INDIAN INSTITUTE OF TECHNOLOGY ROORKEE

NPTEL

NPTEL ONLINE CERTIFICATION COURSE

Biomedical Nanotechnology

Lec – 20
Nanotoxicology

Dr. P. Gopinath
Department of Biotechnology
Indian Institute of Technology Roorkee

Hello everyone I welcome you all to the 20th lecture of this course on nanotoxicology. This is the
last lecture of this course. Till now we have learnt various biomedical applications of the nano
materials. Today we are going to see the other side of nano materials that is toxicity of nano
materials.

(Refer Slide Time: 00:41)

In this lecture we are going to learn what nano toxicology is, what the various exposure scenarios
are and what are the various methodology available to study the toxicity of nano particles. We
are also going to learn what is genotoxicity and how to study the hemocompatibility of the
nanomaterials and also how to study the in vivo assessment of the nano material toxicity.

(Refer Slide Time: 01:02)

Let us see what is nanotoxicology? The nanotoxicology is a study of toxicity of nano materials.
Nanomaterials even when made of inert elements like gold become highly active at nanometer
dimension. As we know that gold is an inert material which we use it for making ornaments or
jewels and when it goes to nano scale it will show different kinds of properties.

The nano toxicology studies are intended to determine whether and to what extent these
properties may pose a threat to the environment and to the human beings. So here this nano
technology is a double edged sword. The same novel properties making nano particles attractive
makes them potentially toxic too. So particular care must be taken in nano medicines since in
this area where greater exposure would be present.

Most of the fields have its own advantage as well as disadvantages. Similarly this nano
technology also like a double edge sword so it has it is own advantages as well as disadvantages,
so we have to select the nano materials wisely for the various biomedical applications.
(Refer Slide Time: 02:08)

Why we have to study the nano toxicology? You can see here these are the various consumer
products where they are using this nano scale materials. For examples so this Loreal Company is
using this nano particles based cosmetics and Motorola is using the CNT based nano emissive
displays. We also have nano care fabrics shirts and carbon nano fiber racquets. In India also we
are having several washing machines which are coated with the silver nano particles. Now a days
in TV advertisements we are seeing some of the soap which are using silver nano particles.

What happens with these silver nano particles? They go into the water bodies. When it goes into
the water body how it is going to affect the living organism in the water bodies. It will also
contaminate the ground water and going to cause toxicity. So in this lecture we try to learn these.
We will learn about the nano toxicity methodology available to study the toxicity of nano
materials.

(Refer Slide Time: 03:10)

So this you might have seen in cricket. Some players used to apply this kind of sun screen. So
this is a bulk form of zinc which is white and opaque. In the picture, in one side he has applied
the same sun screen but it is in the nano zinc. The nano zinc is transparent and the bulk zinc that
is your sun screen it is white and opaque. So when the material goes to nano scale the properties
of the material get changed.

The nano gold can be red and blue and copper nano tubes can connect electricity. So new
applications for familiar materials and but also new risks are associated with them. So the inert
material which we are using day to day, when it goes nano scale it is going to have some
different kinds of properties which may have advantages but at the same time it may have new
risks also.
(Refer Slide Time: 04:03)
There are various companies involved in this nano for example nestle, Pepsi and some of the
other companies. Most of the companies protect their product in the name of trade secret or in
the name of pattern protected laws. So we are not aware about what are the nano materials in the
food products.
(Refer Slide Time: 04:23)
So these are the other nano based products for example sunscreen cosmetic and even the baby
diaper and baby bottle feeding bottle. So in everything they started using this nano materials and
nano coatings but we do not know what will be the long term effects of using them.
(Refer Slide Time: 04:42)

In early 2008 approximately 104 foods and food additives contained nano particles which is on
sale internationally. Some of the analysts suggest that almost 500 nanofoods are available in the
world wide. So almost 500 nanofoods are already in the market and we are not aware about what
are those as there is no labeling. For example if you are using the GM food, there should be a
GM label. Similarly if you are using nano foods or nano coated food, there should be some label
to indicate this. Like these are coated with nano particles or this food is wrapped with nano
package.
(Refer Slide Time: 05:25)
But there is no such labeling. The nano can play a major role in agriculture also. It can make the
agriculture uniform and it can make further automated and industrialized. Because of this the
farmers may lose the farming knowledge but at the same time but it can have the designer
substance which can deliver the nutrients efficiently into the body.

(Refer Slide Time: 05:49)

These are some of the nano foods on sale include cooling oils teas. We can also have the
antibacterial kitchenware, food processing and food packaging materials. So this nano based
food packing materials will prevent the microbial growth. But this nano materials are also
interacting with your foods substance. So when it interacts with the food how much amount of
nano particles releasing into food material and what will be the effects of these are not
understood thoroughly.
(Refer Slide Time: 06:23)

Recently an US based company nanoceuticals made a slim shake chocolate. This is a diet milk
shake that uses silica nano particles coated in cocoa clusters to increase the taste with low cocoa
and sugar content. But the health risk of nano silica is not fully understood and early studies
suggest for the need for caution. So this slim shake is made up of silica nano particles so what is
silica nano particles? Silica nano particles are basically sand particles. So in your childhood if
you eat sand your mom will beat you but the same sand particles you are drinking in the form
silica slim shake. We do not know what will be the long term effects of this silica based
chocolate drinks.

(Refer Slide Time: 07:06)

These are some of the future nano food and agriculture materials. It can be edible nano wrappers
and coatings. We can use the nano biotechnology for manipulation of seeds to increase the
growth and also we can increase the productivity but again what will be the drawback and long
term effects still unknown.

(Refer Slide Time: 07:34)

The nano technology could enable junk foods to be fat free, sugar free and also it can reduce the
carbohydrate content. It can increase the vitamin, protein and fiber contents. Then it can be
marketed as a healthy alternative. But the problem is our relationship with the real food will be
eroded.

(Refer Slide Time: 08:01)

The nanotechnology could displace the workers and erode the forming knowledge because
everything will be automated like automated nano surveillance and management system. So that
could reduce the need for form workers and this could commodify farming knowledge.
(Refer Slide Time: 08:22)
This may lead to some of the multinational companies controlling the food and agriculture.
(Refer Slide Time: 08:28)

Let us see some of the examples of how these nano particles pose some new toxicity risks. The
nano materials are readily inhaled and ingested. At least some will cross the skin and nano
materials gain access to tissues and cells. The inhaled nano particles can cross the blood brain
barrier. Most of the other drugs cannot enter the blood brain barrier but these nano particles can
cross the blood brain barrier.
(Refer Slide Time: 09:01)

So let us see some more examples. The nano sliver is toxic to rodent liver brain and stem cells
and may harm beneficial bacteria also. The nano zinc oxide is toxic to rat and human cells even
at low doses. The nano Silicon dioxide <70nm can cause onset pathology similar to the
neurodegenerative disorders. The nano titanium dioxide can damage DNA in human cells, harm
algae and water fleas especially with help of UV light exposure.

The titanium dioxide is everywhere- in the tooth paste, talcum powder and everywhere. When it
go into the water bodies, it is a very good photo catalyst in presence of sunlight. This can harm
the algae and other living organisms in the water bodies.
(Refer Slide Time: 09:52)
These nano particles can distort our immune system response and can also act like a Trojan
horses. The Trojan horse means the nano particle will be in the body for more time with more
circulation time and suddenly it may cause some new toxic effects. There is a possible link
between the consumption of processed foods and irritable bowl and crown disease. So this nano
materials can also induce this kind of diseases.
(Refer Slide Time: 10:27)
The UK royal society recommended some rules in 2004 for the safety. That is the full safety
assessment of all products that contain nano prior to market release and all nano ingredients to be
labeled. The environmental release of nano materials should be avoided as far as possible and
factories and research laboratories should treat nano materials as if they were hazardous. Now
let us see these properties of nano scale materials.
(Refer Slide Time: 10:54)

The same properties making nano material so interesting can make them potentially harmful. As
I told you earlier, the enhanced reactivity, the increased surface to mass ratio, and enhanced
permeation are the properties which have a wide application in biomedical field. But the same
properties will also have some toxic effects.
(Refer Slide Time: 11:17)
So let us see what the various exposure scenarios are. All substances in the world are toxic to
plants animals and humans at some exposure levels. So there are two roots short term routes and
long term routes. So in the short term routes it is mainly due to inhalation of gas phase, skin
contacts in solution, and also oral ingestion. In the long term routes, they enter mainly due to the
soil absorption, water dissolution and also because of biodegradation issues.
(Refer Slide Time: 11:48)
So the risk evolution for exposure to nano technology product is hindered by the law protected
secrecy of product formulation. There is also a lack of specific regulations on nano technology
products. The reports on toxicity of products are non-mandatory. We are still using the old
criteria and methods that are obsolete. The researchers from the national institute for
occupational safety and health determined the concentration of nano particles in the air while
unloading a reactor for producing the metal oxide nano particles.
(Refer Slide Time: 12:11)

And these are the various international initiative. Recently some agencies have taken some
actions to establish regulations to nano technology. So the International standard organization
and US national nano technology initiative and British standard institute and also the
environmental protection agency are some of them. So all these agencies have published reports
and guidelines related to the handling of nano materials and research approaches to nano
toxicology. But all of them are voluntary to follow and there is no mandatory rules and
regulations.
(Refer Slide Time: 12:47)
So let us see the methodologies for studying the toxicity of nano particles. So if we see this is the
carbon nano tubes. 100 grams of carbon nano tubes looks like 100 kg.
(Refer Slide Time: 12:59)

So there are several points we have to understand to study the toxicity of a nano materials. What
to measure like how much duration and what is the root of exposure and what is the dosage and
formulation of test material. Also what kind of biological species you are going to use to study
the toxicity of nano materials and whether do you have standard references and standard
materials for comparisons, so these are the questions to be considered before we start the nano
toxicology studies.
(Refer Slide Time: 13:31)

The most important thing is how to design a realistic test. So the meaningful results on the
toxicity of nano materials are achieved when the conditions of possible exposure are reproduced
accurately. So let us see how the nano particles would enter the body, the first one is accidently
so it may be due to environmental contamination and work place exposure, the next one is
deliberately introduced. That is your nano material and applications so different methods of
exposure of the nano particle might produce different results. Let us see this with some
examples.
(Refer Slide Time: 14:02)
So the example 1 is insulation of CNT in the rats. The rats that were instilled with high doses of
single wall carbon nano tubes died due to respiratory blockage rather than pulmonary
intoxication. So here they used four kinds of particles including single wall carbon nano tubes by
pressurized intratraqueal installation. The CNT’s was given to the rats and it was the observed
for 24 hours, one week, one month and 3 months. There is inflammation but no cytotoxicity and
this picture is showing that the respiratory airways are mechanically blocked by carbon nano
tubes due to which the rat died.
(Refer Slide Time: 14:46)
The next example is inhalation of carbon nano tubes in rats by exposing the rats to air
contaminated with this carbon nano tubes. This led to the immune suppression. So in the
previous example we have seen that when you inject this carbon nano tubes through intratraqueal
administration the rat died. In this case, we are passing the air contaminated with low
concentrations of carbon nano tubes. After exposure for 6 hours per day for 14 days, the proteins
and immune response in the rats were tracked.
(Refer Slide Time: 15:18)

The researchers observed that signal like TGFß is released when the carbon nano tube is inhaled,
so this was tested by isolating the bronchoalveolar Lavage fluid protein from both exposed and
control rats. It was shown that the proteins from the exposed mice cause immune suppression. So
here there is no death of rat and only the immune suppression. From this example we can
understand that based on the method by you are giving the carbon nano tubes dosage to this
animals the results also varying. So when you instill this carbon nano tubes the rats died but
when the carbon nano tubes are inhaled by the rats only the immune suppression happened.
(Refer Slide Time: 16:03)
The various test assessments and methodologies used led to different conclusions on the toxicity
of carbon nano tubes. Several criteria must be used to select meaningful results like what test
simulates the more realistic exposure and also are these results applicable to all nano tubes? Are
exposure concentration realistic? And do animal subjects respond similar to humans? The
reliable data to answer these questions can make difference between deeming this carbon nano
tubes unsafe or safe.
(Refer Slide Time: 16:34)
In in vitro methods which means you can do it in the laboratory conditions using the bacteria or
cell lines to understand the toxicity and in vivo means inside the living system like living animal
models like mouse or the zebra fish or some other animal models. The ex vivo method is a
combination of in vitro and in vivo so we can study the toxicity of nano materials using the
bacteria and circulatory system in vitro.

We can also study the toxicity of nano materials using the animal models like mouse or rat or
zebra fish. This is the in vivo that is in the living system. The ex vivo is a combination of In vitro
and In vivo. So we can take out the particular organ and we can grow in the lab and we can study
the toxicity of nano materials. The advantages of this method is this is cost effective, time
effective and no ethical issues when you use the In vitro conditions.
(Refer Slide Time: 17:31)

Several studies concluded that response to nano particles is too diverse. For example, dogs and
pigs have a higher immune response to nano particles. Again these nano particle toxicity will
differ depending on the formulation of it. So depending on what kind of nano particles you are
using, and their concentration, pH, the coatings of the nano materials, what are the exposure
mode, exposure time and which is the targeted organ, based on all these the effects may vary.
What kind of animal model you are you going to use, the rat or human cell lines or fish model
also affects. So depending on these parameters like formulation, test and subjection methods the
nano particle toxicity will also vary.
(Refer Slide Time: 18:23)

We can also improve the In vitro techniques by substituting the 2D with 3 dimensional scaffold.
These are created in a hydrogel matrix. In the picture, the liver cells are grey color circles.
(Refer Slide Time: 18:43)
In a 3D dimensional scaffold, the advantages are like it will represent the complex cell-cell and
cell-matrix interaction. Also, with the help of tissue engineering we can develop the artificial
liver and we can easily understand the toxicity of nano materials.
(Refer Slide Time: 19:04)

The researchers have studied the toxicity of quantum dot using the 3 Dimensional as well as 2
dimensional scaffolds.
(Refer Slide Time: 19:13)
They concluded that the 3 dimensional scaffold is exactly mimicking the In vivo conditions. You
can see here this red color are dead cells, so as I explained in the previous lecture so this red cells
are dead cells in the 2D with the same concentration you are seeing more amount of dead cells
and in the 3D culture you are seeing only few cells are dead cells. But the concentration of nano
particle is same. So when you use the 3 dimensional scaffolds, they will exactly mimic like the
In vivo condition.
(Refer Slide Time: 19:47)

The other alternate is like we can also do these modeling in simulation. The toxicity of nano
particle depends on their physical and chemical interactions with the gases, liquids and other
nano particles surrounding them. So this can be studied using the molecular simulations. So the
specific and quantitative knowledge obtained from theory and simulations can help in building
the predictive models and algorithms for assessing the likelihood of toxicity in various natural
environments. So if you know the toxicity of nano materials, we can do the modelling and
simulation and we can accelerate the nano toxicity field.
(Refer Slide Time: 20:26)
Let us see what genotoxicity is. The genotoxicity refers to the ability of a test agent to induce
DNA damage. The cytotoxicity means if it is toxic to the cells and genotoxicity means if it can
damage the genetic material. The genotoxicity assays can detect, quantify and characterize the
DNA damage induced by a substance under investigation. These methods are indicators for the
likely carcinogenic agents. The genotoxin refers to a substance that damages DNA.
(Refer Slide Time: 21:03)
The nano materials release free metal ions which will induce the oxidative stress and this
oxidative stress will damage the DNA. This induces the apoptosis or the inflammation. We have
to understand whether this nano material can induce any damage to DNA or not.
(Refer Slide Time: 21:24)

When you expose the cells to the nano materials, there may be chances for chromosomal
aberrations. If it is a normal chromosome, there is no toxic effect. But if you are identifying any
chromosomal aberration that means it can lead to an abnormal cell growth. It may be the
compound which is tumorigenic. We can understand the genotoxicity by studying the DNA
double stand breakage by using various nuclear stains.
(Refer Slide Time: 21:59)
So these are the various toxic methods available. We can use the Salmonella typhimurium to
understand whether this nano material is inducing any mutation in the genetic material. This
assay is called as Ames assay. We can also use the mammalian cell lines to understand whether it
is inducing the DNA laddering and apoptosis by using the MTT assay and other apoptotic assays.
(Refer Slide Time: 22:31)

What is the Ames test? So here we can use the bacteria called Salmonella which requires
histidine for its growth. We can add a possible mutagen to the bacteria, in this case we are using
the nano particles. The bacteria will be grown in their media with minimal histidine. In the
presence of a possible mutagen, if it cause some mutation, it will cause this histidine negative to
become histidine positive. So this bacteria will grow more on this plate. So if the bacteria are
growing more on this plate that means the nano particle had induced some mutation in the
genetic material.
(Refer Slide Time: 23:11)

Now let us see what hemocompatibility assay is. The hemocompatibility is a very important
factor to decide the application of implantable biomaterials such as artificial blood vessels and
orthopaedic implants. With the development of blood contacting materials or implantable
devices it is necessary to improve the hemocompatibility by surface modification or redesign. So
whenever you make the nano materials or nano particles based coatings, we have to understand
whether it is compatible with the human system. We also have to understand whether it is
compatible with the blood. So by using this hemocompatibility test we can understand the
compatible of the material with the blood.
(Refer Slide Time: 23:52)
So the hemocompatibility test of nano material contains the following assays. It has haemolytic
assay, anticoagulants assay, platelet adhesion and activation assay, blood coagulation time assay
and blood protein adsorption assay.
(Refer Slide Time: 24:05)

We can take the blood from humans or you can collect the blood from goat. In case of human
blood, you have to take the human ethical committee permission. So it is easy to take the blood
from the goat. You can collect the blood from the nearby slaughter house and you can use it for
hemocompatibility studies. When you collect the blood, you have to mix the blood immediately
with the tri potassium EDTA which is an anticoagulant.
(Refer Slide Time: 24:47)

Then you can centrifuge it at 13000 rpm for 10 minutes at 4 °C. When you centrifuge you will
get these kinds of layers and you can use this isolated platelet rich plasma that is PRP for your
further studies.
(Refer Slide Time: 25:03)
This is the polymer film and you want to understand whether this polymer film is
hemocompatible or not. You can cut it into uniform sized circular disks using a simple punching
machine. This can be sterilized by keeping it under the UV light for 30 minutes. Then this can be
transferred to 96 well plate and washed with PBS.
(Refer Slide Time: 25:33)

Then you can add the isolated platelets on the polymer circular disk. You can add 200 µl of PRP
in each well and you can incubate in the incubator for 30 min at 37 °C.
(Refer Slide Time: 25:46)
And you can observe the adhered and activated platelets on the polymer disk surface using the
microscope. You can wash this 3 times with the PBS to remove the non adhered platelets and
view them under a microscope. If there is no change in the shape, that means there is no release
of coagulation factor and it is a good material for implants. But under the microscope if you are
seeing these kinds of shapes that means more adherence and the change in shape is mainly due to
the activated platelets. This means it is releasing more coagulation factor and it is not a good
material for implants.
(Refer Slide Time: 26:34)
The next one is in vivo assessment of nanomaterials toxicity. So why we have to use zebrafish as
a human model? Because it is a small vertebrate tropical water fish. It has a clear and transparent
embryos with short maturation time and functionally homologue with 70% of human disease
genes. The forward and reverse genetic model system is possible here and we can also do the
chemical biology and chemical genetics studies using zebrafish.
(Refer Slide Time: 26:53)

Let us see some of the examples how we can study the toxicity of sliver nanoparticles using this
zebrafish embryos. Researchers have used two different kinds of nanoparticles one is starch
coated sliver nanoparticles and one is BSA coated sliver nanoparticles in this paper.
(Refer Slide Time: 27:11)
We can study the toxicity of the nano materials in terms of mortality rate of zebrafish or hatching
as well as the heart rate and abnormal phenotypes. This is the zebrafish heart beating. You can
measure the heart rate as compared to the control what is the heart rate in treated in zebrafish
embryos.
(Refer Slide Time: 27:35)

So here you can see here so they have used the zebrafish embryo and treated with the different
concentrations of sliver nanoparticles. This is a normal embryo and this is a malformed embryo
and these are the dead embryo in presence of sliver nanoparticles. So based on that you can count
the number of embryo which got the malformed embryo as well as the dead embryo and based
on that you can calculate the toxicity of nanomaterials.
(Refer Slide Time: 28:02)

In this case they have observed that sliver nanoparticles is in most of the mitochondria as well as
the nucleus. This means that the nanoparticles enter the cells through many routes among them
endocytosis through the embryo wall is more likely. Entry into the mitochondria as well as the
nucleus may induce some damage to the DNA also.

(Refer Slide Time: 28:27)

By the elemental analysis of embryo they have observed that sliver nanoparticles is more in the
nucleus. The researchers believe that it may create a cascade of toxic events leading to damage
of the DNA which will leads to various toxic effects.
(Refer Slide Time: 28:42)

The zebrafish embryos treated with different concentrations of the sliver nanoparticles can affect
the heart rate, like the heart rate is going down and the mortality rate is increasing. The
nanoparticle deposition in the central nerve system could have adverse effects in the control of
the cardiac rhythm respiration and body movements so you can see here the heart rate is going
down with respect to concentration. The exposure to sliver nanoparticles resulted in the
accumulation of blood causing edema and necrosis so that is why the mortality rate is increasing.

(Refer Slide Time: 29:15)

We can also use the mouse or rat model for studying the toxicity of nano particles. The
organization for the economic cooperation and development guidelines recommend oral toxicity
and dermal toxicity test to understand the toxicity of nano materials.
(Refer Slide Time: 29:31)
So for the oral toxicity test, colloidal nanomaterials at the limited dose of 5000
milligram/kilogram body weight could be given to the mouse. It can be observed for the toxic
symptoms for the first three hours and after 24 hours, these animals could be maintained and
observed daily for over 14 days for the skin and also behavioral symptoms and toxic signs. Then
we can do the hematological and biochemical testing. These animals are sacrificed after 14 days
and histopathological testing could be made for understanding how much the nanoparticle
deposition in kidneys, spleen and liver is.
(Refer Slide Time: 30:11)
The next one is dermal toxicity test. We can apply these nanomaterials on the surface of the
animal models. The group 1 is treated with the distilled water and group 2 receives 50ppm of
colloidal nanoparticles solution and group 3 we can use a 100ppm of colloidal nanoparticles
solution. So we can increase the concentration depending on the study. So when you treat with
the different kinds of nanoparticles, then you can study the hematological and biochemical
analysis and also the histopathological investigations to understand the toxicity of nano
materials.

As a summary, in this lecture we have learnt what the nanotoxicology is and what the various
exposure scenarios are. We have also learnt what are the various methodologies available to
study the toxicity of nanoparticles. We have also learnt what is genotoxicity and also how to
study the hemocompatibility of your nanomaterials and in vivo assessment for studying the
nanomaterial toxicity. I end my lecture here. I thank you all for listening to this lecture and all
the best for your exams.

For Further Details Contact

Coordinator, Educational Technology Cell
Indian Institute of Technology Roorkee
Roorkee – 247667
E Mail: etcell.iitrke@gmail.com. etcell@iitr.ernet.in
Website: www.iitr.ac.in/centers/ETC, www.nptel.ac.in

Web Operators Pankaj Saini

Dr. Nibedita Bisoyi Arun. S
Neetesh Kumar
Jitender Kumar
Vivek Kumar Camera
Mohan Raj
Production Team
Sarath. K Online Editing
 Jithin. K

Video Editing
Jithin. K

Graphics
Binoy. V. P
 NPTEL Coordinator
Prof. B. K. Gandhi

An Educational Technology cell

IIT Roorkee Production
© Copyright All Rights Reserved