INDIAN INSTITUTE OF TECHNOLOGY ROORKEE

NPTEL

NPTEL ONLINE CERTIFICATION COURSE

Biomedical Nanotechnology

Lec - 11
Nanomaterials for Cancer Diagnosis

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

Hello everyone I welcome you all to this 11th lecture of this course.
(Refer Slide Time: 00:25)

This 11th lecture is on nanomaterials for cancer diagnosis. Let us see what the difference between
cancer and tumor is. The cells that continue to replicate and fail to differentiate into specialized
cells and become immortal are called as cancer cells or tumor cells. The main difference between
the cancer and tumor is the malignancy. A tumor that grows indefinitely and spreads is called as
metastasis. It is also called as cancer and this kills the host.
A benign tumor is a tumor that is not capable of metastasis and does not kill the host. The main
difference between the cancer and tumor is, the tumor cells are localized and it can grow
indefinitely. More numbers of cells grow in a particular location in case of tumor but it could be
removed by surgery. However, in the case of cancer, the cells spread from one location to
another and it is going to kill the host.
(Refer Slide Time: 01:26)

So these are the various types of cancers- carcinoma, which arises from the epithelial tissues
such as glands, breast and skin and it constitutes nearly 90% of all the cancers. Sarcoma is the
solid tumors of muscles and bone, it is 2% of all the cancers. Leukemia is the cancer of the bone
marrow, constituting 3.4% of all cancers. And lymphoma and myeloma this is constituting
approximately 5.4% of all the cancers.
(Refer Slide Time: 01:51)
Let us see the reason for occurrence of cancer. First reason is genetic factors, it may be due to
mutations or it maybe due to translocation, and it may be also due to heredity. The next reason is
environmental factors like UV or chemicals or viral infections. So if a person is working in some
chemical industry he has high chance for getting the cancer. In cancer, the conversion of proto-
oncogenes to oncogenes happens.

All cells are having proto-oncogenes. If the proto-oncogenes are converted into oncogenes, the
normal cell becomes a cancer cell. Any alterations in the tumor suppressor genes also create
cancer. Each cell possesses tumor suppressor genes that suppress the tumor growth, and if there
are mutations in the tumor suppressor genes, then there is a high chance for getting the tumors or
cancer. So this growth promoting proto-oncogenes and growth restricting tumor suppressor
genes should be properly balanced for a control of cell growth.
(Refer Slide Time: 02:47)
The conversion of proto-oncogenes to oncogenes creates an imbalance and it leads to altered
tumor-suppressor genes.
(Refer Slide Time: 02:56)

When this happens, there will be an uncontrolled cell growth, which is called as tumor. This
uncontrolled cell growth is called as a tumor. If the tumor spreads from one location to the other
location, then that is called as cancer. The phenomenon of spreading is called as metastasis.
(Refer Slide Time: 03:17)
The interesting fact about cancer is that at least once a day, your immune system destroys a cell
that would become a cancer cell if it lived. That means everyday at least some cells are trying to
become a cancer cell; if your immune system is strong it can destroy the cancer cells.
(Refer Slide Time: 03:35)
Let us see this animation. The longer cell is the cancer cell, and this one is the T-cell that is your
immune cells. The immune cells are coming and attacking the cancer cell and destroying it. This
shows that if your immune system is strong, it will protect you from developing cancer or tumor.
(Refer Slide Time: 03:53)

What is the major problem in the diagnosis of cancer? The main problem is that we are not able
to diagnose a cancer in its early stage. With the present traditional methods, we are able to
diagnose the cancer only in the later stage and in the later stage we have to go for targeted
treatment or we have to go for radiation and chemotherapy. By using the recent advancements in
nanotechnology, we can go for early detection of cancer and we can protect the patient from
death due to cancer.
(Refer Slide Time: 04:25)
Let us discuss about the status of research in biomarker studies. The PSA means Prostate-
specific antigen. Suppose if the person is having more amount of PSA that means he has high
chance for getting prostate cancer. In future, we will be able to tell you about the expression of
other markers in prostate cancer in addition to the expression of prostate specific antigen. So
based on that we can tell like if it is a BM1 or 2 or 3 and we can give drug according to that. So
here in this example you can see here the marker 1, 2, 3 are in off condition, and marker 4 is
subnormal.
(Refer Slide Time: 04:46)
For example, the expression of the marker 5 is fine, hence there is no metastasis. So we can go
for the treatment accordingly. Each cancer expresses different kinds of markers or receptors on
the surface of the cell. We can easily detect which type of marker is over expressed or which
type of marker is down regulated. So accordingly we can select the drug and we can target and
start the treatment process. This approach will save lot of time as well as we can avoid the side
effects arising out of the excess concentration of drugs. This is called as personalized medicine.
Instead of giving generalized medicines, we can give drug only to address the deregulated
expression of a particular protein. For example, if the marker 1 is over expressed we can give
drug only to suppress the marker 1, instead of giving drugs for all the various markers. This
approach will enhance the therapeutic efficiency.
(Refer Slide Time: 06:13)

Let us see another example. The nano devices can make cancer tests faster and more efficient.
Consider the example, this is patient A and patient B. We can check all the pathways and the
patient’s sensitivity to a drug and we can give the drug according to the patient’s need. For
example, for patient A all the growth pathways are okay, and cell mobility is okay and he is
sensitive for drug A. In case of patient B, he is sensitive to drug B and cell death pathways and
everything is okay. Here, even though both have prostate-cancer, they will be expressing
different kinds of biomarkers. So we can give drugs to address the specific need. This approach
is called as personalized medicine.
(Refer Slide Time: 07:07)

So in cancer diagnosis various nanomaterials like quantum dots play an important role. Quantum
dots are highly fluorescent, nanometer-sized, single crystals of semiconductor materials. The size
of the quantum dots is tunable between 5 to 15 nanometer. Based on its size, the quantum dots
give different fluorescence. This is the advantage of these quantum dots.
(Refer Slide Time: 07:28)
The optical properties of nanoparticles depend greatly on its structure. In particular, the color
emitted by the quantum dot depends on its diameter. The quantum dots can be injected into a
subject and then be detected by exciting them to emit fluorescence. But the quantum dots are
toxic, it is not biocompatible.
(Refer Slide Time: 07:51)

The challenge is in making the quantum dots biocompatible and soluble. So we have to engineer
these quantum dots. You can see here this is your core nano crystal which determines the color
and we have to give an inorganic shell around it. This will improve the brightness and stability
and followed by we have to give the organic coating, so that will provides the water solubility,
and functional groups for conjugation. We can tag any antibody or peptide to these quantum dots
and we can specifically target these quantum dots for cancer diagnosis.
(Refer Slide Time: 08:25)

So you can see here in this example, the quantum dots are not fluorescents. But when you apply
the UV light it will emit light.
(Refer Slide Time: 08:33)
These quantum dots are attached to some antibodies which will specifically go and bind to
cancer cells. So when light is applied to these quantum dots that are bound to the cancer cells,
they emit fluorescence. Here only the cancer cells will emit fluorescence and healthy cells does
not emit fluorescence. So we can easily diagnose the presence of cancer.
(Refer Slide Time: 08:51)

The quantum dots have excellent brightness and photo-stability. In this example, you can see the
difference between the quantum dots and organic dye. So you can see here the exposure time
required for a quantum dot is 0.019 second and for an organic dye it needs 1.22 seconds. So in
this picture the first two pictures is the cancer cell which have high level of expressing of the
particular cancer marker. That means, it is like the cancer cell which is expressing more number
of the markers. When compared to an organic dye, the quantum dots are 50 times brighter. If
there is a cancer cell which is expressing low level of markers, then the organic dye would not
give any signal. However, quantum dots can detect even the lower expression of any particular
marker. That means the person is having cancer but when we use organic dyes for diagnosis,
there won’t be any fluorescence signal. So we may get the result like the person is negative for
cancer but however, the person is having cancer.
(Refer Slide Time: 9:55)

But when we use quantum dots, we can see the fluorescence signals, even when the level of
expression of markers is low.
(Refer Slide Time: 10:09)
Another advantage of using quantum dots is that with respective to size it gives different
fluorescence. When traditional organic dyes are used, there is chance for overlapping of the
fluorescence. In case of quantum dots, there is no overlapping of fluorescence and there is no
cross talk.
(Refer Slide Time: 10:25)
This diagnosis should be multiplex as the cancer cells express multiple bio markers, which
should be detected simultaneously. Based on the expression of the markers, we can identify
whether the person is in the early stage of the cancer or he is in the advantage stage of the cancer.
So let us see the multiplex diagnosis.
(Refer Slide Time: 10:54)

Consider four quantum dots of different diameters and so different colors. They are
functionalized with four different antigens that means for example if the cancer cell is expressing
four different kinds of markers or receptors, these four quantum dots will go and bind to the four
respective markers or receptors.
(Refer Slide Time: 11:08)
If all four receptors are present and it is giving fluorescence, which means the person is in the
advantage stage of the cancer. If, out of the four only two or one is giving fluorescence signal
that means the person is in the early stage of cancer. Hence we can say the difference between
the early stages and advanced stages of cancer using this multiplex diagnosis.
(Refer Slide Time: 11:52)

A research team from quantum Dot Corporation made quantum dots to identify live breast cancer
cells. This technology is currently available for mouse model. We can also use it for various cell
biology applications. However, the use of the same quantum dots in humans require extensive
research to determine the long term effects of administrating the quantum dots. This is because
the quantum dots are made up of heavy metals. So the use of quantum dots for human use
requires a lot of research and clinical trials so that it could be useful for human applications also.
(Refer Slide Time: 12:19)

Let us see another example where quantum dots attached to antibodies are used. These antibody
tagged quantum dots will go and specifically bind to the target site, for example if antibodies
corresponding to prostate cancer is tagged, it will go and bind to the prostate tumor site in the
living mice. They will clump together at that particular site and it will be visible under a simple
mercury lamp. When you apply this light it will give the fluorescence signal and it will be easy
for diagnosing the cancer in the mouse model.
(Refer Slide Time: 12:56)
Let us now discuss about carbon dots. Carbon dots are fluorescent nanomaterials, which has
emerged recently providing an alternative to conventional, metal based toxic quantum dots. The
carbon dots are made up of carbon and it is highly biocompatible and eco-friendly. Carbon dots
are small in size, they are usually 2 to 15 nanometer and it is mainly composed of elemental
carbon. Carbon dots exhibit unique optical properties such as high photo-stability, broad
excitation spectra and size-dependent emission wavelength similar to quantum dots.
(Refer Slide Time: 13:32)
The difference between quantum dots and the carbon dots are that the quantum dots are made up
of the heavy metals and so it is toxic. Whereas, the carbon dots are made from carbon sources.
So it is highly biocompatible. The synthesis of quantum dots is difficult but carbon dots are
synthesized easily. It is difficult to do surface functionalization in quantum dots but carbon dots
can be readily surface functionalized. While quantum dots have poor aqueous solubility, carbon
dots are highly water soluble.
(Refer Slide Time: 14:02)

So let me explain the synthesis of carbon dots by a simple method. We can use chitosan as the
carbon source and poly ethylene glycol 4000 (PEG 4000) as the passivating agent. This could be
mixed in a water plus sulphuric acid solution. Then microwave radiation is applied on this using
a domestic microwave oven. Due to dehydration process, we will get the surface passivated
multicolor carbon dots.
(Refer Slide Time: 14:28)
We can make carbon dots by simple microwave pyrolysis method. So here we are using 0.2 gram
of chitosan as the carbon source which was added to the solution containing 25 ml of water and 4
ml of concentrated sulphuric acid. Then add 0.2 g of poly ethylene glycol 4000 to the above
solution and stir at 500 rpm for 15 minutes. Then subject the solution to microwave irradiation
using a domestic microwave oven. So in this case we have used IFB microwave oven operating
at 100% power level that is 700 watt for different cyclic times. That means 20 seconds on and 10
seconds off and at the end of the reaction we will get brown color solution that has the brown
colored dots. Allow the solution to cool naturally to the room temperature and centrifuge the
obtained dark brown solution at 14000 rpm for 15 minutes to separate the less fluorogenic
insoluble black deposit from the fluorogenic yellowish brown supernatant. This yellowish brown
color is an indication for the formation of the carbon dots. So by using this simple protocol we
can make the fluorescent carbon dots in the lab by using a domestic microwave oven and any
carbon source.
(Refer Slide Time: 15:45)
This is the comparison of the in vivo imaging between carbon dots and quantum dots. So when
carbon dots and quantum dots are injected in the mouse model, both are having equal in their
efficiency to give fluorescence. However, the advantage of using carbon dots is that they are
highly biocompatible. That’s why carbon dots will play a major role in cancer diagnosis in the
near future.
(Refer Slide Time: 16:14)
Let us now discuss about chemical nose. Suppose if you want to identify the rotten apple in a
bunch of apples, you can just smell it and tell which one is rotten, instead of doing chemical
analysis. Similarly we can make a chemical nose. The chemical nose can be injected into the
person and it will bind specifically only to the cancer cells and tell you whether the particular
person is having cancer or not. This chemical nose is made up of three different types of
nanoparticles-nanoparticle 1, 2 and 3. These are functionalized gold nanoparticles and these
nanoparticles are wrapped with a fluorescent reporter polymer.
(Refer Slide Time: 16:50)

If the person is having cancer, he will be having the expression of various markers on the cell
surface. This is the nanoparticle covered with the polymer and here the fluorescence is off. When
this nanoparticle binds to the cancer cell, the polymer will be removed. Once the polymer is
removed, it will give the fluorescence signal or the fluorescence signal will be on. If all the
fluorescence signals are in ON mode, that means the person is in the advance stage of cancer.
Suppose out of these only one particular polymer is giving the fluorescence signal that means we
can say he is in the early stage of the cancer due to the expression of this particular marker. So
accordingly, we can give the drug to that person for cancer therapy.
(Refer Slide Time: 17:52)
We can also diagnosis using nano thermometers. The cancer cells appear to have more elevated
temperature than normal cells. Therefore a local temperature mapping can be used to determine
the spread of tumor. So when compared to a normal cell, the cancer cells will be having more
metabolism and there will be more generation of heat at that particular location. We can make a
gold nano particle functionalized with PEG, that is polyethylene glycol coating and on the top of
that we can add the quantum dots. The emission properties of nano particles change with respect
to temperature due to the stretching and contraction of the polyethylene glycol. So if there is a
change in temperature, the polymer will stretch or it will shrink, so according to that it will give
the different kinds of fluorescence. Further, we can also easily identify whether the person is in
the early stage of cancer or in the advanced stage of cancer.
(Refer Slide Time: 18:58)
Let us see the role of nano materials in the traditional methods like CT scan and or MRI scan and
how we can improve these techniques by using nano materials. The computed tomography that is
CT or CAT scan is a fast and relatively inexpensive way to diagnose a disease. This approach
involves the passage of x-rays through the patient and the x-ray source and the detector are
moved related to a target area in the body. If the iodinated compound localizes in the fluid
surrounding the diseased issue, then the contrast between the diseased and normal issue will be
enhanced. This will help the doctor to find about the state of disease and its progression. To
increase the scattering between the disease and normal issues, patients are often given iodinated
organic compounds.

That means in the traditional methods like CT scan, the patient will be given iodine compounds
and then x-rays will be applied to diagnose the difference between the diseased issue and the
normal issue. Here we can use the gold nano particles coated with the polyethylene glycol.
(Refer Slide Time: 20:10)
The PEG coating will allow the gold nano particle to escape from rapid removal by the
reticuloendothelial system which is responsible for removing the foreign matter from the blood.
Hence when you coat the gold nano particle with the polyethylene glycol, it will enhance the
biocompatibility and also it will allow the nano particles to escape from the host immune system.
When we use the gold nano particle for CT scan, the absorption of x-rays by gold nano particle is
1.9 times greater than any iodine containing organic molecule. So when we use the gold nano
particles for CT scan, it improves the efficiency two times and so this will reduce the exposure of
patients to the x-rays. This will be improving the sensitivity as well.
(Refer Slide Time: 20:55)
Now let us see how these nano particles can improve the MRI Magnetic resonance imaging. The
Gadolinium is an ideal ion for enhancing the contrast in magnetic resonance imaging. Here we
can use the single walled carbon nano tubes, which are usually 1000 nm in length. By pyrolysis,
that means treating the carbon nano tubes at 10000 C, these carbon nano tubes can be broken into
20 to 100 nm long and there will be a pit formation which are the missing carbon atoms on the
surface.
(Refer Slide Time: 21:29)
When we can add the Gadolinium ions to this carbon nanotubes and do sonication, the
Gadolinium ions will go and bind to the pit regions. So the advantage of the gado nanotubes is
that at low concentrations, this Gd nano tubes could be used to bring about the same level of
MRI enhancement as produced by the other agents. Using low concentrations of contrast agents
will be beneficial to the patients.
(Refer Slide Time: 22:00)

(Refer Slide Time: 22:19)

We can also use the magnetic nano particles for MRI contrast enhancement. We can make Iron
Oxide nano particles which are usually negatively charged. We can add a positive charge by
using a polyelectrolyte. The cell membrane is negatively charged, so it can easily bind to the
cells through its positive charge by an electrostatic interaction. This will enhance the MRI
contrast. By adding antibodies to this iron oxide nano particles, we can increase the specificity to
cancer cells and improve the MRI contrast.
(Refer Slide Time: 22:58)

Another example is we can do the gold coating to form core shell morphology of iron oxide nano
particles. Here the iron oxide will be in the middle, followed by the gold coating. The gold
surface can be readily conjugated with various bio molecules and it will be having multiple
functions.
(Refer Slide Time: 23:23)
Let us see how to make a paper based diagnostic device for cancer. Recently, Professor Sangeeta
Bhatia from MIT developed a paper based diagnostic kit, which can detect cancer by identifying
bio markers in the patient’s urine.
(Refer Slide Time: 23:43)

They have taken a nano particle and conjugated it with some peptides. Usually cancer cells
express some amount of protease. These proteases will specifically break the peptides tagged to
this nano particles. The peptides will be broken into small, small pieces which will be excreted
through the urination process. So when a drop of urine is added to the paper based kit having
antibodies specific to these peptides, the peptides come and bind to the antibodies. This will give
a colored line. So if a person is getting these kinds of colored line, that means he is positive for
the cancer.
(Refer Slide Time: 25:33)

And another example is we thought that pregnancy test kit is only for the females. Appearance of
two lines indicate that the person is pregnant and if the person is not pregnant there will be one
line. Recently it was found that if a man takes this test and it two lines appear, then he is most
likely to have testicular cancer. So if a male is getting positive result in this pregnancy test kit
that means he has higher chance for getting testicular cancer. There are several methods
available for cancer diagnosis and everyday some new discoveries are coming in this field for
advancing the early diagnosis of cancer.

So as a summary, in this lecture we have learnt what is cancer and what are the various nano
materials available for cancer diagnosis. We have also learnt what is quantum dots and carbon
dots and how we can improve the CT scan as well as MRI using these nano materials.
I will end the lecture here. I thank you all for listening, I will see you in another interesting
lecture.

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