Video:

Hello everyone. Welcome to another lecture of Drug Delivery Engineering and
Principles. Just a quick recap of what we learned in the last class.
(Refer Slide Time: 00:38)

First we talked about intra articular injections. So, these are injections which are given
into the joint space. So, if this is my femur, this is my tibia. You can directly inject things
in here and that will result in much higher residence time. What we also learned is this
synovial membrane contains lots of blood vessel and lymphatics and they can clear out
the things fairly rapidly, if they are smaller size. So, if you want to inject something to
reside very for long durations, you want to inject bigger particles that can be drugs or the
drugs itself may be big. So, somewhere anywhere between above 1 micron would start to
give a much higher residence time and then anything below that.
Then, we started IV administration. One of the most widely used methods in clinics
specially for things that you want to have instant action on the body. We then discussed
various organs that put the limit on a particle size. So, if you want to do a control through
some bio engineering approaches, we discussed that first of all kidney will clear things

below 6 nanometers so, or below 10 nanometers. So, you want the particle sizes to be
above 10 nanometers, then we said that anything with the size above greater than 200
nanometers, the spleen will clear it away; 200 nanometers plus particles. So, you want
the sizes to be below 200. Of course, the biggest blood vessel is 5 microns, the smallest
blood vessel sorry.
So, you should definitely have less than 5 microns and then we also discussed that liver
is another major organ that is going to clear quite lot of your big particles. So, basically
you want something in the size range of 10 to 200 nanometers okay! Then we talked
about EPR effect in both tumor and inflamed tissues. And what it is? The blood vessels
that are feeding your tumor tissue are fairly dilated whereas, the blood vessels in your
healthy tissue fairly open.
So, this is healthy tissue, this is tumor tissue and these pores are about 100 nanometers in
tumors. And they become larger as a tumor grows as well as there are also pores in
inflamed tissue when the vessel dilates to allow more cells and more nutrients to go in.
And because of that if you have size ranges in between 10 to 100 nanometers, you will
have a much higher efficiency of delivery and this is called the enhanced permeation
because they will go here, but not here. And then this tumor micro environment also has
very poor lymphatic system. So, it cannot really clear things away once they go in and
that is the enhance retention so, which is nothing, but EPR effect. So, that is something
you can utilize as well; utilize quite a bit in the literature ok.

(Refer Slide Time: 04:06)

So, let us continue our discussion on the IV route that we were talking about and we are
going to talk about drug called Doxil. And this is similar to the EPR effect. It utilizes that
effect and is an approved nano carrier for cancer drug delivery and doxil contains
doxorubicin as the major effective drug. The doxorubicin is a very effective anti cancer
drug. In fact, most initial cancer studies are done with doxorubicin and but the problem is
it has a very high cardio toxicity. So, cardiotoxicity means that something little to the
heart. So, it starts to kill heart cells and then; obviously, when you inject something IV, it
is definitely going to go to the heart as well.
So, that is a problem and that limits the amount of dose or dox solution you can give to
your patient. Because eventually at a certain dose all the demon cells will die, but before
that dose is reached the patient may die because of all this cardiotoxicity and even other
types of toxicity. So, that puts a limitation on the dose of doxorubicin that can be
administered. So, to counter that what was done is the doxorubicin was taken, it was
encapsulated into a liposome; this was done through reverse loading. I hope you guys
remember what reverse loading is. So, reverse loading is nothing, but using the property
of molecules to diffuse into the lipid membrane if they are non ionic at a certain pH.
And once they go into the membrane, you make the liposome such that and the inner
environment has a different pH and they will become ionic. So, that will cause the
movement of the drug to go into the liposomes and effectively build up a very high

concentration in the liposome. So, that is what was done and then these were actually
further PEGylated. So, again, I hope you remember PEGylation.
PEGylation prevents the interaction of various cells in protein with your biomaterial
surface and because of that it will not lead this particular liposome get adsorbed with
several proteins or get cleared away by the immune system. So, it increases the residence
time and this resulted in quite a bit effectiveness of this. It was used very heavily for
ovarian cancer, still used and it also reduced the cardiotoxicity and because it was using
that EPR effect.
So, what happened is this was made of size of about 100 nanometers and because of that
the vessels in the heart, are fairly healthy in tumor tissues. So, this liposome was not
going in releasing doxorubicin in the to the heart cells. But then in the tumor areas where
the vessels were fairly dilated and these particles can go and permeate and these were
accumulating in the tumor areas. And actually, effectively what you did is you let us say,
if you had a ratio of drug in tumor versus the drug in healthy tissue that for your free
drug was much less than for your doxil. And because of that this therapy took off and
was used in the clinics as well and it and is still used in the clinics.
(Refer Slide Time: 07:55)

So, here is also showing that not only that you are actually improved the
pharmacokinetics also because doxorubicin is a very small molecule much less than 6
nanometers.

So, doxorubicin gets cleared away from the system very quickly as well. So, you have to
continuously infuse doxorubicin in the patient if you want any therapy whereas, doxil
since its now about hundred nanometers, it has residence time that is very high and it
slowly releases the doxorubicin into the system. So, as you can see the concentration of
dox was more than 2 orders of magnitude higher than what you had in case of free drug.
(Refer Slide Time: 08:44)

So, here is another example. So, in this example, the product name is Abraxane and this
is basically one of the vials you can find in the market. And what it is? It is carrying a
drug called paclitaxel again very effective against lots of cancer cells especially breast
cancer, but it actually has a very low solubility in water. So, what that means, it is fairly
small molecule. So, what that means, is you cannot really inject too much because it will
precipitate out and again may clog your heart vessels or your brain vessels causing heart
attack or stroke respectively.
So, what was done is this paclitaxel molecule was conjugated to albumin protein which
is one of the most abundant protein in our blood. And it then self-assembled into a
structure, because paclitaxel is fairly hydrophobic and it did not really want to interact.
So, it self-assembles into a structure like this where paclitaxel ended up being in the core,
and albumin changes conformation to reflect on the outside and interact with water with
a mean size about one 130 nanometers. Again, close to that EPR effect range that we

wanted and that resulted in very enhanced and increased transport and accumulation in
tumors.
So, this drug was approved in 2005, had bumper sale that year and still is being used
quite a lot. So, as I said its nothing, but a protein bound drug that you are using and
forming a nano particle out of that and using the EPR effect that we talked about.
(Refer Slide Time: 10:33)

Yes, another example. This in this case, we are looking not a tumor, but at inflamed sites
or infectious site. So, the product is called AmBisome and what it is? Again, a liposomal
formulation in which the drug being used is amphotericin which is an antifungal drug.
And, but then this drug causes lots of side effects in nephron basically, kidney.
So, that was causing limitation of how much drug you can use, but what these folks did
is they encapsulated this or actually attached it to the surface of the liposome into the
lipid membrane layer that caused first of all increase in the size. So, it did not really
accumulate in the kidney and the secondly, controlled release as the liposomes release
this drug out. So, this was the product and there was approved in 1999 and again was
used quite often.

(Refer Slide Time: 11:39)

So, this concludes our session on the route specific delivery. We are now going to
change and start a different module which is immune response to materials and so let us
get started on this. So, if you have any material that you are putting in and we have
talked about this briefly as we went down through this course but if you are introducing
something new, the body is going to identify this as foreign materials and there will be
some immune response against it.
So, what we will do is, we will try to understand what kind of immune response builds
up in these circumstances and how do we tackle this, how do we make sure that this
immune response is not rejecting a material they will be putting in, and it is not affecting
the performance or whatever the function the material was supposed to do. So, this is
what we are going to learn. So, before we learn that, we have to learn a little bit
immunology that will be required to understand this module.

(Refer Slide Time: 12:37)

So, let us start with this. So, this is a very classic figure. This is taken from Janeway and
this is showing different cells of the immune system. So, you have a bone marrow which
is located in the long bones. It is the cavity within the long bones that you can find and
that is a site which is extremely rich in pluripotent, hematopoietic stem cells. And what
does this pluripotent mean? That means, that these stem cells can give rise to several
different cell types.
And so, these give rise to several different cell types as I mentioned. Some are called
common lymphoid progenitor; some are called common myeloid progenitor. The
myeloid progenitor goes in and further differentiates into granulocytes and macrophage
progenitors including some of the blood cells as well and then they further go in and
differentiate into blood cells and platelets.
Whereas the common lymphoid progenitors have a much more complex route they go
and differentiate into B cells and T cells. Again, there will be lot of biology, but bear
with me and we will describe these as we go along. So, you have B cells and T cells and
then they also give rise to some of these granulocytes which was from the myeloid
progenitor will also give rise to neutrophils, eosinophils, basophils and there are some
unknown precursors as well monocytes, dendritic cells. These are all different types of
immune cells which are responsible for different functions and we will describe some of
them as we go along through this module.

The B cells and T cells can then have different types as well; they can have effector cells
or they can have helper cells. So, both B and T type of cells can have both effector and
helper cells. So, some of these B cells are responsible for secreting antibodies something
that I am sure you must of all of heard by now and then there are T cells which are
directly responsible for killing any rogue cells and any cell that is not functioning well
and immune system is not liking it.
And some of these monocytes and granulocytes derived things can then go in and
become mast cell macrophages and dendritic cells. And these are cells that are more
responsible for presentation of anything to the immune system which the thing is foreign.
And then there are lymph nodes which are nothing, but they are these so, I said that there
are blood vessels. Then along the blood vessel, these are another parallel network that is
called lymphatics.
So, let me take another color for that. So, along with the blood vessels there is another
parallel circulatory system that is called lymphatics at a much lower flow rate, but
nonetheless flowing throughout our body. And this lymphatics from time to time and
from different places actually harbors secondary lymphoid tissue which is called lymph
nodes. And what these tissues are? They are depot of these immune cells which has a
sampling around. So, obviously, this is tissue. So, what it is? They are sampling
whatever is coming out from this tissue going into the lymph node where immune cells
are present in very high numbers.
All these B cells, T cells and other types of macrophages and dendritic cells are present
in a very high number and they are continuously signaling as to if there is something
foreign. So, let us say there is a bacterial protein that is going to come in here. It is going
to be picked up by one of these immune cells particularly dendritic cells and
macrophage. And it is going to be then presented to one of these effector cells or
memory cells or the helper cells which will then mount an effective immune response
against it.
And again, if you are unaware of this, do not be worried; we will describe it in a little
more detail as we go along. So, these are some of these common terms cells of the
immune system. Most of them are actually fairly important for this module. So, it will be
good if you can start to remember some of these cells.

(Refer Slide Time: 17:08)

And so, let us talk about function of these cells. So, macrophages which, again we have
discussed before already; these are cells that take up foreign objects, foreign bacteria
foreign viruses, their major job is to first of all do phagocytosis which is a process
through which large particles and large foreign cells are being taken up. And then they
get activated and start secreting lot of bactericidal enzymes and bactericidal proteins that
kill them. And then they are also responsible for antigen presentation which means that
they are the ones who even when they kill them or even when they harbor them, they
will present it to the rest of the immune system, saying that hey look! there is something
foreign here.
So, that is the major rule for these. Dendritic cells like macrophages are again very
similar and they are also responsible for phagocytosis and antigen presentation.
Neutrophils are slightly different in that regards, they do a lot of phagocytosis too;
however, they are not as involved in antigen presentation as the macrophages and
dendritic cells, but they are mainly responsible for killing the bacteria. So, they even act
as a kind of a suicide bomber where they carry lots and lots of harmful bactericidal
molecules. And when they find that there are bacteria in the surrounding, they will just
burst and release all that near the bacteria vicinity; killing the bacteria in the process.
Then we have Eosinophils and one of the function responsible for them is killing of the
antibody coated parasites. So, they are more responsible for big worms like pathogens

and they can even be effective against some of the smaller pathogens too. And these
basophil, mast cells are not as widely studied and understood probably their function in
the immune system is either not very understood very well or they do not really play a
very major role. But their major function is to cause inflammatory reactions and they are
actually also responsible for allergies. The same with the mast cells, they also release
granules containing histamine and other active agents to kill the surrounding
environment bacteria and pathogens.
(Refer Slide Time: 19:33)

So, how does the inflammation start? So, this is important because we need to
understand how does it starts that and we put our material, we must make sure that this
does not happen. So, let us learn first of all how it does it start. So, again the immune
system is very vast and contains lots and lots of cells and they are surveying pretty much
every part of the body that we have.
So, all tissues that we have contains some one or other type of the immune cell. So, what
happens if there is some injury ok? These cells become very active. So, they will then go
to that injury site, they will sample that to see if there is anything that is foreign that
caused this injury, why did the injury happen. And if they do fine, they will continue to
digest this foreign antigen and end the cells that are dying there because; obviously, if
there is some injury then you must have some dead cells, your skin may have ruptured.
So, skin cells are dead, some of the blood vessels may have a ruptured. So, those

endothelial cells may be dead and if they do find something that is causing this, they will
secrete cytokines.
Sometime, I mean most of times they will even do that without any the initial injury is
enough for them to start secreting some cytokine. So, let us say this was macrophage that
was sampling that area which is one of the major cells responsible for also sampling
tissues. So, then they will start secreting several classes of these cytokines which are and
we will talk about in the next slide, but which are nothing, but protein-based molecules
involved in signaling and amplifying the immune system.
So, some of them being IL-1, TNF-alpha, IL-6, IL-8, IL-12; these are some
inflammatory cytokines and they all have their own functions, which some of them are
overlapping. Some of them are systemic so, they will actually involve the whole immune
system of the body, some of them are local which is more at a localized level.
But what will basically happen is like an IL-1, if it is released, it is going to cause
activation of the vascular endothelium. It is going to activate any kind of lymphocyte,
which is a type of immune cell in the surrounding, causing the local destruction of the
tissue and will even cause they increase in the effector cells which are the cells which are
going to kill anything foreign or going to help in an aggregation of that to come into the
side. So, another class of molecule is TNF-alpha similar to IL-1. It is fairly
inflammatory; it activates again the vascular endothelium and increases the permeability.
So, as I if you remember in the tumors part of the talk, I was talking about, that if you
have inflamed tissue, you have again vessels that are more permeable. They are not as
tight as they are in healthy homeostatic tissue and that is because these factors are being
released, which are signaling these cells to increase a junction diameter. And what that
does is it causes more cells to come in, more growth factors, more immune cells, more
cytokines to come in and that is how the inflammation is leakier, then inflamed sites are
leakier.
And so, that causes entry of antibodies complement different cells into the site. Then we
have IL-6 which are again responsible; all of these IL-6, IL-8, IL-12. These are
responsible for again activating different immune cells; some of them NK cells, some of
them causing antibody production and Th1 which is the T helper cells. They are all
essentially accumulating at the site.

(Refer Slide Time: 23:21)

So, I mention cytokines and chemokines. Let us quickly define what these are. So,
cytokines are small proteins of typically about anything between 25 to 30 kDa released
by various immune cells. So, typically this is immune production although other cells
can also release them and they say they are usually released in response to a certain
stimulus that might be given. And then they induce responsible news responses by
binding to a certain receptor.
So, lots of cells have receptors for these cytokines and they go and bind to that receptor
which then signals the cell to either get activated, to start migration, to start causing the
vessel dilation. So, all of this is controlled by these cytokines in the several classes of
them. So, one thing as we are going along, I will I like to point out is immune system is
fairly dynamic research topic every 3-4 years, the whole immune system understanding
changes by quite a bit amount.
So, what I am giving you now is what is currently well accepted and well hypothesized
and, in the books, but all of these are fairly lucid definitions and they may change as the
time goes on. So, these cytokines connect in an autocrine manner which basically means
that they can act on the cells that are actually releasing them. So, let us say if this is an
immune cell and it releases this cytokine out into the media.
These cells themselves have receptors for these cytokines and when these cytokines bind
to the same cell, they will activate the cell further. So, this is kind of a positive feedback

loop where cell is getting activated, it is losing this cytokine and these cytokines are
binding to the cell itself and activating them further. Or they can act in a paracrine
manner where once let us say these cells have released it. There is another cell in the
surrounding, that goes and binds to it and this cell now gets activated. So, now the signal
is amplifying because the neighboring cell is also becoming inflamed.
(Refer Slide Time: 25:55)

So, and then some of these cytokines connect in an endocrine manner where; that means,
that let us say a cell has released in its certain organ, that cytokine, is now going into the
blood vessel and that blood vessel is taking it everywhere in the body where to even
distant parts of the body; it is causing this thing to activate the cells. However; obviously,
since now this is going to involve some time as well as exposure to lots of different
proteins. This will depend on the ability of the cytokine to first of all enter the circulation
and then finally, once the end of the circulation then how stable their what is there half
life in the circulation because if the half life is very low, then they will not be able to go
ahead and do activation in a distant organ.
And then there are chemokines. Chemokines like cytokines are also secretary by immune
cells. They are actually a type of cytokines. So, chemokines are part of a cytokine and
then they act as a chemo attractant property. What that means, is let us say if there is in
problem at this site or this cell and its secreting some of these cytokines which are then
building up in the surrounding. So; obviously, what will happen is near the cell, you will

have quite a bit away from the cell it is going to become less and less. So, let us say there
is another cell bit far away from this cell, but as it secretes that more and more cytokines
are coming from this direction.
This cell will then move and get closer to this. So, it is acting as a signal to call more
immune cells to come to the site. So, that is what chemokines do.
(Refer Slide Time: 27:39)

So, let us talk about some physiological events. So, let us say if there is a tissue injury
and some foreign entity is also present. So, what is the first thing that is going to happen?
The tissue macrophages and the neutrophils will get activated; so, neutrophils are not
early resident whole lot, but they are in quite high numbers in the blood. And if there is a
tissue injury, then you have also ruptured the blood vessels and the neutrophils are
present at the site. So, they start doing phagocytosis to whatever was damaged and out in
the surrounding. If they get activated, they will start to secrete cytokines.
Now, that they are secreting cytokine this local cytokine release is going to trigger a
cascade of events right because as we discussed in the previous example, first of all these
cytokines are going to act in feedback loop mechanism either on in the same cell or in
the neighboring cells and amplify their production. They will start calling immune cells
from the surrounding. So, more immune cells are now coming in so, more immune
response is being developed and some of them will actually go far away and start
activating the immune cells there as well.

And some of the other cascade of event is first of all vasodilation. So, now, they will also
dilate your blood vessel which means that more immune cells can come into the site and
that is why you see actually that site becomes fairly red, that is because the blood
circulation is increased there.
Then you have influx of protein rich fluid and the cells are coming in. So, you now you
see swelling as well now you have lot more fluid into the environment where the injury
has happened. So, that is why you see swelling and that causes pain as well because
nerve cells also get involved.
And then you have influx of leukocytes and phagocytic cells into the tissue. So, because
of this vessel dilation, all these phagocytic cells are also again which was circulating in
the blood are also coming into the tissue. And then if the next step is if you are able to
clear whatever was a foreign entity and the body thinks if this is fairly compatible, then
the next step is healing and tissue regeneration; which in cases of bio-material is what we
want. So, more new vessels will form and maybe the scar tissue will form if it is a deep
wound and all and the healing process will start.
(Refer Slide Time: 30:07)

So, what are the systemic effects of the cytokines? So, the released cytokines can act on
distant tissues to further facilitate immune response. So, this is on the systemic effects of
it. So, some of these proteins like IL-1, IL-6 and TNF-alpha can potentially do that. So, if

they go to the liver, they will activate some other proteins like C-reactive protein and
mannan binding lectin. This will lead to an activation of a complement system.
We have not talked about complement system yet I think, we will talk about it in the next
few classes. So, this just hold on to this, but it activates another class of an immune
response that we have not talking about yet. The other thing that they will do is in the
bone marrow, they can activate the bone marrow endothelium and they will start
mobilizing more neutrophils. So, as I said bone marrow is a site where all these immune
cells are being produced.
So, they will start the production of neutrophils and start to cause them to come to the
side. Then they can also go and act on hypothalamus which is a brain organ and that is
responsible for increasing the body temperature. And the reason it is done is because our
immune cells are actually in our own cells are actually much more capable of handling
higher temperature than let us say pathogenic cell. So, if you increase the body
temperature. So, from 37 let us say if you go to 39, the body is at a more advantage at
that point, than these pathogenic cells.
Then similarly the fat and muscle tissue also get these signals and they start, because
now you will need some energy to maintain that much heat to have that much influx of
cell to have more cells come in. So, that will start happening and I mean both of this will
cause decreased in viral and bacterial replication and then more dendritic cells are being
called in again; these are also cells that are going to do phagocytosis and make sure that
your bacteria are being killed. Okay! We will stop here and we will continue in the next
class.