limited for large-scale genetic studies1.As the most
numerous and phylogenetically diverse group of
vertebrates, fish teach us important principles about
fundamental processes in vertebrate evolution,
development and disease processes2. Fish have served as
useful sentinels to detect environmental hazards and as
efficient, cost effective model systems for mechanistic
toxicology and risk assessment for many decades3.
Received on 09.05.2017 Modified on 31.05.2017
Accepted on 30.07.2017 © RJPT All right reserved
Research J. Pharm. and Tech. 2017; 10(7): 2221-2225.
DOI: 10.5958/0974-360X.2017.00392.4
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During the past decade zebra fish have gained a pivotal
place in embryology and developmental genetics. Zebra
fish and other aquarium fish species have distinct
advantages as models for biomedical research including
much lower husbandry costs than mammals4.
The zebrafish, Danio rerio, has emerged as a popular
vertebrate model in different areas of research such as
developmental biology, genetics, pharmacology, and
toxicology in recent years5,6. Due to transparent embryos
and rapid organogenesis, zebrafish were recognized as a
tool for developmental biologists in the 1970s. In the
1990s, zebrafish were used for the first vertebrate large
scale mutagenesis screen, yielding thousands of
mutations, some of which recapitulated human diseases.
Several characteristics make zebrafish a convincing tool
for drug discovery7,8. The zebrafish has several features
that make it attractive as an animal model. As an
oviparous species, zebrafish have external fertilization
and development, which allow for easy detection of
morphological alterations and manipulation of the
 Research J. Pharm. and Tech. 10(7): July 2017

transparent embryos9. Zebrafish has rapid growth (with
all major organs formed within 2-4 days), high egg yield,
and a short generation time. Their genetics and
developmental biology have been well documented. The
sequenced zebrafish genome is being assembled and
annotated, and many molecular techniques have been
developed to study gene function in zebrafish, including
the production of transgenic and mutant fish and the use
of transient antisense gene-knockdown methods All
these features render the zebrafish embryos/larvae as a
convenient platform for pharmacological assessment and
screening for potential adverse effects of drugs,
particularly with regard to their potential adverse effects
during the developmental process10,11. Recently,
zebrafish- based assays have been developed for testing
toxicity, including acute toxicity (LC50), organ- specific
toxicity and a developmental toxicity 12. Zebrafish also
exhibit similar responses to xenobiotics chemicals as
mammals, including induction of xenobiotic enzymes
and generation of oxidative stress.
Evaluation of gut microbion:
Fish stocks:
Fish (Danio rerio) were obtained from local market
kolathur, Chennai. The fish were maintained in five
different tanks of 150 L capacity filled with natural
aerated saline water and the water was replaced every 2
days. Each tank was with dimensions 18x12 inches
Temperature was maintained at 30–32°C with a natural
light–dark cycle. During acclimation period, Zebra fish
were fed once a day, 5 g/100g body wt. throughout the
experiment. The pH, dissolved oxygen and total
alkalinity were in the range of 7.31–7.49, 7.71–8.12 and
31.3–37.13 ppm, respectively. The fish were
acclimatized in the laboratory for a week before the
experiments were initiated.
Determination of LD50:
Acute toxicity can be determined by the calculation of
LD50, i.e., the dose that will kill 50% of animals of a
particular species.

Currently, nanotechnology is used in the various fields Estimation of the dose range and percentage of
of science and technology. Hence, nanosized organic and mortality:
inorganic particles are finding increasing attention in An approximate LD50 can be initially determined as a
medical applications due to their amenability to pilot study by a so called ‘staircase method’ using a
biological functionalization. Based on enhanced small number of fishes and increasing the doses of the
effectiveness, the new age drugs re-nanoparticles of nanoparticle. Six doses can be chosen for determination
polymers, metals or ceramics, which can combat of LD50 starting from no death to 100% mortality. In
conditions like cancer and fight human pathogens like our study for estimation of LD50, 6 doses were mixed in
bacteria13. Increasing numbers of commercial products, water to respective groups of fishes, 10 in each group.
from cosmetics to medicine, incorporate manufactured The animals were observed for 24 hours and then 48
nanomaterials (MNMs) that can be accidentally or hours for any toxic symptoms. After 48 hours, the
incidentally released to the environment14. Concern over number of deceased fishes was counted in each group
the potentially harmful effects of such nanoparticles has and percentage of mortality calculated.
stimulated the advent of nanotoxicology as a unique and
Nanoparticles exposure:
significant research discipline. In the present study, toxic
effect of free and polymer coated silver and gold Experiments were carried out in glass aquaria. After
acclimation, fish were divided into five groups each
nanoparticles has been evaluated against gut microbiota
of zebra fish. group containing 10 fish. One of the groups was
maintained as a control in natural tap water. The others
MATERIALS AND METHODS: were exposed to LD 50 of respective nanoparticles per
Synthesis of free silver and gold nanoparticles: litre of water. The experiment was carried out for
Silver and gold nanoparticles were synthesized by 21days. Experimental aquaria were aerated and test
chemical reduction of 0.1 M silver nitrate with 0.1 M media were replaced every day and there was no fish
sodium borohydride and 0.1 M HAucl4 with trisodium mortality during these exposures.
citrate respectively Synthesized nanoparticles were Microbial count determination:
characterized by suitable analytical techniques as At each sampling time, five fishes per tank were killed
described in our previous studies15. by decapitation Fish gut tissues were dissected under
Synthesis and characterization of chitosan stabilized aseptic condition at every time intervals. Isolated guts
nanoparticles: were individually washed several times with fresh
Chitosan stabilized nanoparticles were synthesized by phosphate buffered saline to minimize the possible
the method as described in our earlier studies15 and microbial contamination and used for the study. The gut
synthesized polymer stabilized nanoparticles were was homogenized with sterile insect ringer’s solution
characterized by suitable techniques. (IRS) in mortar and pestle. The homogenate was filtered
through Whatman filter paper no. 1 and the pH was
measured using pH meter (Mic Datal and Co., Chennai).
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 Research J. Pharm. and Tech. 10(7): July 2017

The filtrate was serially diluted in sterile saline and 0.1 Table 3:LD50 for free AuNps
ml of aliquote was plated on nutrient agar (NA), Group Dose(ug/lit) Log dose % Dead Corrected %
1 5 1 0 0.1
sabouraud agar (SDA). The seeded NA plates were 2 10 1.2 1 0.5
incubated at 37°C for 24 - 40 hours whereas the SDA 3 25 1.5 3.5 7
plates were incubated at 28°C for 7 - 10 days 4 50 1.7 15 15
respectively. Microbial colonies appeared after the 5 75 1.8 51 51
incubation period was enumerated and the numbers of 6 100 2.1 53.2 53.2
colony forming units were expressed as dry weight of Table 4:LD50 for CS AuNps
the gut. Group Dose(ug/lit) Log dose % Dead Corrected %
1 5 1 0 0.1
Identification of microflora: 2 10 12 1 0.6
Different morphological microbial colonies were 3 25 1.5 3.6 7.2
selected, subcultured and stored at 4°C on respective 4 50 1.7 15.1 15.1
agar slants. Bacterial strains were identified based on 5 75 1.8 51.1 51.1
morphological, cultural, biochemical characteristics. 6 100 2.1 53.4 53.4

RESULTS AND DISCUSSION: Study groups:

In the present study, gut microbiota mediated stress on Group 1: Control group.
zebrafish using free and polymer coated metallic Group 2: Fishes treated with free AgNp
nanoparticles has been carried out. Synthesis of free and Group 3 Fishes treated with CS AgNp
polymer coated nanoparticles were synthesized by Group 4 Fishes treated with free AuNp
chemical reduction and stabilization of respective metal Group 5. Fishes treated with CS- AuNp
precursor by suitable reducer and polymer. Respective
Among the various nanoparticles treatment, both free
nanoparticles thus prepared reveals highly stabilized
and chitosan stabilized metallic nanoparticles exhibited
monodispersive particles with the nano range described
high level of gut microbial stress which was confirmed
in our previous studies15..Synthesized nanoparticles thus
by drastic reduction of bacterial count in nanoparticles
obtained were lyophilized and the lyophilized
treatment. The status of the confirmed environment is
preparation was evaluated against gut microbiota of
reflected in the large and varied microbial communities
zebra fish. The zebrafish has several features that make it
inhabiting the gut. The indigenous gut bacteria is
attractive as an animal model. As an oviparous species,
regarded as a valuable metabolic resource to the nutrition
zebrafish have external fertilization and development,
of the host by improving the ability to live on sub-
which allow for easy detection of morphological
optimal diets, improved digestion efficiency, acquisition
alterations and manipulation of the transparent embryos9.
of digestive enzymes and provision of vitamins16. The
Zebrafish has rapid growth (with all major organs
contribution of gut microbiota to the nutrition and
formed within 2-4 days), high egg yield, and a short
disease suppression was also studied by Gut microflora
generation time. In this study, nanoparticles mediated
act as a reflection of the environment and incidence of
stress on gut microbiota has been studied. Initially
entomopathogens.
different concentration of nanoparticles was tested to
determine LD 50. Table 1-4 reveals LD 50 of respective
nanoparticles against zebra fish which was used to study
Inhibitory Effect (%) of free AgNp
the gut microbiota specially aerobic microbiota which
Inhibitory Effect (%)
confirmed by changes in bacterial count. 100
90
Time of Exposure (hrs)

Table 1:LD50 for free AgNps

Group Dose(ug/lit) Log dose % Dead Corrected % 80
1 5 1 0 2.5 70
2 10 1.1 12 12 60
3 25 1.4 23 23
50
4 50 1.6 43 43
5 75 1.8 65 65 40
6 100 2 100 97.5 30
20
Table 2:LD50 for CS-AgNps
10
Group Dose(ug/lit) Log dose % Dead Corrected %
1 5 1 0 2.5 0
2 10 1.2 12 12 1 6 12 18 24 30 36 42 48 54 60 66 72
3 25 1.5 24 24 Inhibitory Effect (%)
4 50 1.7 45 45
5 75 1.8 65 65 Fig 1.Inhibitory effect (%) of F-AgNps on gut bacterial count at
6 100 2.1 100 98 different time periods (hour)

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 Research J. Pharm. and Tech. 10(7): July 2017

Inhibitory Effect (%) Inhibitory Effect (%)

Inhibitory Effect (%)
Inhibitory Effect (%)
100 9
90 8
80 7

Time of Exposure
Time of Exposure

70 6
60
5
50
4
40
3
30
20 2
10 1
0 0
1 6 12 18 24 30 36 42 48 54 60 66 72 1 6 12 18 24 30 36 42 48 54 60 66 72
Inhibitory Effect (%) Inhibitory Effect (%)
Figure 2.Inhibitory effect (%) of CS-AgNps on gut bacterial count Figure 3.Inhibitory effect (%) of F-AuNps on gut bacterial count at
at different time periods (hour) different time periods (hour)

Figure 4.Inhibitory effect (%) of CS-AuNps on gut bacterial count at different time periods (hour)

Inhibitory effect of bacterial count was gradually which confirms antibacterial activity. Interestingly, both
reduced with respect to the increasing time periods (Fig free gold and polymer stabilized gold nanoparticles
1-4). shows less inhibitory effect at all the tested time periods.
Bacterial count was not affected as in the control during
A gradual increase in inhibitory effect was observed all the tested time periods. Bacterial species that were
from initial exposure time and maximum reduction in isolated from gut of control group reveals the presence
increased incubation time. Both free and chitosan of Bacillus sp, Pseudomonas sp, Micrococcus sp,
stabilized silver nanoparticles shows remarkable Enterobacter sp, Alcaligenes sp Bacterial colonies
reduction in the bacterial count at increased period of obtained in plate count were purified and morphological
exposure.92.0 and 92.5% of inhibitory effect on bacterial different colonies were selected. Pure cultures were
count was inferred from free silver and chitosan maintained on nutrient agr slant. Identification of the
stabilized nanoparticles treatment during 72 hours of bacterial species was carried out by morphological,
exposure. Among the various nanoparticles, silver has cultural and biochemical characteristics which all shows
the potential activities like anti bacterial and anti cancer the occurrence of the above mentioned bacterial species.
activities. There are many commercially available silver In treatment group of free AgNps and CS-AgNps
based anti bacterial agents available in the various parts category, Bacillus sp and Pseudomonas sp were recorded
of the world and the various biological mechanism (Table 5).

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 Research J. Pharm. and Tech. 10(7): July 2017

Table 5.Effect of nanoparticles treatment on the bacterial composition

Bacterial species Treatment
C F-AgNp CS-AgNp F-AuNp CS-AuNp
Bacillus sp + + + + +
Pseudomonas sp + + + + +
Alcaligenes sp + - - + +
Enterobacter sp + - - + +
Micrococcus sp + - - + +
+ present
-Absent

No changes in species composition of bacteria were ofloxacin nanodrug conjugate AgNp-AZ, OF and AuNp-AZ, OF)
against blood cells. Der Pharmacia Lettera, 8(2); 421-424
recorded in both free AuNps and CS-AuNps treatment.
Further studies based on culture independent methods
will helpful to explore bacterial communities associated
with gut of zebrafish and their changes to the
environmental stress.

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