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    Study Reminders


    Hello everyone, welcome to another lecture for Drug Delivery Engineering and
    Principles. I am going to talk about further on protein adsorption.
    (Refer Slide Time: 00:36)

    So, let us just do a quick recap of what we learned in the last class. So, in the last class
    we talked about pre-adsorbing proteins on surfaces, which basically means that if I want
    a certain kind of response from a surface, let us say if I wanted to bind those cells, then
    what I can do, I can add ligands proteins onto the surface even before I put it with the
    cells, so what will happen is, these proteins will adsorb on the surface and will directly
    start interacting with the cell with their certain cognate receptor.
    So, the advantage here is, I get a lot more control over my surfaces because now they are
    interacting the way I want them to interact. And then the certain there are lots of
    applications of this that you can vary to sort of modulate this response that you are
    getting. Then we talked about a model that is being used to study protein adsorption one
    of that was a monolayer model. So, what it is? It means that let us say if I have a surface,
    it just means that there will be only one layer of protein, that will adsorb on the surface to

    cover whatever is exposed area and once that layer has a adsorb no more preabsorption
    will happen.
    So, that is the assumption in this monolayer model and again we will discuss today that
    this is actually not true, but for the sake of this model, it will be an orientation like this
    there will be no more protein that is going to come in adsorb here. And then at the same
    time we discussed about hard and soft proteins. So, what we are saying is, there are some
    proteins which are fairly hard meaning with a fairly rigid they do not change the
    structure very easily the structure is very stable and then there is some proteins which are
    very prone to denaturation, their structure is easily changeable. So, these are called soft
    And then we talked about protein orientation and structure essentially saying that if I
    again have a surface which is exposed to low concentration of protein. So, what will
    happen is, in the protein that is initially getting adsorbed will have time to expand on the
    surface because it still has this open side next to it. So, it will keep on expanding until
    either the site gets filled or the protein reaches its happy state in terms of the equilibrium.
    So this is a case in a low concentration setting whereas, if I have high concentration then
    what will happen is, the protein that gets initially adsorbed will not have space left
    because of the high concentration as several other proteins have also adsorb and it will
    tend to be at a higher amount on a given unit area or the given sort of the structure of the
    protein will also remain more or less rigid or it will not really change a whole lot.
    And then we talked about wettability of the surface itself. So, what we were saying is if
    we have hydrophobic surface, meaning it has low wettability; then it will tend to form
    very strong bonds with proteins, because the outside water environment is not competing
    with it and the hydrophobic domains will tend to interact with this hydrophobic surface
    as well the surface also does not want to interact with water. So, there is an energetically
    driven process, which causes a lot more interaction between protein and hydrophobic
    surface compared to let us say this is a very hydrophilic surface and then the proteins
    will cell adsorb to it, but their affinity to the surface will be much lower.

    (Refer Slide Time: 04:18)

    So, now having done all this, as I briefly mentioned this monolayer model is a model that
    is not really well accepted at this point of time because more and more research has
    shown that there actually does form multiple layers of protein on a surface. So, to explain
    this its simply another model is proposed this is a multilayer model.
    And what it essentially means is that there is a 3D structure to a protein this interface. So,
    even if I have a surface, there could be several layers of proteins that is going to come
    and attach to it. And essentially what will happen is its defined as sort of a hard and a
    soft corona. So, a hard corona is defined as something that is fairly stable on a surface, it
    is very difficult to rip this protein out from the surface. So, something it is directly
    interacting with the material and it is very close to the material is typically called a hard
    corona and something which then comes and then interacts with this hard corona is
    called a soft corona.
    And the whole reason for the soft corona to happen and this could be nanoparticle or the
    implant, it does not have to be a particle it could be a bulk device and the reason that this
    soft corona start interacting the hard corona because this hard corona has actually a
    change in the structure. So, this is not depicted in this image, but let us say this circular
    protein has now become elliptical where its adsorb. So, it has now exposed new sites to
    the body which were never exposed earlier. So, now, there could be an another protein
    that comes and sort of attaches to these exposed sites and has some affinity to them. So,

    that is why you get the soft corona and may not be a very strong interaction at that point,
    but they cell some interaction to it.
    So, as again is depicted here also, so you have a physical surface, then you have some
    amount of protein that comes and then there is some other protein that comes on top of
    those proteins and this essentially creates a whole corona of protein around a particle.
    (Refer Slide Time: 06:34)

    And then there is another concept called competitive adsorption. So, what this is, is the
    equilibrium adsorbed protein in composition is defined by how much is the total protein
    first at the site and then what is the relative protein concentration. So, this is very
    complex phenomena and very very poorly understood for most of the materials that we
    use and then the kinetics of this has also become important. So, what it essentially is
    saying is, is again if I have a surface, first of all the amount of protein that is coming in
    depends on the total protein amount that is getting adsorbed.
    It depends on how many types of proteins are there, then it will also depend on what are
    their concentrations. So, all of course, not all proteins will have the same concentration -
    some will have very high, some will have very low and then the kinetics also becomes
    important. So, if let us say one protein even though it is a lower concentration, it gets
    completely adsorbed on the surface very very quickly, then those will dominate or
    otherwise vice versa will happen for other kinds of situations.

    So, the kinetics of these protein adsorption and again the kinetics of different proteins
    will be different and then it is also dependent on time and location of the implant and
    why am I saying that is because what will happen over time is let us say the environment
    is changing, there more cells coming in, they’re secreting more proteins and the
    concentrations of protein is changing, the types of proteins are changing and because of
    that what will happen is this equilibrium which is being maintained is going to constantly
    change or if the implant actually is moving or let us its a particle which is traversing
    through the body and then of course, you will have different effects taking place at that
    So, essentially saying that the adsorbed layer of this composition is fairly dynamic and
    typically as I previously described the hard corona is still fairly stable, but it can be
    dynamic and what can also happen is that initially the proteins that are adsorbed can be
    displaced by other proteins over time with very high affinity. So, let us say if I have a
    protein adsorbing on a surface with a certain affinity x and then I have another protein
    with an affinity of 4 x, even though this protein is at a lower concentration and maybe
    has taken time to diffuse to the surface and x is already adsorbed to it, what will happen
    is this protein will eventually be able to slowly and slowly be able to displace this and
    get adsorbed there because a equilibrium says the higher affinity will dominate over the
    lower affinity. So, to this particular phenomena where essentially the higher affinity
    proteins get to replace the lower affinity proteins from the surface is also known as
    Vroman effect -you will see that we used quite a bit in the literature. So, just something
    to remember.

    (Refer Slide Time: 10:08)

    So, again just to summarize this protein adsorption, so we are saying that synthetic
    foreign materials acquire bioreactivity only after first interacting with dissolved proteins.
    So, I mean even if I have a material that has no sites that can bind to the cell, what will
    happen is the proteins will adsorb to it, these proteins will have sites that can bind to cell
    and that is how they can mediate cell interaction and gives bioreactivity to the surface.
    And any kind of cell interaction or enzyme interaction that is going to happen will be
    mediated by this protein layer. And then there are some principles of protein adsorption.
    There are limited adsorption site and so, there is a lot of competition that happens
    between the proteins to sort of get represented on the surface and then there are driving
    forces this could be several things- could be the hydrophobicity and all.
    The surfaces themselves are going to change; I mean if I am using PLGA versus PEG
    versus let us say PLA versus PCL, all of these have different hydrophobicity,
    hydrophilicity, have different surface groups, have different interaction with the proteins.
    So, the type of proteins that can absorb on these surfaces is going to be very different and
    then again the biological activity of the adsorbed protein varies on different surfaces as
    we just discussed.
    So, once the protein adsorb, the more it changes conformation the lesser will be its
    activity. So, if a protein that has a structure like this, gets absorbed and becomes a
    structure like this, it has changed quite a lot of structure. So, its activity may get severely

    damaged or might not be even active at all whereas, if the same thing becomes like this
    its fairly similar. So, you can think of a scenario where this may be still 70-80 percent
    active than when it was in solution, so these things can also vary.
    (Refer Slide Time: 12:11)

    So, again this is just showing how a nanoparticle actually interacting with the cell, the
    cell is not actually seen the nanoparticle surface directly because it is already adsorbed
    protein in the surface within less than milliseconds and its actually interacting through
    the adsorbed protein. So, it becomes very important to study protein adsorption.
    And then another interesting thing is, you can conjugate a particle with various ligands
    you think that they may be used to interact with the cell surface, but look what happened
    once you put it in the media there is a whole lot of protein adsorption layer that has come
    in and this particular targeting ligand is not even accessible to the cell. It may be
    accessible in some cases depends on the surface and the ligand itself, but this is
    something to consider while you put these ligands that they may not be actually directly
    interact with the cell.

    (Refer Slide Time: 13:08)

    And let us talk about how can we study this quantification of protein on the surface. So,
    one thing is to do an SDS PAGE. And so, it is fairly simple what you do is you put your
    implant or particle and incubate with let us say serum and so, what will happen is, what
    whatever different proteins are there will come and adsorb and serum is just an example
    you can put it in some other fluid, maybe if you are going to give it to lungs to
    innovation, you are going to put it in lung fluid or lavage, if you are going to give it
    through orally you can maybe put it with saliva.
    So, there is all different kinds of biological fluids that can be used - depends on the
    application. So, once these proteins have coated on this implant, you take this implant I
    mean again as I said this is a matter of milliseconds we are talking about. So, you can
    leave them for a few minutes and take the implant you centrifuge to remove anything
    that is external and resuspend it let us say in water and once you have done that, you can
    use an SDS PAGE.
    So, SDS is again a very strong detergent. So, what it does is this completely denatures
    the protein and interacts with the protein in such a high affinity that it comes off from
    this implant both the hard corona and the soft corona. And then you can just run a PAGE
    gel which is nothing, but as gel that separates the protein out on the basis of its size or
    molecular weight and in that regards then you can study what all proteins are there
    through western blotting.

    So, I hope everybody knows western blotting - all it is to transfer it to a membrane and
    then once its transferred to a membrane let us say now this is the membrane itself you
    can come with antibodies against known proteins. So, let us say if I am predicting
    fibronectin is one of the protein that is getting involved, I will come in with the
    fibronectin antibody and incubate with this. So, if fibronectin is one of the protein that
    was on the implant, then this empty body is going to go and bind to fibronectin and I can
    tag this antibody with some fluorophore initially, so that is going to start fluorescing or I
    can put some enzyme and allow some color around this band.
    So, that way I can know that fibronectin present in this milieu or its not present in this
    milieu and similarly you can do it for several other proteins. So, again this way is fairly
    semi-quantitative, its not very quantitative. Another good way to do this is using
    something well a surface plasmon resonance which is again very widely used. So, its
    highly sensitive and it depends on the dielectric of the surface. So, what you can do is,
    you can take your surface that you want to test. So, that is here; you can then coat it with
    some metal layer. So, in this case let us say if we have coated with gold.
    So, once the proteins adsorb to it, the dielectric of this particular protein layer is going to
    be different from your initial surface and because its different you can shine a light - its
    going to scatter differently and you can read it through a detector and that is going to
    give you a surface plasmon effect because the dielectric is changed and through that you
    can then quantify by running some standards of known amounts, you can then quantify
    whether the protein is first of all adsorbing and interacting with the surface.
    And then secondly, if it is then what is the amount of protein that comes on the surface
    because this dielectric will be directly related with the amount of the protein that has
    come and adsorb on the surface. So, that is one way you can study the protein adsorption.

    (Refer Slide Time: 17:23)

    So, what we will do is, we will we will take an example from the literature - this is just to
    sort of give you an idea of how the research is happening. So, this is a paper that was
    published quite a while back now in 2010 and it is about how nanoparticle can induce
    unfolding of a protein called fibrinogen and this unfolding then results in signalling
    through a receptor called Mac- 1 receptor and that causes inflammation. So, here is just
    an example of how a foreign material that is introduced causes inflammation. So, its
    more a mechanistic table.
    (Refer Slide Time: 17:59)

    So, what the authors have done in this paper is they have taken gold nanoparticles. So,
    this is gold nanoparticle, they have coated this gold nanoparticles with polymers- during
    synthesis it uses various polymers and one of them is PAA which is Poly Acrylic Acid.
    So, what they’ve done? They have coated the gold with poly acrylic acid and then they
    have incubated it with a protein called fibrinogen- its very widely available protein and it
    is found quite an abundant amount in our serum as well. So, and what they have shown
    in this paper is well this adsorption happens, it activates Mac-1 on immune cells or
    actually on other mammalian cells.
    And once it activates Mac- 1- Mac- 1 goes into the nucleus that signalling goes into the
    nucleus and causes the up regulation of NF-kB pathway which is a very known to be a
    master regulator for inflammation and that causes the release of lots of inflammatory
    cytokines and that can cause some reactions in the body, you can start getting fever- very
    similar response to when we get sick.
    (Refer Slide Time: 19:41)

    So, let us go a little more deeper, so what the did is they make three different sizes of
    particles. So, 5,10 and 20 nanometers in size -this is the diameter and then what they did
    is, they incubated this with serum and then the same process that I just described with the
    SDS-PAGE. So, this is just a SDS-PAGE. So, what you can see here is several types of
    proteins have come in and adsorb on these ones and what you see here is 5 nanometer
    has quite a bit of protein and then 20 nanometer has less protein for the same amount of

    the gold. And what they primarily observed was that the 65, 55 and 45 kDa chains of
    fibrinogen were very abundant.
    So, essentially when I talk about this, I am looking at 65 which is this guy, the 55 which
    is this guy and then the 45 which is this guy. So, these three bands were very prominent
    every time they did this experiment. So, then they found out that this is actually nothing,
    but three different chains of fibrinogen.
    (Refer Slide Time: 20:48)

    So, that is what they are describing here. So, fibrinogen comprises of three protein chains
    the alpha, beta and gamma and this is the confirmation - fairly large molecule 45
    nanometer and it’s a dimer. So, what they are further shown is that this C terminus which
    is fairly covered in a normal protein structure as you can see here has a binding site for a
    receptor called Mac- 1 on a cell.
    So, Mac- 1 receptor can come and bind to this site although the site is not typically
    exposed. So, the Mac- 1 does not bind to fibrinogen by itself unless the fibrinogen
    structure is somewhat damaged or denatured, so that this Mac- 1 site is exposed. And
    then what this showed is that the addition of these PAA coated gold nanoparticle resulted
    in the loss of the protein secondary structure. So, as you can see here, so what they have
    shown is, this is a circular dichroism its a its a method that measures the secondary
    structures in the protein. So, proteins when they fold they have structures like alpha helix

    and beta sheets and others as well. So, these alpha helix and beta sheets have a certain
    wavelength through which they give signals.
    So, if this is a normal protein structure which the C in this case. So, this red line is a
    normal protein, so which means that if you are getting this red line the protein is in this
    particular configuration; but as you add more and more gold nanoparticles to your
    solution what you find is that the structure is actually changing and you can see this
    signal that you get from the secondary structure is actually decreasing as you go forward.
    So, this is supposed to be the highest concentration. So, at this point you are talking
    about 10 microgram per ml of fibrinogen and give it to this 40 microgram per ml of your
    gold particles.
    And just to show that the gold itself does not have any secondary structure they will just
    run the gold alone. But so, what you can see is there is quite a bit of change in the
    structure or loss in the secondary structure, which is actually proving the point that once
    this protein goes on to the particles, it changes structure.
    (Refer Slide Time: 23:24)

    Then they have shown that this fibrinogen has selective binding for your Mac- 1
    receptors. So, what they have shown is they have used THP-1 cells which are a human
    monocytes; and this THP-1 has a Mac- 1 receptor. And then another one they have use
    HL-60 which does not have a Mac- 1 receptor.

    So, what this see here is if you just put protein, the amount of protein that gets bound to
    the cell surface is fairly low, but once you put protein plus gold nanoparticle you get

    quite a lot of protein that goes on the cell and the reason for that is now because the Mac-
    1 receptor site is now accessible, the Mac- 1 receptor on the cell surface now can bind to

    Whereas, in the negative cell line you do not see that and if you incubate with albumin
    also you do not see that, because already the albumin has coated the particle. So, the
    particle cannot bind to the fibrinogen. So, just describing what I just said that causes the
    unfolding and that is why it can interact with the Mac receptor.
    (Refer Slide Time: 24:47)

    And then these guys further went ahead and show that the experiments it would done
    with 20 nanometer did not show any significant bound protein. So what they have done
    is this is again your bound protein on these THP-1 cells and what to see if you have only
    fibrinogen its structure is preserved. So, that does not cause anything. If you have 5
    nanometers just like the last figure it causes a lot of binding of the protein. If you use 20
    nanometer, which we showed earlier, it does not bind and change the structure that
    much, you see again there is a reduction in that protein that is getting adsorbed.

    (Refer Slide Time: 25:27)

    And then they showed that you can use some inhibitor of this particular Mac- 1 receptor
    and so, if you put receptors that binds to the Mac- 1, then your fibrinogen coated protein
    on the particle cannot bind to this and so that is what they have shown here. So, the
    bound protein is actually decreasing whereas, if you use another peptide which has no
    affinity, it does not do anything. So, it is a further confirmation that this is Mac-1
    receptor that is actually interacting with your protein.
    (Refer Slide Time: 26:02)

    And then they have gone ahead and showed that the binding of the fibrinogen activates
    NF-kB signalling. So, if you take this last example what they have done is they have

    added all fibrinogen ,PAA gold nanoparticles, as well as antibody that binds to the NF-
    kB P65 domain and so, what you can see is because of that you get a big shift.

    So, if NF-kB let us say was for an example x kDa and let us say in a normal gel the x kD
    will come here. Once it binds to the antibody now its molecular weight is increased by x
    plus the antibody molecular weight, so that has now gone and shifted up there. So,
    further confirmation that actually this gold nanoparticle bound fibrinogen not only is
    activating Mac-1, but that are causing further signalling in is actually in causing a NF-kB
    (Refer Slide Time: 27:09)

    And then again as I said NF-kB is a master regulator. So, it will secrete lots of cytokines.
    So, in this case you are seeing IL-6, IL-8 and TNF alpha which are both inflammatory
    cytokines. So, their expression level goes up when you have NF-kB signalling and which
    is again a major cause of the inflammation that is happening. So, again the THP-1 cells
    were used here which showed that the secretion of IL-8 and TNF alpha.

    (Refer Slide Time: 27:38)

    And so, does the surface characteristic actually influence the protein binding? So, what
    they did is, now the change the surface.
    (Refer Slide Time: 27:45)

    So, in this case what they did is when they made these gold particles they kept on
    decreasing the PAA concentration, the PAA, as I said is Polyacrylic Acid, which is
    highly negatively charged. So, when use 100 percent PAA you get a fairly highly
    negative charged particle the zeta potential is a way to measure the charge and as you
    decrease this PAA amount the charge increases because this negative charge is going

    down and so, what they further show now is that your protein binding is actually
    decreasing to the gold particles as you are changing the charge.
    So, they have replaced PAA with this PDHA and they are further showing you that
    charge is what is responsible for binding of this protein to the to these cells and if you
    have lower charge, then your fibrinogen is either not binding to your gold particle or
    even if its binding, it is not exposing Mac- 1. So, this as a surface charge density is
    critical or fibrinogen binding in this case. So, I think that is where we will stop and we
    will take it further in the next class.
    Thank you.