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Dec 3, 2012 (4 years and 7 months ago)

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Toxic effects of Nanoparticles on healthy cells and biomolecules in in
-
vitro conditions




Page
1




Toxic effects of nanoparticles on healthy cells and Biomolecules in
in
-
vitro

conditions

Charanraj.S.Rathod
*
, Gautham. N, Siddharth Ghosh
, Prof V. Sridhara (Dept of BT, New Horizon College
of Engineering, Bangalore), Prof. Mahadeva Raju

G K (Dept of Chemical Engineering, Dayananda sagar
College of Engineering, Bangalore)

gautham.n@live.com

,
charanraj.rathod@yahoo.co.in



Abstract:

Nanoparticles
, as an important industrial material is widely used in Healthcare and Pharmaceuticals,
Biotechnology Research and Cosmetics. Although the small size of

nanoparticle is useful in various applications, the
biosafety of this material needs to be evaluated. T
he potential toxicity of the nanomaterials is not well understood.
The aim of this Nanotoxicology study is to analyze the factor affecting the biological beings. Analysis of four
Nanoparticles Zinc oxide, Titanium dioxide, MWCNT (Multiwalled Carbon Nanotub
es) was carried out. Alumina, Zinc
oxide (ZnO) and Titanium dioxide (TiO
2
) are two chemical compounds with very wide industrial and commercial
applications, particularly as pigments. Due to their physical properties, both compounds are also used as sunscre
en
ingredients for protection from UV radiation. At the nanoscale, ZnO and TiO
2

have proven to have a similar level of
protection compared to normal
-
scale sunscreen particles but without any white residues left behind. In this study,
Mouse fibroblast cells, Plant seeds, Protein macromolecules, were used to evaluate the cytotoxicity o
f the
Nanoparticle. Carbon nanotubes have been proposed as a possible gene and drug delivery vehicle. We evaluated
the cytotoxicity of nano
-
TiO
2

and nano
-
ZnO on mouse fibroblast cell culture. Cytotoxicity endpoints including IC50
(50% inhibitory concentrat
ion) of the test materials were determined. Exposure to both ZnO and TiO
2

nanoparticles
indicated a range of cytotoxicity responses on mice skin fibroblast cells. The results indicate that at 4 hour exposure
present only a mild adverse effect to mouse cell

fibroblasts. However, at 24 hour exposure both nanoparticles had a
substantial toxic impact to cells. The effect of nanoparticles on protein molecules and their activity was also
determined by incubating the enzyme Amylase with the above nanoparticles and

then determining its enzyme activity
to find out that all the Nanoparticles were inhibiting the enzyme activity of the amylase. The most was by TiO
2
. It was
regarded that the inhibitory action was due to possibility of the protein being degraded due to

the reactivity of the
Nanoparticles, The allosteric site being blocked by the Nanoparticle thereby inhibiting the enzyme from forming the
product, The enzyme may have gone inside the Nanoparticle in case of MWCNT, and therefore the substrate is not
able
to encounter the enzyme or the enzyme is immobilized to the surface of the Nanoparticle in such a manner that
the enzyme active site is not exposed. Human blood serum proteins were incubated with the nanoparticles and then
subjected to SDS PAGE to observ
e that after exposing with Nanoparticles the bands start disappearing with increase
in the concentration. It was observed at 1% concentration of Nanoparticle of MWCNT, Alumina, ZnO and TiO
2
shows
increasing order of the band disappea
rance and

at 2% concent
ration of ZnO and TiO
2

there were no bands inferring
that the degradation of peptide bonds in protein is the reason here for the nanotoxicity. Growth of Green gram
(
Phaseolus radiatus
), Peanuts
(
Arachis hypogaea
), Pea (
Pisum sativum
) and White chickpeas or

Kabuli chana (
Cicer
arietinum
) seed’s shoots and roots are quantified with the entire four Nanoparticles, with different concentrations.
Characterizations of all Nanoparticles by Transmission Electron Microscopy (TEM) showed that ZnO is the smallest
parti
cle, followed by Alumina. ZnO exhibited considerable toxicity in all the cases. One of the reasons is the small
size of the particles. Result of this studies shows that certain optimization has to be done to use Nanoparticle
s to
minimize
its

toxicity. The
future work should be done from the prospect of size, atomic configuration and reactivity of
Nanoparticles to reduce the toxicity, so that the human society can use Nanomedicine effectively compare to the
normal pharmaceutical drugs.


1. Introduction

Nanotechnology is the understanding and
control of matter at dimensions between
approximately 1 and 100 nanometers, where
unique phenomena enable novel applications. A
nanometer is one
-
billionth of a meter and any
particles having their dimensions between
1 to
Toxic effects of Nanoparticles on healthy cells and biomolecules in in
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100 nanometers are known as nanoparticles, as
the particle decreases unusual physical,
chemical, and biological properties can emerge
in materials at the nanoscale. These properties
may differ in important ways from the properties
of bulk materials an
d single atoms or molecules.
Nanoparticle seeks to deliver a valuable set of
research tools and clinically helpful devices in
the near future. New commercial applications in
the pharmaceutical industry including advanced
drug delivery systems, new therapie
s, and
in
vivo
imaging are being developed. As the
Nanomedicine industry continues to grow, it is
expected to have a significant impact on the
economy. The novel application of Nanoparticle
in medical and biotechnology application can be
a miracle, but the

extent of toxicity of
Nanoparticle is the most disadvantageous part
of Nanotechnology. Nanotoxicology is the study
of the toxicity of nanomaterials. Because of
quantum size effects and large surface area,
nanomaterials have unique properties compared
with

their larger counterparts [2]. The
nanomaterials, even when they are made of
inert elements like gold, become very active at a
nanometer range. Nanotoxicological studies are
intended to determine whether and to what
extent these may pose a threat to the
e
nvironment and to human beings.

The smaller a particle is, the greater it’s
surface area to volume ratio and the higher its
chemical reactivity and biological activity. The
greater chemical reactivity of nanomaterials
results in increased production of re
active
oxygen species (ROS), including free radicals. It
may result in oxidative stress, inflammation, and
consequent damage to proteins, membranes
and DNA thereby imparting toxicity. [4] A large
number of particles could overload the body's
phagocytes, th
ereby triggering stress reactions
that lead to inflammation and weaken the body’s
defense against other pathogens. By adsorbing
onto their surface some of the macromolecules
they encounter affecting the regulatory
mechanisms of enzymes and other proteins.
The nanoparticles can cross the membrane
barriers easily and enter the blood stream to
later on get accumulated in the vital organs

Other properties of nanomaterials that
influence toxicity include: chemical composition,
shape, surface structure, surface
charge,
aggregation and solubility, and the presence or
absence of functional groups of other chemicals.
The large number of variables influencing
toxicity means that it is difficult to
generalize

about health risks associated with exposure to
nanomaterials


each new
nanomaterial

must be
assessed individually and all material properties
must be taken into account.



2. Materials and methods

2.1. Nanoparticles


Four different types of nanoparticles are
used: Multiwalled Carbon Nanotube (MWCNT),
Alumina (Al2O3), Titanium dioxide (TiO2) were
bought from Amorphous Nano, USA. Zinc Oxide
(ZnO2) was synthesized by us using the college
lab facility. Zinc Sulphate (ZnSO
4
.7H
2
O)
(Qualigens Chemicals), Deionized Water.


2.2. Plant Material


Green gram (
Phaseolus radiatus
),
Peanuts (
Arachis hypogaea
), Pea (
Pisum
sativum
) and White chickpeas or Kabuli chana
(
Cicer arietinum
) were bought from the

market.
For each experiment, growth index was
measured by taking these 5 seeds on a

Petri
dish
.


2.3. Animal Mat
erial


Fibroblast cells were taken from white
mice and normal mice. We used Indian Institute
of Science, Bangalore’s central facility of animal
house. Blood samples were taken from three
volunteers.


2.4. Chemicals


2.4.1. Enzyme activity


Diastase (1, 4
-
α
-
D
-
Glucan
-
glucanohydrolase;
α
-
Amylase) REF
-

RM638
-
100G manufactured by Himedia, CAS No. 9001


19


8, minimum assay: 1:2000 I.P. units.
Potassium Sodium Tartarate, Sodium
Hydroxide, Phosphate buffer solution pH 6.8
(Buffer tablets
), Soluble Starch and
Gl
ucose.


2.4.2. Fibroblast Culture


High
-
sugar Dulbecco’s modified Eagle’s
medium (DMEM) and Fetal Bovine Serum (FBS)
from Hyclone. Penicillin, Streptomycin from
Sangon. Trypsin, MTT, propidium iodide, and
RNase from Sigma
-
Aldrich.

DMEM/F12
(Dulbecco's modified essential medium/Ham's
12 nutrient mixture), supplemented with 5% (v/v)
Toxic effects of Nanoparticles on healthy cells and biomolecules in in
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fetal calf serum, and 1% (v/v) antibiotic (2 mM L
-
glutamine, 100 U/mL Penicillin and 0.1 mg/mL
Streptomycin). HBSS (Hank's Balanced Salt
Solution), Tryps
in/EDTA, Trypan blue (0.4%
(w
/v))

and
Promega 96 well for Nonoradioactive
cell proliferation kits.


2.4.3. SDS PAGE


GeNei
TM

SDS
-
PAGE Kit, catalog no.
:106167. Molecular weight marker, SDS7B2
Prestained Molecular Weight Marker (M.W.
27,000
-
180,000), from S
igma Aldrich.
Coomassie Violet R 200 bought from Sigma
Aldrich
.


2.5. Cytotoxicity testing on Mice Fibroblast cells



The tail skin biopsy was obtained from a
mouse which was processed and used for
fibroblast culture using the standard procedure.
After 6 d
ays from seeding, the cells reach
confluence . Then the culture medium is
removed from the culture flasks and the cells
were rinsed with sterile HBSS. Confluent cell
layers were enzymatically removed using
Trypsin/EDTA and resuspended in culture
medium. Ce
ll viability was assessed by vital
staining with trypan blue (0.4%(w/v)), MTS
assay was carried out and cell number was
determined using light microscope


Nanoparticle suspension was produced
by individually suspending TiO2 and ZnO in the
culture medium at

concentration of 5000 ppm
and dispersed by ultrasonic vibration for 15 min.
In order to ensure the uniform suspension, they
were stirred on vortex agitation (1 min) before
every use.

Cytotoxicity testing was performed using
the Promega CellTiter 96 Aqueou
s Non
-
Radioactive Cell Proliferation (MTS) assay to
determine the number of viable cells in culture
(Promega, 2005). The test protocol for
cytotoxicity evaluation was adopted from
previously published papers and
manufacturer’s

instructions (Malich

et al., 1997; Hayes and
Markovic, 1999; Bakand et al., 2005a; Bakand et
al., 2005b; Lestari et al., 2006; Hayes et al.,
2007). [11]

A standard plot of Cell viability v/s Absorbance
at 490nm was obtained after subjecting the
serially diluted fibroblast sus
pension to MTS
assay.

Nanoparticles were suspended in
culture media, serially diluted across 96
-
well
microtiter plates (100μL), and incubated at 37ºC
with 5% CO2. Two sets of exposure times were
carried out. These included 4h and 24 h
exposure periods. Fo
ur hours prior to the end of
each exposure period, an MTS mixture (20 μL/
well) was added. After the completion of
exposure period, the plates were placed on a
microwell plate reader (Biorad microplate
reader), shaken for 10 s and the absorbance of
the for
mazan product was read at 490 nm. Each
experiment was repeated on three separate
occasions. Two internal controls were set up for
each experiment: (1) an IC0 consisting of cells
only and (2) IC100 consisting of medium only.
Background absorbance due to the

non
-
specific
reaction between test compounds and the MTS
reagent was deducted from exposed cell values
(Hayes and Markovic, 2002) [12].


2.6. Evaluation of Phytotoxicity


Stock suspensions for all the
Nanoparticle of 2% concentration by using
Deionized wa
ter were prepared. They were
properly mixed with a magnetic stirrer at
maximum RPM, for 30mins.


2.6.1. Seed Germination


Seeds were immersed in a 10% Sodium
Hypochlorite so
lution for 10 min to ensure the
surface sterility and then soaked in DI water for
control an
d nanoparticle suspension for 2
hours
[12]. The seeds were soaked in the nanoparticle
susp
ensions in a shaker incubator at
a
temperature of 37°C at a minimum possib
le
RPM for 2 hours to avoid the
agglomeration of
nanoparticles. All the Petri dish
es were sterilized
with 100% Ethanol

and dried. One small piece of
filter paper of same diameter was placed inside
all the Petri dishes. A bed was made inside the
Petri dish with hydrophilic cotton and the seeds
were put in an arranged manner .Seeds of the

same kind were soaked in each Nanoparticle
suspension and were put into the Petri dishes
and the test medium was poured into the
absorbent cotton. The Petri dishes were covered
and sealed with parafilm and placed in a growth
chamber with the parameters me
ntioned in the
figure 12. After 5 days, the shoot and root length
were measured. All the seeds of the control
Toxic effects of Nanoparticles on healthy cells and biomolecules in in
-
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were germinated and then the germination was
halted. Firstly, we experimented with all the
seeds where we put one seed per plate. After
checking t
he effect per plate five seeds were
added and the experiment was carried out the
same way.




2.7.

Effect on proteins

2.7.1. Effect on Enzyme activity

Diastase, a commercially available
enzyme is used to find the action of
nanoparticles on enzyme activity. One of the
compound of diastase is
α
-
Amylase (1, 4
-
α
-
D
-
Glucan
-
glucanohydrolase;
α
-
Amylase), so we
checked amylase activity by DNS

(Dinitro
Salicylic a
cid)
colorimetric assay where starch
was the substrate for the assay to form glucose.

The enzyme was incubated with 2%
nanoparticle suspensions for 30 min at 37C
before checking for its activity.


2.7.2.
Effect on Serum Proteins

Precipitation of the serum
proteins was
carried out using ethanol precipitation method.
1:9 volume of sample to ethanol (100% chilled at
-
20°C) was mixed and kept at
-
20 C for 60
minutes. It was centrifuged for 15 minutes at 4°C
at a maximum speed of 15000 RPM.
Supernatant was disch
arged to retain the pellet
formed, the tube was dried by inverting it on a
tissue paper. The pellet retained in the tube was
washed with cold ethanol (90%) kept at
-
20°C.
Ethanol residue was removed by air drying. The
pellet was resuspended in 5 ml PBS (pH

6.8) by
vortexing. The resuspended protein was then
used for the experiment. 1:1 ratio of protein to
1% and 2% different ultrasonicated nanoparticle
suspensions were incubated for 30 min at 37°C.
The total volume of the sample was 2 ml. These
samples were

then prepared for the
electrophoresis by mixing with the loading buffer
in 1:2 ratios. Then the treated sample is
subjected to SDS
-
PAGE.


2.8
Characterization

of nanoparticles


After evaluating the toxic effect of
nanoparticles, we needed to do the
characterization of the particles to confirm our
analysis. As we have mentioned earlier we have
worked with TiO2, Al2O3, MWCNT and ZnO
Nanoparticle. We followed characterization of
nan
oparticle by Transmission Electron
Microscopy (TEM) to find out the physical
dimension and property of the nanoparticles.


3. Results


3.1
.

Effect of Nanoparticles on Fibroblast cells.


Dose response curves were plotted for
the test chemicals after correction by subtracting
the background absorbance from the controls.
IC50 values were extrapolated graphically from
the plotted absorbance data. Relationship
between test chemical (ZnO and Ti
O2)
concentration (ppm) and cell viability (%) after 4
and 24 h exposures using the MTS
assay are
presented in Figure (1) and (2)
respectively.

Cell
viability was significantly reduced in a dose
-
dependent manner after exposure of mice
fibroblasts to nanoparticles using the MTS
assay. The experimental data for IC50 values on
the cells are summarized in Table no. 1 with
data presented as mean value
s ± standard
deviations. The experiment was conducted
following 4 and 24 h exposure time. Slight cell
viability reduction was observed after 4 h
exposure time but by increasing the exposure
time to 24 hours the cytotoxicity of both
nanoparticles increased
substantially. Following
24 h exposure time the IC50 of ZnO was 50.66
±
12.93 ppm, and for TiO2 the IC50 was 80.12
±
9
ppm.


Fig
ure(1)


Toxic effects of Nanoparticles on healthy cells and biomolecules in in
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5





Figu
re(2)



Table (1)
: IC50 values for ZnO & TiO
2



3.2
.

Effect of nanoparticles on germination of
seeds


It was
observed that peas (
Pisum
sativum
) and green gram (
Phaseolus radiatus
)
has the inhibitory effect with MWCNT and
Alumina, significantly with ZnO

Nanoparticle. 3
different concentrations of Nanoparticles were
used viz., 0.5%, 1% and 2% for the germination
of the seeds. The nanoparticles can be reported
with minimal toxicity on the test plants if it has no
negative effects on the seed germination an
d
root growth at such a high concentration
according to USEPA guidelines, 1996.

Effect of Nanoparticles on peas and
green gram was taken into consideration, since
these 2 seeds are significantly affected rather
than Peanut or Groundnut (
Arachis hypogaea
)
a
nd White chickpeas or Kabuli channa (
Cicer
arietinum
). Growth rate of all the seeds are
inhibited in ZnO Nanoparticle, MWCNT
suspension, Nano alumina particles suspension
had very less cytotoxicity and somewhere TiO2
growth is not inhibited rather induced,

(Kabuli
channa (
Cicer arietinum
)). In this work, all
germinated with cotyledons sprouting out of
seed coat and 1 mm was used as the minimum
length to be called root. Nanoparticle of ZnO
showed the greatest cytotoxicity among the 4
types of Nanoparticles.



Figure (3)



Figure (4)



Figure (5)

Toxic effects of Nanoparticles on healthy cells and biomolecules in in
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Figure (6)


3.3
.

Effect of Nanoparticles on Enzyme activity


We first evaluated normal α
-
Amylase
activity to understand its activity with starch in
optimum reaction environment
-

pH 6.8,
incubation temperature
37C showed the activity
of α
-
Amylase is almost linear. The evaluation of
nanoparticle suspension under the same
reaction conditions was carried out. Figure __
shows that it does not have the similarity with
the control reaction that is the activity of α
-
Am
ylase without any nanoparticles. The
discussion section deals with the analysis of
these effects.


Figure (7)


3.4. Effect of Nanoparticles on Serum proteins


In the gel after SDS
-
PAGE, the band
formed shows
with reference to the Molecular
weight markers
that the serum protein contains
differen
t proteins of varying molecular
weights.
The significant proteins are of the molecular
we
ights between 200 to 55.4, 21.5
and 14.4

kD.
We can see in the figure __
. Ctrl is the contr
ol
which is the plain untreated
bloo
d serum protein
and A,B,C,D,E,F,G,H are the samp
les treated
with 1% Alumina, 1%
CNT, 1% Zinc , 1% TiO
2
,
2% Alumina, 2% CNT, 2%Zinc and 2% TiO
2

respectively.



Figure (8)



3.5.
Characterization


TEM showed that ZnO

is the smallest
particle, followed by Alumina. ZnO has
shown
MWCNT

showed proper walls in the high
resolution images. The core diameter of the
Carbon nanotube is approximately 10nm making
it easier for the protein or DNA molecules
to
enter

the MWCNT and
t
hereby

undergoing
inhibition of activity. TiO
2

nanoparticle is
approximately 10
-
30 nm, making it easier for the
particles to enter the cells through the cellular
pores. At higher resolution, TiO
2

shows larger
surface area

Fig
ure (9)

: TEM images of Nanoparticles




Toxic effects of Nanoparticles on healthy cells and biomolecules in in
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4. Discussion


Exposure to both ZnO and TiO
2

nanoparticles indicated a range of cytotoxic
responses to human skin fibroblast cells.
However, the toxic endpoint of IC50 could not be
measured for 4 hr exposure due to incompl
ete
dose response curves. These results indicate
that at 4 hrs exposure, both nanoparticles
present only a mild adverse effect to human cell
fibroblasts. However, when the exposure time
was increased to 24 hrs, both nanoparticles had
a substantial toxic im
pact to cells. This time
dependent result is standard dose response
behavior
. These results are supported by
previously published results which indicated that
longer (3 day) exposure generated a greater
toxicity to human bronchial epithelial cells (IC50
(T
iO2) 6.5
μ
g/mL) using the MTT assay (Gurr
et
al.,
2005). Results of this study demonstrated
that the MTS assay could be implemented as an
effective and sensitive tool to assess cytotoxicity
of ZnO and TiO
2

nanoparticles on human skin
fibroblasts. Recent published
in vivo
studies
have shown significant adverse effects to mice
using nano
-
scale Zn metal powder and TiO
2

at 5
g/kg body weight (Wang
et al.,
2006; Wang
et
al.,
2007). Pulmonary toxicity has also be
en
observed in rats after 24 hr exposure to low
concentration (5 mg/kg) of nano
-
scale TiO2
particles (Warheit
et al.,
2007).
In vitro
toxicity
assessment has become widely used for recent
toxicity studies.


Such assays provide rapid, cost
effective and reli
able results (Hayes and
Markovic,

1999). Toxicity results of ZnO nanoparticles in
Chinese hamster ovary cells indicated IC50 at
340
μ
g/mL, respectively (Dufour
et al.,
2006).
Nanomaterial usage will continue to increase
rapidly and widely in areas such as
cosmetics,
pharmaceuticals and other industrial
applications. Accurately assessing the toxicity
and safety of these nanomaterials to human
health is of utmost importance. Toxicity data
generated in this study can potentially be used
to assess human topical

risk exposure to
nanomaterials.

Future studies should be
focused on investigating the potential risk of
nanomaterials to human health at the
microscopic cellular level by implementing
appropriate microscopic techniques such as
TEM (transmission electroni
c microscope) to
reveal general mechanisms of toxicity and
characterizing exposure to nanomaterials. To
verify it, we evaluated the toxic effects of
nanoparticles on enzyme activity and serum
proteins.


Even though we got toxic respose of
nanoparticles

on
germinating seeds, it was not
in conformation with the results found in the
earlier paper published by Daohui Lin, Baoshan
Xing [12]. This was because we used a
magnetic stirrer instead of an ultrasonicator to
disperse the nanoparticles. Further studies h
ave
to be done without such kind of experimental
error.


Enzyme assay
shows that

all the
Nanoparticles are inhibiting the enzymatic
activity of the amylase. The most prominent was
Titanium dioxide. The inhibitory action is due to
few possibilities. Firstly
, the protein degrades
due the reactivity of the nanoparticle. Secondly,
the allosteric site is being blocked by the
nanoparticle to inhibit the enzyme to produce the
product. Thirdly, the enzyme may have entered
inside the nanoparticle in case of CNT, and

therefore the substrate is not able to encounter
the enzyme and lastly it is immobilized to the
surface of the nanoparticle in such a manner
that the enzyme active site is not exposed. To
verify all these
possibilities we undertook
some
biophysical test o
f protein which is gel
electrophoresis

by SDS PAGE.


The serum protein
-
nanoparticle
interaction shows that the after exposing with
nanoparticles, bands started disappearing with
increasing concentrations of nanoparticles.
Bands fully disappeared and came a
t end of the
gel, which infers that all the protein was
degraded and denatured into small peptides or
amino acids resulting in the molecules travelling
to the end of the gel. It was observed that at 1%
concentration of nanoparticle of MWCNT,
Alumina, ZnO a
nd TiO
2

showed increasing
order
of the band disappearing. And at 2%
concentration of ZnO and TiO
2
, there were no
form
ation of bands (Figure (8)
). So,
degradation
of peptide bonds in protein is the reason here for
the Nanotoxicology.


5. Conclusion


This
work was done in various parts of
Science and Technology and various branches
of Biology. Our work was basically focused on
Nanoparticles, which have been implemented
widely in the field of Biotechnology,
Pharmaceuticals and Cosmetic industry to
ameliorate

the process of disease diagnostics
and disease curing, in the 21
st

century. Our aim
was to check the novel material


Nanoparticle’s
Toxic effects of Nanoparticles on healthy cells and biomolecules in in
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ill
-
effect on healthy cells from a macroscopic
view since it is used
in vivo
. While carrying out
studies on the cellular p
henomenon of
Nanotoxicology, we plumbed deep into the
molecular level of toxicity of Nanoparticle by
checking the effect of Nanoparticle on the
biomolecules, which are the building blocks of
cells, to understand the over all process of
Nanotoxicology. We h
ad taken Nanoparticles
like MWCNT, Alumina, Titanium dioxide, Zinc
and Zinc Oxide nanoparticle in the consideration
for checking the cytotoxicity of cells. Ill
-
effects of
nanoparticles on the cellular and molecular level
was identified throughout the study

by checking
the parameters like IC50, Cell Viability, Growth
rate, Biochemical and Biophysical activity of
biological elements. Based on these
parameters, we optimized the various conditions
for Nanoparticle usage like exposure timing to
TiO
2

and ZnO (whi
ch is used directly on skin to
avoid the Ultraviolet radiation of Sun), the
exposure timing in this case should be less than
or equal to 4 hours for mice cell. In future, our
aim is continue this experiment on human
fibroblast cells. In the case of plants,

this study
did not find
much toxic effect

due to
experimental error in making nanoparticle
suspension without using ultrasonication, which
leads agglomeration and precipitation of
nanoparticle. We experimented at the molecular
level of Nanotoxicology by c
hecking the protein
molecules’ activity, like enzyme activity of
Amylase which showed that few materials like
Carbon Nanotubes are only moderately toxic
compared to others, and from the results of
various biophysical experiments like SDS
PAGE, we identi
fied the massive degradation of
protein molecules under the effect of
nanoparticles, which was revealed by the
absence of bands in the 2% concentration of
Nanoparticles. Comparing the same
concentration with different particle composition,
we found TiO
2

to

be more toxic than ZnO. The
future work should be done from the prospect of
size, atomic configuration and reactivity of
Nanoparticle to reduce the toxicity, so that the
human society can use the Nanomedicine
effectively compare to the normal
pharmaceutic
al drugs. Nanoparticles are being
continuously produced in nature in various forms
with contributions made by numerous human
activities as well. These novel particles have
emerged as a next generation tool showing
successful implementation in wide areas of

medicine
,
diagnostics, drug delivery and also in
industrial fields like Biotechnology, Mechanical,
Electronics and Chemical Engineering.
However, the pros and cons of nanoparticles
need to be thoroughly studied before mass
implementation, mainly their tox
ic effects on
human, animals, plants and the environment,
which requires wide research on the optimum
quantity of nanoparticles used for various
applications for a better future in
Nanobiotechnology.


6. Acknowledgement


We are highly obliged to Prof.
Sridhara.
V, of Department of Biotechnology, New
Horizon
College of Engineering
(NHCE)
,

Bangalore f
or
his
guidance. And

we also like to thank all the
faculty and staff of Dept of BT, NHCE and We
are thankful to Prof. Mahadev Raju and other
faculty members
of the Departme
nt of Chemical
Engineering, Dayananda Sagar College of
Engineering

for their guidance and
encouragement. We

also acknowledge the
valuable assistance of Prof. R.R.Dighe,
Chairman and Professor of Department of
Molecular Reproduction Developme
nt and
Growth of Indian Institute of Science, Bangalore
for providing facility to
carry

out our animal cell
culture. We are thankful to Indian Institute of
Science, Bangalore
-
Central Animal Facility to
collection of animal cell sample.


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[19] Figure Caption: Molecular Imaging and
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