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Pre-Absorbed Proteins

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Video:

Hello everyone, welcome to another lecture for Drug Delivery Engineering and
Principles. Just a quick recap of what we learned in the last class.
(Refer Slide Time: 00:37)

So, were talking about protein adsorption -why we are doing this is because we want to
understand what happens when you put any foreign material in the body or in contact
with any sort of body fluid. Let us say if this is my body that I am putting in, and the first
thing is going to come in contact with of course, the fluid and the major part of the fluid
in our body is essentially nothing, but water and then the next major component is
proteins.
So, all those proteins that are present in a fluids will start to interact with this particular
surface and then depending on how the surface is- whether it is hydrophobic or
hydrophilic these proteins will then tend do adsorb on different areas and a different
conformation on this surface and only then the cells which are again in the vicinity will
come in and they will start interacting with the surface through these adsorbed proteins.

So, the cells actually do not really see the surface very well. What they see is actually the
adsorbed protein layer through which they will start interacting.
So, that is why it becomes very important because whatever the cell is seeing is actually
nothing, but the absorbed protein. So, we need to study what actually this absorb protein
is and how can it be modulated to get that desired function. And so then we talked about
in the process of understanding what protein absorption is first is what a surface? So,
what defines a surface? How do we know that it is a surface it is an interface between
whatever the two medium has been presented to it. We talked about what are surfactants.
So, again what are surfactant? Surfactants are nothing, but molecules that have both
hydrophilic and hydrophobic domains and they can phase separate out hydrophilic and
hydrophobic domains and essentially let us say if I say this is oil, this is water then these
surfactants will essentially line up this interface with the hydrophobic domain going
towards the oil phase and the hydrophilic domain going towards the water phase.
And so again this becomes important because what we are saying is proteins are mild
surfactants because they contain several amino acids about nearly all proteins with large
structures will have about 20 amino acids, all 20 amino acids. And then these 20 amino
acids some are hydrophilic, some are hydrophobic even within the structure of the amino
acid there are a hydrophilic and hydrophobic domains and because of that these proteins
act as mild surfactants and their structure will change depending on what medium they
are. So, if they find any hydrophobic domains or hydrophobic surfaces what will happen
is the hydrophobic domains will start to come out and interact with those hydrophobic
surfaces.
So, that is why we studied surfactants. Then we talked about protein folding which is
again a very related thing. So, in natural process let us say if we are talking in cells
which is nothing, but an aqueous media. Let’s say this is the unfolded protein. So, the
protein folding will be in such a way that all these external domains will be hydrophilic
and the reason for that is because these external domains will have to interact with the
water that is present in the surrounding.
So, they like to interact with the water. So, that is why all these hydrophilic domains will
come outside and then all this inner domain will be hydrophobic and again the same

reason that these domains do not really want to interact with the water that is present
outside.
So, they want to bury themselves and prevent any sort of interaction happening with the
water outside. So, that is why protein folding becomes important because now what we
are saying is when this particular proteins now suddenly start seeing a hydrophobic
surface, these domains will tend to come out and then these domains will tend to go
away and that is why the protein folding will change.
So, that is why we studied protein folding. Just in very very brief terms I mean protein
folding it itself is a very complex phenomena in a whole course can be designed on it,
but what we talked about is just some general concepts of the way these proteins are
going to fold.
(Refer Slide Time: 05:06)

So, having done all that and having established that these surfaces can modulate the
protein folding and protein adsorption, another concept that is now being used in the
field quite a lot is the pre-adsorption of these proteins to surfaces and why would you
need to do that? So, let us say if I have a surface and I want this surface to signal the
cells in a certain way. So, I mean if I have a cell that is going to interact with it and I
want this particular surface to interact with the cell ligand , x.

So, for that to happen I want to make sure that whatever protein gets coated on the
surface has some affinity for this ligand x or this receptor x. So, this is a receptor and let
us say I am putting some ligands on the surface.
So, I may or may not put ligands, but essentially I want this surface to interact with the
cells through this x. So, one of the strategy the use for that is to pre-adsorb proteins that
we know is going to interact with this receptor x and that is one way that I can control
the signaling that is going to happen through the surface. See the reason for that is
because once I put it in the body or once I put it in contact with the body fluid there are
all kinds of proteins in the body fluid, I mean we are talking about thousands and
thousands of them and so the probability that let us say a certain protein will come and
adsorb to it is fairly low.
So, to prevent it what we do is we first treat it with a solution containing only this ligand
L and that will ensure that at least some of the ligand L will remain on the surface and
can interact with the cell. So, some of the proteins that are very widely used for this is
fibronectin, fibrinogen, vitronectin and these are nothing, but these are proteins that
contains a adhesive sites.
And so if I intubate my biomaterial with these proteins for some time what will happen is
these proteins will then coat the surface completely and then I can put this implant with
cells or in the body. And these proteins may still come off and were going to talk about
this in next few slides, but it increases the probability that one of these proteins will now
start interacting with this cell receptor x and give the signaling that I want this surface to
give.

(Refer Slide Time: 07:33)

So, whereas, the pre-adsorbing with a non-cell adhesive it can also be used. So, let us say
if I want to put an implant which I do not really want to interact with the cell. Let us say
I have a implant that is carrying a drug D and all I want to do from this implant is for it
to release the drug constantly and not have any sort of unknown proteins coming and
absorbing making a layer causing this diffusion of the drug to be difficult from this
implant. So, we know that proteins will adsorb to it we cannot avoid it. So, why not let
us just absorb something that does not interact with cells.
So, let us say if I directly implanted what will happen? all kind of random proteins will
come and essentially adsorb on the surface and once they do that I am not sure that what
will that signal to the cell whether they cause a massive cell layer to form on the surface
and then that can again lead to cascade of events that can interact with another cell and
make a big big layer.
So, that is the last thing I want if I want the drug D to release in the system because now
what is happening is not only the drug has to diffuse out from a matrix, but now it has to
diffuse through this protein layer and through multiple cell layers which is going to be
very challenging and it is something that I cannot control right. Because I initially
designed this let us say to release x milligram per minute or something, but now once
this sort of cell layer and protein layer has been formed I do not know whether it is going

to release x milligram per minute maybe it is going to come down to X by 2 ,maybe is
by x by 4.
So, as a clinician I am very worried now because I do not know how much dose I am
giving. So, to prevent that what you can do is you can essentially coat it with a known
protein that you know is not going to interact with cells. So, even though you do have a
protein layer now, what you do is you prevent the cells from coming in and adsorbing on
it. So, that way atleast I know that what is the diffusion of this drug from this matrix
through this protein layer and that way I have some good idea as to how much drug is
getting released per unit time or per day or whatever it might be.

So, this is one application I am giving you there could be several other applications-
maybe the implant is such that we do not want the immune cells to interact, maybe it is

carrying cells inside and we do not want immune cells to come in and sort of kill those
cells away. So, these are several applications that you can think of in that direction and
then several other strategies apart from pre-adsorbing proteins and all of this we are
going to talk about in the future classes in this course, but that is just one example that I
am giving you.
So, these are things that you can play around with protein adsorption it itself to get some
desired result. And then next thing is that often you should define the material signaling
in-vivo as well. So, you can prevent non-specific protein adsorption. So, one thing is to
prevent the cell and then another thing is to prevent any kind of protein to adsorb on it.
So, let us say if some enzymes absorb to my surfaces and they start degrading these

surfaces or they start degrading the drug itself. So, again you can control that by pre-
adsorbing under proteins and that can somewhat either delay or at least completely

abrogate any of this process from happening.

(Refer Slide Time: 11:15)

So, let us talk about some kinetics of protein adsorption. So, I initially said in the
introduction of this class that the first thing that the implant is going to interact with is
water because that is the most abundant fluid around and then the next is going to be
proteins and the cells are going to interact through that protein. So, why is that and then
the reason for that is that the protein adsorption is a very rapid phenomenon. So, the only
thing that limits protein adsorption is the diffusion of the protein from the fluid to the
surface of your implant. So, what essentially that means, is if I am putting an implant and
I am looking at the implant after a few seconds or few minutes that implant at this point,
I believe, would be completely coated with your proteins that are present in the
surrounding media.
So, let us say this is your surrounding media, you have proteins. The only limitation of
protein adsorption is essentially the diffusion of this protein to reach the surface. Once it
reaches the surface we are talking about less than milliseconds for these proteins to
adsorb onto the surface. And then what will happen is once proteins have come in and
adsorbed, the initial layer of the protein, then let us say another protein is now trying to
come - this protein has no space to directly interact with this particular surface it can start
interacting with the proteins that are coated on the surface because these proteins may
also have some domains which are now getting exposed which was not earlier present,
but then this particular protein which is arriving late or the slower phase will have to
either remove these proteins from the surface or will not be able to interact the surface

directly and we will have to interact with the already coated layer. So, they will find the
empty slot maybe these proteins are large and there is a small protein that can diffuse
into these empty slots.
So, those will be able to go in, but then eventually all of the surface will be covered
fairly rapidly and it will be very difficult for any further protein to come and interact
with it.
(Refer Slide Time: 13:29)

So, then there are several models that are being used to sort of study this protein
adsorption. So, one is a monolayer model which is a fairly simple model and what it is it
assumes with the protein adsorption is limited to a monolayer. So, what it is saying is let
us saying is- if I have a surface. So, what this monolayer model assumes is let us say if
there are proteins in the vicinity these proteins will adsorb onto the surface and if there is
another protein that needs to come in let us say I have another protein that wants to
come and adsorb on the surface, it cannot come and adsorb on top of the surface.
So, this is no and the only thing that can happen is this comes in it, removes one of the
protein unit and then goes and start to interact with that empty space that is being
created. So, that is a simple monolayer model and if you go by this what you are saying
is you have let us say a certain protein concentration being adsorbed. So, as you increase
the protein concentration the amount of protein adsorbed is going to increase and we will

come and discuss as to why this will increase and why this will not be constant and just
bear with me for a couple of slides.
(Refer Slide Time: 14:58)

Then there is another concept which is hard and soft proteins. So, what does that mean
so; that means, that some of the proteins could be fairly hard meaning they are not very
flexible. So, some of these examples are given here ribonuclease, lysozyme and other
proteins they are considered to be very hard. So, they have very high internal stability
which means that they will not really change the structure a whole lot they will still
interact with your surface, but the structure of the protein will still remain and it really is
not going to change a whole lot from what the initial structure was. And then the other
one is the soft protein and which ,as the name suggests, is nothing, but their very low
internal stability which means that if they find a new surface the structure can change
quite a lot depending on the contribution of the surface hydrophobicity and
hydrophilicity.
So, some of those examples are IgG, beta-casein, haemoglobin and several other
examples actually most protein will find are soft proteins and their structures will easily
change.

(Refer Slide Time: 16:01)

So, next thing that I was initially talking about was this protein orientation and what
essentially; that means, is this a few examples here. So, you can have a globular protein
essentially meaning some sort of a big large protein with a spherical like structure that
comes and there is some change in the structure of this as you can see these spheres are
now become more like ellipse and it coats on a surface the other model could be -
depending on the concentration that you have used, let us say this ellipse shape protein
comes in you can have a configuration where very little amount of protein as adsorbed,
but still covers the surface compared to an orientation where a lot of protein has adsorbed
and onto the surface. So, what you typically find in the literature is and during the
experiments is that this keeps on varying depending on the concentration you are using.
So, let us say if I have a surface and I come with one microgram per ml concentration of
a protein and so what will happen is since I am talking about very low concentrations of
protein in the solution it is sort of diffusion limitation as to how much protein can come
in and adsorb on the surface. What you will find is let us say I have a protein which has
this 8 structure. So, it will come it will sort of start interacting with it and what will
happen over time is it will start to expand on it because it can find more space. So, this 8
structure gets elongated and elongated.
Hence the structure has changed quite a bit plus a single protein is occupied quite a large
space. Now consider a case with the same example where let us instead of 1 microgram

per ml I come in with 100 microgram per ml, now on this particular surface. So, now,
what will happen now I have a lot more protein concentration in the surroundings. So,
the fusion will not be as limited. So, the proteins will come fairly quickly.
So, let us say a protein gets adsorbed. It is able to change the surface a bit because it still
has some more space to which it can absorb to, but by the time it goes to this
configuration all the rest of the sites are occupied because th quite a high concentration
of protein that is present in the vicinity. So, all of them are coming in and essentially
occupying the surface. It cannot really expand do something like this state that is not
happening and the same thing will happen if I go at a even higher concentration.
So, what you will find is not only the protein adsorption is dependent on the type of
proteins that are present in the surrounding, it is also dependent on the concentration,
because the concentration can change the orientation of the protein orientation as well as
the structure of the protein. So, if you look at this structure this is different than the
structure which is here, even though you use the same protein to start with, but at
different concentrations. And then now if I go back to two slides earlier what I was
saying. So, let us say if I now have a protein at a concentration of 0.5 mg per ml.
So, the amount of protein that is going to absorb is going to be different than let us say at
2 mg per ml or 3 mg per ml and the reason for that is the proteins that are coming to
adsorb on the surface have time to change their orientation and still occupy further space.
So, you will have a scenario where what you will get is essentially at lower
concentrations you have quite a little protein on a surface whereas, at a higher
concentration you may have quite a bit of protein that is getting adsorbed because there
is enough protein in the vicinity and there is not enough time for the protein to change it
is orientation.
So, that is why you see a curve like this even though it is the same protein ,fibrinogen,
that was given to the surface, but depending on the initial concentration you get this sort
of increase and then once you reach a saturation concentration where diffusion is no
longer a limitation there is enough molecules in the vicinity that will completely saturate
the surface, then it does not matter. So, in this particular case this is shown to be more at
around 2 mg per ml going to it different for each protein that you are handling with, but

all proteins will typically show a structure like this and this of course, is a assumption
with a monolayer model and we will talk about other models as well in this case.
(Refer Slide Time: 21:04)

So, what about the effect of wettability? So, the general trends if you assume this model
about protein adsorption is that proteins will adsorb quite tightly and strongly to
hydrophobic surfaces and that is very obvious. Because let us say if I have a surface
which is fairly hydrophobic then the proteins can change the structure quite a bit on this.

Let us say this is a protein that has come and started interacting and there is lots of van-
der waals interactions that is going to happen between the protein and the surface and

there is really no competing force for this hydrophobic surface because everywhere in
the surrounding the fluid contains water and that is hydrophilic.
So, these hydrophobic domains will be very happy interacting with this hydrophobic
surface and they do not really want to change whatever is the structure because the
surrounding is hydrophilic. So, typically what is seen is that very tight and strong
interaction of proteins with hydrophobic surfaces. The conformation and the extent of
denaturation will also depend on the water wettability again first of all it will depend
whether the protein is soft or hard.
So, typically hard proteins will not change the structure, the soft proteins will change the
structure, but quite a bit and then the more hydrophobic is the surface the more the
change in structure and then again the reason for that is very similar because earlier the

proteins were situated in water and all they are hydrophilic domains were outside
whereas, to interact with the hydrophobic surface their structure almost has to go to a
complete overhaul where all the inner hydrophobic domains are going to come out and
start extracting interacting with the surface.
So, you will see that quite a bit of change that will happen in this structure. And then
again as I said it is very energetically favorable for them to displace water from the
surface point of view as well right the surface also does not really want to interact with
the water. So, surface is also very happy when it comes in contact with these
hydrophobic domains. So, we will stop right here in this lecture and we will continue
further in the next class.