Hello everyone! Welcome to another lecture for Drug Delivery Engineering and
Principles. My name is Rachit and we are discussing the Immune Response module at
this point. So, let us do a quick recap what we learned in the last class.
(Refer Slide Time: 00:39)
So, in the last class, as I just mentioned, we were looking at immune response to
materials, and specifically, we were talking about looking at the blood response. So, we
said that there are two ways in which the blood can respond, one is the complement
system, which contains several proteins that initiates it. This is already circulating in
serum and there are various ways in which this can be activated. The three major ways
are named as classical, alternative, and lectin pathway. And, what we also discussed was
that, alternative pathway is the major response that is causing the activation against the
And there are few ways by which we can prevent that: one is to coat the heparin, which
is going to inactivate the cascade, another is making your surface less nucleophilic. So,
making it such that they do not activate these complement proteins and then you can also
bind it to C5a, so that will also result in prevention of activation. Then, next we talked
about blood clotting. So, how does the blood clot? This is mainly mediated by platelets
and they eventually lead to the formation of a fibrin mesh. So, if it is a surface or let us
say this is a blood vessel and somehow the blood vessels are ruptured, these platelets just
go in and form this aggregate of cells and then they reinforce it with a polymer mesh to
plug this hole.
So, this prevents any kind of blood to leach out, but the problem is that, these platelets
gets activated at any kind of surfaces which is not endothelial surface through various
proteins that gets absorbed on these surfaces. So, once they are exposed to protein coated
substrate, they will also do this and this can also lead to fouling of a surface. Maybe it is
not going to perform the function that you wanted it to perform.
(Refer Slide Time: 03:01)
So, let us look at the blood coagulation pathway. So, just like the complement system
and this is also divided into three common pathways, which is extrinsic pathway,
intrinsic pathway, and common pathway.
(Refer Slide Time: 03:12)
So, let us look at the extrinsic pathway. So, this is again a very simplified form that I am
showing you. Just listing out the major proteins and major components that are involved.
It is actually having huge lots of proteins are involved with this, so let us see how this
happens. So, you have some vessel or tissue injury that has happened: this is because
maybe you put in a needle, maybe you put in an implant or maybe there is some accident
that the patient suffered.
So, that causes lots of tissue factors to get activated. This will then combine with several
proteins that are present in the serum. And, eventually for the purpose of this talk what
you have to remember is eventually that leads to formation of factor Xa, so that is a
protein that gets generated.
(Refer Slide Time: 04:03)
Intrinsic pathway is again same, so you have some vessel injury: this is the one that is
also responsible for biomaterial surface, so this may be some biomaterial surface. So,
because of this vessel injury to the basement membrane that is below the endothelial
cells. This membrane is made up of primarily collagen. And so, again the platelets never
really see collagen in a healthy vessel, but when there is an injury or there is a
biomaterial surface, this biomaterial can also code collagen on to it is surface.
So, now these platelets get exposed to collagen. Once that happens, then you have again
cascade of event where factor XII activates factor XIIa, this gets further cascading into
factor XIa. And further and further it goes on eventually leading to the same molecule
factor Xa to be generated on the surface.
(Refer Slide Time: 05:05)
And then there is a common pathway which goes and picks up from the factor Xa and
factor Xa then activates prothrombin. So, prothrombin is a protein that is circulating at
very high amounts in a blood and results in formation of thrombin, and then this
thrombin is another enzyme that cleaves fibrinogen or polymerized fibrinogen into
fibrin. Fibrinogen is a monomer and thrombin itself is an enzyme which is causing
essentially polymerization of fibrinogen causing long fibers of fibrin to form. This will
then use some more serum proteins to get cross linked as well.
At the end of this, you will have a fibrin that has been cross linked. If again this is a
material surface, first you will have aggregation of these cells and then you have fibrin.
So, first you have fibrin polymers and then eventually cross linking of them as well, so a
very tight network is formed. So, for all practical purposes this is nothing but a hydrogel
carrying platelets and blood cells encapsulated and plugging the hole, where the blood
was leaking out of, so that is the major pathway.
So, this is of course, required for functioning of a healthy human because if you have any
defects anywhere this will cause problems. This is because the moment the person gets
injured there is a heavy risk of them bleeding out. So, this is of course, a very essential
pathway, but the problem is that if we are trying to do any kind of biomaterial-based
therapies and these things will occur on the biomaterial, and will not let the therapy to
succeed. So, what is the way around?
(Refer Slide Time: 07:05)
Once you are activating this continuously even the serum proteins and the levels of the
serum of all these enzymes that are causing clotting to happen are increasing. Here, you
are seeing a blood vessel and you can see these fibrin clots, that are actually floating in
the blood vessels. And, this could be a big problem because once you have all these, they
can actually clog the smaller blood vessels. This takes us back to previous discussion
wherein, it causes stroke or heart attack or anything can happen causing loss of function
of a particular tissue.
(Refer Slide Time: 07:47)
So now the clot forms. What is the normal way through which the body then removes the
clot? Let us say this is the healthy blood vessel; this got injured and now you have a
blood vessel which has a hole in it. Now, you have this clot that is formed with the
process that I just described: aggregates of cell with a polymer. And now, eventually the
body wants to return back to this original blood vessel, because this is still obstruction
from cells to the cells that are flowing through them.
So, in a steady state you want it back to a vessel like this, which is fairly well formed and
has no hole in it and no obstruction either. Then the body also has mechanism by which
it can break down fibrin. Once the healing has happened, these endothelial cells which
are at the surrounding have proliferated and migrated. This will eventually lead to a very
nice covering of these endothelial cells and no hole that is remaining. But for that to
happen there is again a cascade of events that causes the lysis of this cross-linked fibrin.
Let us quickly look at that.
(Refer Slide Time: 09:02)
So, as I said, once hemostasis is restored and the tissue is repaired; the clot must be
removed from the injured tissue. So, there are fibrinolytic pathway, as the name suggests
to break down fibrin, and the end product of this pathway and just like those all these
previous pathways there are few major proteins that are involved. In this case there is an
enzyme called plasmin, and this is extremely important in terms of cleaving this fibrin
clot and it has fairly broad-spectrum activity.
Plasmin is formed by the activation of a pro-enzyme which is called plasminogen. First
this plasminogen which is already present in the serum gets activated either by the
plasma or the tissue activators and then these tissue plasminogen activators are also
found in most tissues except liver and the placenta. And these are typically synthesized
by endothelial cells and that is why they are concentrated in the walls of the blood
vessels. So, it helps because now these endothelial cells, so let us say this is the
obstruction and these endothelial cells are now migrating towards this obstruction, they
will themselves secrete this fibrinogen activators or plasminogen activators to cause the
degradation of this clot here.
Some of the well characterized activators out there are urokinase and some tissue
plasminogen activator called tPA. So, these things are actually very well characterized
and actually used in humans too, and these can be used to coat materials to prevent clot
formation. So, now, what you can do, is you can take your material you can pre-coat
with these activators. So, if the activators are present on the surface then the first time the
fibrin comes in it is going to get cleaved off.
(Refer Slide Time: 11:13)
Thus, you would not let the clot to form. Here is an example: you have plasminogen
activators that is going to change plasminogen to plasmin which is the major enzyme and
then this plasmin is going to take this fibrin and degrade into fibrin fragments. So, there
are always some steady state levels any damaged endothelium releases these activators
as well as plasminogen.
(Refer Slide Time: 11:47)
So, these can be then used to degrade the clot. This is again a complex figure I do not
really want you to remember all this, but just want to show you how it is typically done.
So, you can have an artificial surface or you can have a vessel wall. In this vessel wall
these endothelial cells are secreting all kinds of enzymes and making sure that this is not
So, you have these endothelial cells coated over basement membrane which could be
collagen and thrombin of course, plays a major role. Plasminogen, plasmin we have all
talked about. The tissue plasminogen activator again is a major role and this is essentially
what caused what is the pathway that happens. Again, as I said you do not really need to
remember this, but there are some key players such as factor Xa, plasmin, plasminogen,
fibrin, fibrinogen these are some things that you should remember.
(Refer Slide Time: 12:41)
So, here is some example say we are talking about hemo-compatibility or MEMs
component. So, what a MEMs? These are devices which are used as electronic devices
which are used for measurement and mostly for diagnostic purposes in human body and
what you are seeing here is you have different choice of materials. So, mostly silicon
based, but some other materials as well and what you are seeing is, if you incubate them
with platelets how many platelets are seen per centimeter square or per millimeter square
in this case.
And so, what you find is different substrates will have different affinity for platelets and
that may have to do with that proteins that are getting absorbed, the type of proteins and
the amount of protein, and as you can appreciate that the different materials can have
different effects. So, maybe if your application it does not matter whether you use
polyurethane or silicon, then polyurethane may be the better choice just because it is
going to cause less adherence of platelets and thereby less clot formation over its surface.
(Refer Slide Time: 13:43)
So, just one more SEM image; I told you that these platelets when they get attached to a
surface, they lose their round morphology and actually become spiny and start to a
spread around. So, that is what you are seeing here in this SEM image, and in this image,
you are looking at three, four different types of surfaces at a higher magnification. And
again, you can appreciate that all of these are causing attachment of platelets some at a
higher or some at a lower amount which was seen in the previous slide. But this is what
if you then give the whole serum do it these things can start forming and following the
surface with blood clot.
(Refer Slide Time: 14:28)
So, what are the implications in drug delivery, what are the effects of this in the drug
delivery? First is there is a fibrous capsule that is formed consisting of fibrin clot. Again
not very good: your molecule release it will get altered. So, it is causing diffusion
limitation because earlier what you had done is you had modeled let us say this was
PLGA particles and you thought that is going to degrade at a certain rate or the drugs are
going to diffuse out from these pores at a certain rate. And, come into the system
immediately, but that is not going to happen anymore, because now this is coated with a
thick layer of the blood clot which is going to change the diffusion.
So, that is a parameter that can cause no diffusion to happen. So, that is a parameter that
you had not accounted for and that is going to cause the failure in the therapy you have
limited exchange of fluid. So, let us say if this was carrying cells and was used for tissue
engineering. So, let us say I have this material that is encapsulating cells within it.
Now, these cells cannot get external glucose, oxygen because there is a same thing: there
is a blood clot that is preventing the diffusion from happening and neither they can get
the base products out. So, all of these have been blocked off and that will eventually
result in the death of these cells. So, again not ideal or it may be an osmotic pump.
So, that will not work, not only that, the things that are coming in is going to decrease,
but the things that are going out as well. So, since the water is not coming in as much
amount as you had hoped, the degradation may change, the hydrolysis may change. So,
these are some of the things that will start to change the whole kinetics of your device.
So, all of this leads to slower rate of drug release, when we are talking about drug
delivery applications. And it is a danger to patient, maybe because that that amount of
drug is required for the beating of the heart or for the functioning of the brain and those
things are fairly sensitive for even small minor changes. So, any of this can cause serious
complications to patient.
(Refer Slide Time: 16:57)
Now, let us talk about systemic toxicity. So, so far what we have talked about is when it
comes to the surface both the complement and blood system are involved. So, the
complement proteins are coming in binding to the surface and then on the platelets where
they are coming in binding the surface and causing falling of the surface. So, what about
systemic effects? Systemic effects refer to reactions that are far away from the site of
injection or implantation.
So, if I let us say put something in my arm what happens to the rest of the body? So, arm
maybe gets inflamed, but does the rest of the body also suffers from this? And so, this is
more looking at a global response rather than just locally at the arm or whatever the site
of injection or implantation may be. So, lots of systemic toxicity is seen and this can be
due to several reasons, first is maybe the chemical is small and can diffuse and it is toxic.
So, there is a direct chemical toxicity that is causing this systemic toxicity to happen, or
maybe whatever you put in the degradation products are toxic. So, we only talked about
the PLGA degrading into PLA and PGA or lactic acid and glycolic acid: although those
are not toxic. But they are causing drop in pH, but then products themselves can be toxic
maybe it is some metal that is releasing out leaching out metal ions that is causing
toxicity. So, could be anything maybe it is causing free radical to generate. These are
oxide species that are highly reactive and will react with lots of surfaces on lots of
So, maybe these are being generated and then they are circulating out into the system,
because these are small species and you have generation of vasoactive compounds from
complement activation. So, again the complement has binding to the surface, then it is
going to start activating and releasing lots of molecules that can cause the immune
system to build up.
And, then immune system is going to travel everywhere in the body and your body will
become fairly immunogenic at that point and then you have other immune reaction that
can also happen. And, some of the symptom that you will see once this has happened is
you can start seeing joint pain, you can see swelling in different regions, you can see
allergic reactions. So, maybe it has happened a lot of the time, because if your body is
allergic it may suddenly start to react with a piece of cloth that earlier you had no
problems wearing with. This changes the blood chemistry whole together your lymph
node gets swelled, so I am sure you might have heard about your lymph nodes getting
So, sometime you go to the hospitals and doctors of your choice and they will try to
touch your back a lower neck to see if you have any lymph nodes getting swelled up.
Resulting quite a lot of immune cells now floating around in the blood that may cause
several other side effects. So, this is a part of systemic toxicity of how this is a global
response rather than just localizing at this point.
(Refer Slide Time: 19:59)
So, we are going to now talk about adaptive immune response, so far, we were looking at
innate immunity and some blood response. This was something if you remember and this
is something that is nonspecific for the most part where it does not really distinguish
between let us say a bacterium or a virus. It just recognizes some patterns and on the
basis of that it causes amplification of immune response to try to kill it off immediately.
But what happens if it does not happen what happens? if the material remains in the body
that the body does not like, or what happens if the bacteria is able to colonize the surface
and maybe form bio-film or maybe persist due to various other evolutionary advantages
that it has. And then how does the body tackle it? that is where the adaptive immune
response comes in.
(Refer Slide Time: 20:49)
So, before we go to the adaptive immune response let us talk about what are the different
cell types that are present in the adaptive immune response. So, by far the most you will
hear here about are these dendritic cells and macrophages, you have already heard about
them; these act as sort of a link between the innate and adaptive immunity through these
DCs and macrophages.
And why I am saying is that because; obviously, we know that DCs and macrophages
can engulf anything foreign that they find. So, and that is a part of an innate response
maybe it is not really a very specific there is a just engulfing happening or this could be
an adaptive response that will come in just a bit. So, these for adaptive immune response
they act as an antigen presenting cell. What does that mean? Anything foreign is an
antigen which an immune system can recognize.
So, these cells DCs and macrophages they can process this and then they can present it to
the rest of the immune system. See here take a look at this: This is the foreign material
that is there and the cells are present in very large number in the tissue and they survey
the surrounding especially macrophages that most tissues have their own distinct
macrophages. If we have already learned about that lungs having quite a bit of them
when we were talking about inhalation base route delivery; we had learned that liver
cells have Kupffer cells which are nothing but liver macrophages. So, all of that is
present in very high numbers.
Then the next start T cells; so, these are leukocytes. they are purely adaptive immune
response and there could be several types of them. There is a helper T cell which helps in
boosting the adaptive immune response for a particular antigen. There are cytotoxic T
cells which will go and directly kill a mutant cell or a cell that the body thinks is not
normal, and then there are regulatory T cells. And these are involved in tolerance which
basically means that you do not really want what happens, if let us say immune cell like
a T cell for some reason for due to some abnormal signaling starts to recognize your eyes
So, the last thing you want is your own body cells to kill off your eye tissues. So, then
we also have the body as also mechanism for these regulatory T cells that involved in
tolerance. So, maybe these will go to the eyes site and we will make sure that they are
not letting these cytotoxic or helper t cells to kill off the eye tissue. And, then the other
major leukocytes are B cells and like DCs and macrophages they are also antigen
presenting cell. So, they can also present foreign antigens to the immune system, but the
major function that they are known for is the antibody generation.
So, all the antibodies that are generated these are generated through B cells they are
mainly involved for external pathogens these antibodies. And that is how once the
antibody binds to a pathogen that is a sort of a flagging a pathogen saying that this is the
position here is an antibody and all of these immune cells or most of these immune cells
have receptors for that antibody it is called Fc receptors. And we will we will talk about
that in a moment, but that is what it causes them to recognition by the immune system
(Refer Slide Time: 24:28)
So, let us look at the physiology of the primary immune response. So, let us say you have
a peripheral tissue, I will change color, so let us say you have a peripheral tissue and
there is some injury or there is some pathogen that has come in. So, because of this
pathogen and the injury what will happen to you may have pathogen or external proteins,
antigens in that case they represent the site then you have these tissue macrophages that
are going to take up these antigens.
So, these macrophages will then go to a secondary lymphoid organ, which are the
secondary immune response organs; one of them is called lymph node. So we have
lymphatics here. So, these things will go through the lymphatics they will go and then
present whatever antigen that they have acquired to these leukocytes the T cells and the
B cells that will cause activation of the leukocytes at those lymph nodes.
Once these cells get activated, they will start moving around in the blood and again get to
the site where the injury has happened because these things are leaky, so extravasation
will happen. And now, you have more and more leukocytes coming in which are actually
trained on how to handle pathogens that are this particular antigen.
So, then they will come in and start to clear away whatever pathogen or whatever foreign
material is present. That is how this adaptive immune response kicks in and this is the
physiology of it. We will stop here and we will continue our discussion with the adaptive
immune response in the next class, it is fairly complex and quite exciting also. We will
look at that to let us to how this builds up, how the body then differentiates between self
and non self and what do we do with our materials, so that we can prevent the immune
system from activating ok.