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    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
    materials.
    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
    happening.
    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
    proteins.
    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
    swelled up.
    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
    as foreign.
    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
    and clearance.

    (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.
    Thank you.