INDIAN INSTITUTE OF TECHNOLOGY ROORKEE

NPTEL

NPTEL ONLINE CERTIFICATION COURSE

Biomedical Nanotechnology

Lec-19
In Vitro Methods of Study Antibacterial and Anticancer
Properties of Nanomaterials

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

Hello everyone I welcome you all to this 19th lecture of this course. This 19th lecture is on in-
vitro methods to study the antibacterial and anticancer properties of nanomaterials.

(Refer Slide Time: 00:36)

So in this lecture we are going to learn various In Vitro methods to study the antibacterial and
anticancer properties of nanomaterials.
(Refer Slide Time: 00:39)

First we will study about the various methods available to study the antibacterial properties of
nanomaterials.

(Refer Slide Time: 00:46)

The first experiment is turbidity assay. So in this we are going to visually monitor the turbidity of
the bacterial growth. Whenever you make nanoparticles the first thing is you have to select the
suitable bacteria to study the antibacterial efficiency. The ideal choices include one gram positive
bacteria and one gram negative bacteria.

So in this experiment we have selected S. aureus as a gram positive model and E. coli as a gram
negative model. We have treated with different concentrations of copper, zinc, nanofibers. So
here you will be taking equal amount of bacteria in the test tube and you will be adding different
concentrations of your nanomaterials to study the antibacterial efficiency of the nanomaterials.

Nanomaterials are not added in the control and so you can see here the growth of bacteria is
visible by the turbidity. With respect to increasing concentrations, you can see here the turbidity
is going down. So using this visual turbidity assay we can easily identify the minimal inhibitory
concentration as well as minimal killing concentration or minimal bactericidal concentration.

(Refer Slide Time: 02:03)

I will explain you how to determine the minimal inhibitory concentration and minimal killing
concentration or minimal bactericidal concentration. So for this you will be selecting one gram
positive bacteria and one gram negative bacteria. When we add these nanoparticles to this gram
positive bacteria and gram negative bacteria we can identify whether your nanomaterial is having
antibacterial efficiency against the gram positive as well as gram negative. We can identify
whether it is having a broad antibacterial efficiency or not.

Here we have taken gram positive bacteria S. aureus, and gram negative bacteria E. coli. For
identifying the MIC concentration, we have to add equal concentration of bacteria to the test tube
with the nutrient broth or the LB broth. We can add 107 CFU that is Colony forming units. So we
are adding equal concentration of bacteria to various test tubes and you will be adding different
concentration of your nanoparticles.

The first one is the control, where there are no nanoparticles. In other tubes, you will add
different concentration of nanoparticles, for example you are adding one microgram and here
you are adding two microgram, three microgram, four and five microgram. After that you will be
incubating for 12 hours that is overnight incubation. After incubating you will see complete
turbidity in the control where there is no nanoparticle, and you will be able to see that the
turbidity is going down with respect to concentration in other tubes.

At some concentration you won’t see any bacterial growth. So if you are not seeing any bacterial
growth at this concentration, so that is your MIC or MKC. So how do you confirm that it is MIC
or MBC, so from this test tube you have to inoculate into a fresh test tube. So from the test tube
where you don’t see any growth from there you will take the inoculum and add it to the fresh
tube, and you incubate for 12 hours. After 12 hours if we are able to see the growth that means
this concentration is called as minimal inhibitory concentration. If there is no growth that means
this concentration is called as minimal bactericidal concentration.

So you are inoculating equal amount of bacteria and you are adding different concentration of
nanomaterials. In the test tube where you do not find any growth from there you are taking these
inoculum and adding to the fresh tube with the medium. If you are able to see the growth that
concentration is called as inhibitory concentration. So from the another test tube where there is
no growth you are inoculating to the fresh tube and if you are seeing there is no growth that
means this concentration is called as minimal bactericidal concentration.

(Refer Slide Time: 05:40)

This minimal inhibitory concentration and minimal bactericidal concentration can be also studied
by optical density measurement that is called as OD okay. So whenever you estimate the DNA or
protein will be measuring that absorbance at 260 or absorbance at 280 nanometer, because your
DNA or protein is absorbing. But whenever you use that bacteria we are using the term OD, that
is optical density, because here it is not absorbing, it is scattering the light. This is the difference
between absorbance and optical density.

So here this is the S. aureus bacteria, untreated S. aureus bacteria and it is treated with a different
concentration of nanomaterials. So in this case it is Ag-ZnO nanocomposite. You can see here
with respect to concentration the bacterial growth is going down for gram positive bacteria, and
for the gram negative bacteria E. coli. So you can see here this minimal inhibitory concentration
as well as the minimal bactericidal concentration is different for both the bacteria.
Depending on the type of bacteria, the antibacterial concentration will vary. Similarly it depends
on the bacterial strains also, the antibacterial concentration or efficiency will vary. In this
example you can see here, 60 microgram is the MIC concentration for gram positive bacteria,
and 70 microgram is the MBC, that is minimal bactericidal concentration.

And in case of E. coli it is 550 microgram as MIC and 600 microgram as a MBC value. Here we
are measuring the values of three experiments (n=3). That means this experiment is repeated for
three times and we get this kind of bar chart. So you will plot the average of the values and also
the standard deviation error bar.

(Refer Slide Time: 07:30)

In this slide we can see that the effect of various concentration nanoparticle varies with respect to
different time points. In the control where there is no nanomaterial, the growth is gradually
increasing. During the preparation of silver nanoparticles, we have used the sodium borohydride
as reducing agent. But the reducing agent is not toxic, you can see here where we added the
reducing agent there is no inhibition of growth, it is also growing similar to the control. And you
can see this one, this one is the low concentration of silver nanoparticles that is 5.66 you can see
here in presence of low concentration of silver nanoparticle, the growth is gradually decreased.
And when we use the MIC or MKC concentration you can see that there is no growth, the
growth is completely inhibited or arrested.

(Refer Slide Time: 08:19)

In our experiment we have used Green Fluorescent protein expressing E. coil. So the advantage
of using this green fluorescent protein expressing E. coil is when you use the wild type bacteria,
to monitor the effect of the nanomaterials, you have to stain the bacteria using gram stain. When
you use the gram stain, there is a lot of washing steps, so there is a chance for artifacts or false
positive or false negative results. So to avoid that we can use this green fluorescent protein
expressing E. coli. Here you can easily monitor the control vs silver nanoparticle treated E. coli.
In control, the morphology is normal and in the treated one the cell is damaged.

(Refer Slide Time: 08:57)

We can also do the time dependent studies, so at zero hour you can see here control and silver
nanoparticle different concentration you can see here uniform amount of bacteria. So with
respect to time, in control where there is no nanomaterials, the growth is gradually increased but
where there is nanoparticle the growth is inhibited. Another advantage of using the GFP E. coli
is, we can also estimate the fluorescent intensity. It is another way to confirm the antibacterial
efficiency. With respect to different concentration of nanoparticle the fluorescent intensity is also
going down. So as I told you that every experiment you have to repeat for 3-5 times and you will
get the average then you have to make the standard deviation error bar.

The stars on the top of the graph denotes the statistical analysis. If you have more stars that
means your data is more statistically significant, a simple term to understand. You can see here,
in control the bacteria is more and with respect to concentration of nanoparticles the bacteria
number is going down.

(Refer Slide Time: 10:09)

The next method is antibacterial assay with the disc diffusion method. Here in the bacterial plate,
you will be adding a lawn of bacteria, by spread plate method. So once you made a lawn of
bacteria, then you will be adding this uniform size of nanofiber disc or if you are having a
nanoparticle solution you can make a uniform sized sterile whatman paper. You can dip it into
the nanoparticle solution and you can place it on the top of the bacterial plate, where there is a
lawn of bacteria.

Allow this to stay for overnight incubation. After overnight incubation, you can see a clear zone
of inhibition. That means these are nanofiber or the nanoparticle loaded disc it is inhibiting the
growth of the bacteria, so this is called as zone of inhibition. By measuring the length of the zone
of inhibition, we can calculate the antibacterial efficiency of the nanomaterial.

(Refer Slide Time: 11:09)

Another method is colony counting method. It is the same as the visual turbidity method. From
the tube you can spread the culture into the bacterial plate. By spread plating technique you can
spread that bacterial solution on the top of this plate, and here you will be incubating the plate for
overnight then you can count the number of colonies. You can see here with respect to increasing
concentration, the numbers of bacterial colonies are going down.

(Refer Slide Time: 11:45)

This is the control where there are huge numbers of colonies and with increasing concentration
of nanoparticles, the number of bacterial colony is going down. So we can easily count that
bacterial colony and you can plot it.

(Refer Slide Time: 11:50)

We can also observe the morphology of the treated bacteria by using scanning electron
microscope. In the control untreated, the bacteria is having normal morphology that is rod shape,
E. coli. In the case of treated one you can see here the morphology is damaged, the E. coli is
damaged and it shows confirm the death of the bacteria.

(Refer Slide Time: 12:15)

We can also use the transmission electron microscope to understand the bacterial cell death
mechanism. So you can see here this is a single bacterial cell and this is the expanded view of the
bacterial cell. The black color spots are silver nanoparticles. The silver nanoparticles are binding
to the cell wall and it is damaging the cell wall of the bacteria and killing the bacteria.

(Refer Slide Time: 12:32)

Based on the literature, this is the possible antibacterial mechanism. So, most of the metal
nanoparticles will be having positive charge and our cell bacterial cell is having negative charge.
So, this positively charged nanoparticle will come and bind through electrostatic interaction to
the bacteria and it will induce the formation of reactive oxygen species. Some of the
nanoparticles will go inside the bacteria and bind to the DNA. This will inhibit the DNA
replication as well as they inhibit the protein synthesis. And finally, it will damage their cell
wall. So, these are the various mechanisms nanoparticles will follow to kill the bacteria.

(Refer Slide Time: 13:20)

Now let us see the various methods available to study the anticancer properties of nanomaterials.

(Refer Slide Time: 13:26)

Before we study the anticancer properties of nanomaterials, we have to understand two types of
cell death; apoptosis and necrosis. Apoptosis is the normal or programmed cell death. It is the
physiological process and necrosis or the accidental cell death is the pathological process.
Apoptosis is similar to a normal death of a person and the necrosis is similar to the accidental
death of a person.

(Refer Slide Time: 13:49)

Let us see the difference between apoptosis and necrosis. So, in apoptosis membrane blebbing
will occur and there would not be any loss of integrity. It begins with the shrinking of cytoplasm
and condensation of nucleus and its ends with fragmentation of cells into smaller bodies. And,
non-random mono and oligonucleosomal length fragmentation of DNA will happen in this
apoptosis. In necrosis there will be loss of membrane integrity and it begins with swelling of
cytoplasm and mitochondria and ends with total lyses of the cell. Here there will be random
digestion of DNA, and so you will find smear of DNA in the gel electrophoresis.

(Refer Slide Time: 14:27)

So, let us see these in detail. This is your viable cell. So, during apoptosis what will happen the
cell will shrink and the chromatin will condense, it forms a budding and this apoptotic body will
be formed, which will be phagocytosed. There would not be any inflammation, but in case of
necrosis cell will be swelling and it becomes leaky. So, all the cellular contents will be released
abruptly and it will cause inflammation. The cellular and nuclear lysis will lead to inflammation.

The simple example is like if you want to break a wall you can use the simple hammer and break
the wall and you can reuse the bricks to build the new wall, it is similar to your apoptosis. In case
of necrosis for breaking the wall as if you are using your bomb blast and you are completely
destroying the particular environment as well as the wall ok. So, it is the simple example to
understand the apoptosis and necrosis.
(Refer Slide Time: 15:19)

So, let us see: what are the various methodologies available to study the apoptotic cells. So, we
will see all these methodologies in detail one by one.

(Refer Slide Time: 15:25)

The first one is simple dye based cell viability assay. So, it would not tell you whether cell is
following apoptosis and necrosis, but it will tell you the number of viable cells and number of
dead cells. Here we will be using the dye called trypan blue. The trypan blue dye will stain only
the dead cells. So, the viable cells would not uptake the dye and it will exclude the dye. So, that
is why this method is also called as dye exclusion method. So only the dead cells where the cell
membrane integrity is lost will uptake this dye and the dead cells will appear like a blue colour.
So, we can easily count the blue colour cells and we can calculate the number of dead cells. So,
when you treat the cells with the different concentration of nano particles and add this trypan
blue stain and you can count the number of dead cells.

(Refer Slide Time: 16:23)

The next method is MTT assay or cell viability assay. Here we will be using the MTT solution
and this MTT usually it will be a yellow in colour. But in the presence of mitochondrial
dehydrogenase enzyme this will convert this yellow colour into formazan crystals that is a purple
colour. If you have more cells viable then you will have more purple colour. So, with respect to
concentration you can easily monitor the cell viability and this can be measured using a
microplate reader.

(Refer Slide Time: 16:56)

We can plot the cell viability based on the reading from MTT assay. So, here in control there is
no nanoparticle and with respect to different concentration of nanomaterials the cell growth is
going down. We can check this on different cell lines like BHK 21 and HT 29 cell lines. So, with
respect to cell lines and the anti cancer or the toxicity effect will be varied. So, let us see how we
can calculate the IC 50 concentration.

(Refer Slide Time: 17:27)

By using the MTT assay we can calculate the IC50 concentration that is similar to your MIC,
MKC only. So, the IC50 means the concentration required to inhibit the 50 percentage growth of
cells. In the MTT you will be measuring this cell viability percentage. By dividing the
absorbance of your treated sample by absorbance of your control that is untreated sample and
then multiplying it by 100 will give you the cell viability percentage and how to calculate the
IC50.

(Refer Slide Time: 18:35)

In the graph, first one is your control that is the 100 percentage. The control sample is untreated
For treated samples we get values less than 100 that is 80 percentage, 50 percentage, 30
percentage etc., The IC50 is the concentration required to inhibit 50 percent growth. You can
check the concentration at which it is inhibiting 50 percent of the growth.

We have to repeat all the experiments at least 3 to 5 times and we have to plot the graph with
standard deviation error bar after performing the statistical analysis. The stars in the graph
indicates the statistical significance. That is if you are having more stars that means the data is
more statistically significant and the error rate will be less and the reproducibility will be more.

(Refer Slide Time: 20:10)

In this example you can see here we have used A459 which is a lung cancer cell line as well as
the MCF-7 which is a breast cancer cell line. We have used an anticancer drug niclosamide and
this is not a water soluble drug. So, when you add it to this it is not taken up by the cells. Hence,
there is no cell death, but when you put this anticancer drug into the albumin nanoparticles, these
nanoparticles are taken up by the cells and it is inducing the anticancer effect. So when
niclosamide is given along with the BSA nanoparticles, we can see that the growth of the cells
are going down.

We can calculate the IC50 concentration using these values. The IC50 values will vary
depending on the cell type even though you are using the same kind of nanoparticles loaded with
same anticancer drug. That is why whenever you make any nanocarrier or nanomaterial you have
to study the toxicity of nanoparticles on various cell lines.

(Refer Slide Time: 21:22)

Another simple experiment to understand whether your nanoparticles are inducing apoptosis or
necrosis, we can use this acridine orange and ethidium bromide that is AO/EB staining method.
So, here in this acridine orange is a fluorescent DNA intercalating dye and it is permeable to all
cell nuclei and it will give green colour fluorescence. The ethidium bromide is a red colour dye.
This is a fluorescent DNA intercalating dye and it will enter only the membrane compromised
nucleus to emit orange red fluorescence.

(Refer Slide Time: 21:55)

So the live cells will give a normal green colour fluorescence and in the case of early apoptosis
due to the chromatin condensation, there will be a bright green fluorescence. And in case of late
apoptosis the combination of red and green you will get the orange colour. In case of necrosis
there won’t be any nuclear condensation as only the membrane will be damaged. So, you will be
getting a red colour nucleus.

Here you can see here this is your control. It is completely green colour and in the treated one
due to nuclear condensation, you can see here the bright green colour spot that is your early
apoptotic cells. In case of late apoptotic, orange colour is seen due to the combination of green
plus red colour.

(Refer Slide Time: 22:55)

The next method is a Hoechst and rhodamine staining. So, using this stain the advantage is we
can do the live cell monitoring. Hoechst stain is a membrane permeable nuclear stain, which will
give blue colour fluorescence when it binds to the double stranded DNA. This is a nuclear stain
and its used to differentiate condensed pycnotic nuclei that is nuclei with condensed chromatin
from the normal nuclei.
The rhodamine is a membrane permeable dye that stains the mitochondria and cytoplasmic
compartments. So, the rhodamine will be staining the cytoplasm and it will give this orange
colour fluorescence and this Hoechst is the blue colour stain that will stain the nucleus.

(Refer Slide Time: 23:40)

Using this we can do the time dependent study. So, you can see here this is the untreated one and
these are the treated with different concentration of nanomaterials and we can see here at
different time points at 6 hour time point, at 12 hour and 24 hour so, at different time points we
can study the effect of the nanoparticles. The white arrows indicate the chromatin condensation
and this yellow arrow point towards the cytoskeleton compaction. So, you can easily monitor the
nuclear condensation as well as the cytoplasmic constriction using this Hoechst and rhodamine
staining.

(Refer Slide Time: 24:24)

We can also examine the morphology of the untreated and treated cells by using the scanning
electron microscopy. So, this MCF-7 is your breast cancer cell line and A549 is a lung cancer
cell line. So, these are untreated. So, this untreated cells is having a spindle shaped cells it is well
attached to your cell culture dish and it has an intact membrane morphology. But here in the
IC50 concentration treated cells the cells are shrunk and it became rounded in shape. These cells
are loosely attached and this are called as membrane blebbing. These are the hallmarks of the
apoptotic cell death.

When the cells are alive, what will happen it attached to the plastic surface very tightly and it
will have a spindle shape. When the cells die what happens it will detach from this cell culture
dish and it will become a rounded cells. There will be the formation of apoptotic membrane
blebbing on the top of the cells. These can be easily monitored using the scanning electron
microscope.

(Refer Slide Time: 25:26)

We can also use the atomic force microscope to understand the cell morphology. So, this is your
control you can see here this is your like a long fibroblast cells and this is your nucleus. In the
untreated the cell morphology is perfect, but in the treated one you can see these cells are
undergoing early stage apoptosis you can see here that membrane blebbing has started forming.
In the late apoptotic cells the cells become rounded.

(Refer Slide Time: 25:54)

We can also use the atomic force microscope to study the surface roughness of the control versus
treated cells. In the control cells, there is a smooth surface and whereas, in treated cells you can
see here the roughness is also changed. So, by measuring the roughness also we can understand
the status of the cell membrane and everything.

(Refer Slide Time: 26:15)

The next method is cellular DNA fragmentation ELISA. So, here we will be incubating the cells
with BrdU that is bromodeoxyuridine. Here the uracil will be incorporated into the DNA. So, this
BrdU will be incorporated into a DNA. And when you treat with the nanoparticles, there will be
fragmentations of DNA. These fragments of DNA can be added to the ELISA plate where the
plate is coated with anti DNA antibody. When you add another antibody which is specific for the
BrdU, it will come and bind and form a sandwich ELISA.

(Refer Slide Time: 26:56)

So, here we will be adding the BrdU to the cells. The DNA will be labelled with this BrdU and
when you treat this BrdU labelled cells with different concentration of nanoparticles, due to this
nanoparticle treatment there will be fragmentations of DNA into the cell that is the indication of
apoptosis. So, we will be taking this ELISA plate or 96 well plate and will be coating this plate
with anti DNA antibody. This will be briefly treated in microwave and due to this, the DNA will
be opened up. We will be adding another antibody which is specific for the BrdU and which is
having this enzyme labelled antibody ok. So, when you add the substrate, it will produce the
colour formation. This is similar to the sandwich ELISA. Here the DNA is sandwiched between
two antibodies and when you add the substrate there will be a formation of colour. So, if you
have a more colour formation; that means, it indicates that that is the more apoptotic induced by
the particular nanoparticle.

(Refer Slide Time: 28:45)

So, you can see here with the increasing concentrations of nanoparticle treatment, there is an
increasing amount of DNA fragmentation, which denotes induction of more apoptosis.

(Refer Slide Time: 28:56)

The next method is apoptotic DNA laddering. This is the nucleosome core and this nucleosome
is connected by the linker DNA. The distance between each nucleosome is 180 base pairs
approximately. So, during this apoptosis the caspase will activate the endonuclease. This
endonuclease enzyme will cut this nucleosome at different sites randomly. This generates the
mono or oligo nucleosomes. The size of each mono nucleosome is approximately 180 base pairs.

(Refer Slide Time: 29:53)

Here we will be getting the multiples of 180 base pairs. So it can be mono nucleosome or it can
be dinucleosome or it can be oligo nucleosomes. When we run this in a gel and you will find this
kind of ladder pattern. So, you can see here, so, this is your control genome where you are
having only single band. And, this is the apoptotic DNA where you can see here a ladder
formation this is called as DNA laddering.

The caspase-3 enzyme will be activated and it will lead to cleavage of chromosomal DNA. This
oligonucleosomal DNA fragments result in distinct laddering pattern. By using a confocal laser
scanning microscope, we can see here with respect to concentration the amount of cells is going
down. In MTT assay, with respect to different concentrations the amount of viable cells are
going down. In the case of DNA fragmentation ELISA you can see here this is the apoptotic
induction is more. So, this is correlating with your MTT data.

(Refer Slide Time: 30:45)

The next is Fluorescence Activated Cell Sorting method to understand the apoptosis. So, in this
the cells are labelled with various fluorescent markers. The detector will detect the cells
according to the fluorescent signal. So, you can see here this red is going to here yellow is going
to here. So, we can easily sort out the cells based on the fluorescent labelling.

(Refer Slide Time: 31:12)

So, using this flow cytometer we can understand the apoptosis. The cells can be labelled with
different kinds of antibodies specific for the apoptosis and necrosis. The fluorescent detector will
detect the cells according to the fluorescent signals which can be plotted and analyzed.

The control, apoptotic and necrotic cells will fall in different regions. The apoptotic cells or
necrotic cells will have different kind of markers. So, we can label those markers with the
fluorescent tag and by using the flow cytometer we can understand the apoptosis pathway. So,
here the flow cytometry measures the light scattering properties of cells and fluorescent
emissions of the molecules attached to the cells.

(Refer Slide Time: 31:58)

Let us see how we can use this flow cytometric analysis to understand the apoptosis. Usually in
the live cell, the plasma membrane we will be having phosphatidylserine. This
phosphatidylserine will be facing towards the nucleus, but in case of apoptotic cells what will
happen there will be a flip flop. So, due to that this phosphatidylserine will be exposed to
outside. When it is exposed to outside we can make the antibodies specific for that and this
antibody can be labelled with fluorescence dye green fluorescence dye. In case of late apoptotic
cells, due to the membrane perforation, the ethidium bromide or PI stain can directly enter the
nucleus and stain the nucleus into red colour.
Here you are having green fluorescence in apoptotic cells and you are having red fluorescence in
late apoptotic cells. In case of necrotic cells, there won’t be any green colour fluorescence; you
will be having only red colour fluorescence. So, when you do the flow cytometer, the cells will
be sorted according to the signals.

The live cells will be here in this first box and the cells which is in the early apoptotic so, that is
your green fluorescent cells that will be captured in this and the late apoptotic cells where there
is the combination of green plus red so, that will be captured here and the necrotic cells where
you will be getting only the red colour signal that will be captured here. So, based on the amount
of the percentage you can see here the control one the percentage is only 17.7 that is a late
apoptotic cells in the treated one you can see here it is approximately 38.9 percentage; that
means, with the treating with the nanoparticle the apoptotic percentage is increased.

(Refer Slide Time: 33:39)

Another method to understand the toxicity of material is the ROS assay. In the ROS assay, we
will be using the DCFH-DA, a non-fluorescent dye and when you add it to the cells and cellular
esterase will convert this non-fluorescent into fluorescence and in presence of reactive oxygen
species this non fluorescence DCFH will convert into DCF that is fluorescence. So, based on the
fluorescence signal we will understand the amount of ROS production.
(Refer Slide Time: 34:11)

So, here you will be using this excitation at 495 and emission at 529 nanometer and this non
fluorescent population can be gated into R2 channel and fluorescent cells can be in the R3.

(Refer Slide Time: 34:26)

Here the untreated cells will be taken into R2 channel that is your non fluorescent channel and
with respect to different concentration you can see here the fluorescent signal is increased. So,
due to production of reactive oxygen species, the non fluorescent DCFDA is converted into DCF
fluorescence. With respect to concentration this is your 50 percentage or your IC50
concentration. Here, the ROS production is 8.4 percentage and when you use the IC50
concentration the ROS production is 24.1, and when we use double the IC50 concentration, you
can see here the ROS production also doubles.

(Refer Slide Time: 35:17)

By using this simple assay we can measure the reactive oxygen species production. This is
another data to show the ROS estimation by DCFDA. After 20 hours’ time point there is no
fluorescence the reactive oxygen species is produced less and with increasing the time and
increase in concentration you can see here the fluorescence is more; that means, the reactive
oxygen species production is more.

(Refer Slide Time: 35:41)

We can also analyse the fluctuation in the mitochondria membrane potential by using this
rhodamine 123 stain, which is also one of the indicators of apoptosis. So, here the change in the
mitochondrial membrane permeability with loss in mitochondrial membrane potential is an
indicator of early apoptotic events. And, here we will be using this rhodamine 123 dye it is a
cationic dye which can rapidly diffuse inside the mitochondrial interior. And, it can reflect the
changes in the mitochondrial membrane potential from a to c1. In untreated cells, there is no loss
of fluorescence and here you can see here d1 and e1 you can see here significant decline in the
fluorescence due to the loss of mitochondrial membrane potential. So, this result shows that there
is a remarkable decline in the mitochondrial membrane potential, which confirms the induction
of apoptosis by the drug loaded nanocarrier.

(Refer Slide Time: 36:44)

We can also study the gene expression by RT-PCR that is the reverse transcriptase PCR. The
beta actin is the internal control or the house keeping gene. These are the various pro apoptotic
genes and anti-apoptotic genes. The bcl-2 is an anti-apoptotic gene. So, you can see here the
down regulation of anti-apoptotic genes.

When compared to the untreated, the expression of bcl-2 gene is going down. The expression of
the anti-apoptotic genes are going down and the expression of apoptotic genes are increasing.
This confirms the induction of the apoptotic pathway.

(Refer Slide Time: 37:51)

We will be isolating the RNA from the cells both treated as well as the control cells. The RNA
will be converted into cDNA by an enzyme called reverse transcriptase enzyme. This CDNA
will be amplified by PCR. This is called as semi quantitative PCR. So, when we amplify that
control vs treated and we will be running a gel to understand the gene expression. For example,
so, this is your caspase 3 gene and this is your control sample, this is your treated. So, in control
we are seeing less amount of amplification, in the treated one we are seeing more amount of
amplification. So, this shows that this caspase 3 expression is more, which leads to induction of
apoptosis. The gene expression intensity can be calculated using a software called image J. we
can plot the difference in the gene expression intensity when compared to control how much fold
the gene expression is increased or decreased.

(Refer Slide Time: 39:46)

Based on the pro apoptotic and anti-apoptotic genes we can make a schematic illustration of the
events. In this example, we are using the AgZnO nanoparticles. It is binding to the cell
membrane and it is inducing the production of reactive oxygen species. The production of
reactive oxygen species can be studied using DCFH-DA and this ROS production will induce the
expression of p53, which will induce the expression of bax bad genes. These are the pro
apoptotic genes in the mitochondria. It will also down regulate the expression of anti apoptotic
genes like bcl-xl and bcl-2. The pro apoptotic genes will induce the expression of caspase 3.

The caspase 3 will induce the caspase 3 activated endonuclease. This will fragment the genomic
DNA into small-small fragments and all this events combine and induce the cellular apoptosis.
So, upon cellular uptake of the nano composites which will induce the oxidative stress by
inducing this reactive oxygen species and it will trigger the p53 mediated apoptotic pathway.
This will leads to induction the various events cascade of events like mitochondrial, membrane
permeability and also it will also changes the up-regulation of various pro apoptotic genes, which
will lead to the execution of apoptotic pathway.

So, as a summary in this lecture we have learnt what are the various methods available to study
the antibacterial as well as anti cancer properties of nano materials. I will end my lecture here, I
thank you all for listening this lecture; I will see you all in another interesting lecture.