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Hello, everyone. Welcome to another lecture for the Drug Delivery Engineering and
Principles. So, let us just do a quick recap of what we learned in the last class.
(Refer Slide Time: 00:38)

So, in the last class we talked about various particle properties; we had talked about size
and shape before this and then we continued the discussion and further talked about
charge. So, we said that typically positively charged particles are better for cell uptake,
but if we want longer circulation and in-vivo applications, we want neutral or slightly
negatively charged particles.
And what are the reasons for this? Of course, that the cell membrane itself is slightly
negatively charged. So, there is a electrostatic interaction and that is why it will get
closer to the cells for it to uptake it whereas, in circulation you have several serum
proteins which are also negatively charged. So, because of that these serum proteins will
adsorb onto your particles and will not let them flow longer because they might get
recognized by some other cell.

Then we talked about elasticity and in general what we said elasticity that low modulus
is good for low modulus circulation. We gave several examples of how to make these
low modulus particles somewhat most of them trying to mimic RBC and then the reason
we say that these low modulus is good is because spleen will clear any rigid particles if
they are big enough. If they are above 200, 250 nanometer then spleen will be able to just
clear these up. But, if they are they have low modulus they can squeeze through those
spleen gaps and vessels and then they can continue to circulate longer.
Then we talked about metal particles. In this case we talked about both synthesis and
applications. So, applications we are mainly talking about imaging or contrast agent we
also talked about photo thermal therapy and then you can even conjugate the drugs on
the surface. So, essentially drug delivery can also be done and then finally, we talked
another sort of sidetrack about how you can use particles both particle hitch hiking. So,
you can conjugate it to RBC’s or you can conjugate it to other immune cells like T-cells
and B-cells and because of that the body will not be able to recognize them and they can
circulate or reach wherever these particular cells are growing.
So, this sort of concludes our major particle discussion for this course. We are going to
come back to it in future classes and for different type of applications, but this was just a
basic concept I wanted to give you on particles. Now, we are moving into tissue
engineering and learn how various aspects of these polymers and these drugs can be used
for better drug delivery for good tissue engineering and both of these things go hand and
hand in - all tissues in application requires some sort of drug to be given for better
efficacy and better retention of the tissue, better healing of the tissue.
So, let us talk about some concepts of tissue engineering, but before we do that we are
going to talk about protein adsorption which is again an integral part of tissue
engineering. So, that is what this class is going to be about. This is going to be about
protein adsorption.

(Refer Slide Time: 05:02)

So, before we talk about adsorption let us talk about what is the surface and what is an
interface? So, any outermost region of a material so, if let us say if I have this material.
So, any material will have a bulk region and a surface region. So, when I say surface, the
surface is a region which is going to be slightly different from the bulk because it is
going to be exposed to different environment than the bulk- like if I take a bulk volume
here, all sides of this bulk volume is exposed to a very same environment. But, as you
get closer and closer to the surface, that is not true.
So, that is how you can distinguish between a surface and a bulk that the outermost
region of the material will be chemically or energetically different from the rest of the
bulk just because it is at the boundary. So, the interface, this could be water outside, this
could be air outside or this could be some other medium outside but, the surface will be
slightly different from the bulk.
Then what is the adsorption? So, adsorption is nothing, but it is formally defined as a
partitioning of a chemical species between a bulk phase and an interface. So, let us say
now I have this solid and let us say some air or some liquid here. So, how does this air or
this liquid or any molecule in that air or that liquid partitions between the air and the
solid at this interface?
So, for most purposes let us say we are talking about a very thin layer here. So, air in the
surrounding area will be fairly uniform. The solid below the surface will be fairly

uniform, but right at the interface the air may like to sit or attached to the solid interface
and that will create some sort of a difference in the gradient of the air it could be higher
or it would be lower than the outside, but this will change and so, this physical
adsorption or this physical partitioning of a chemical species between the bulk and the
interface is termed as adsorption.
(Refer Slide Time: 07:23)

So, it should not be confused with absorptions remember the only difference is this one
word here you have d and here the absorption is b. So, absorption is a bulk phenomenon.
So, when I say adsorption that is surface whereas, absorption is a bulk phenomena. And
both these phenomena are actually relevant to biomaterials and both of these will be
widely used as we go along and so, let us quickly give an example.
So, let us say when I have a dry crosslinked polymer network. So, let us say a hydrogel
for example; so, I told you right that there is a hydrogel which will have a certain
network and where you have a very high tendency to absorb water. So, what that
essentially means is the water is going to go throughout this network and will cause
swelling of this network. So, this is absorption of water.
Whereas, the other example is proteins will adsorb to the biomaterial surface. Let us say
if I have a material which is solid in which the water cannot pass through and the outside
water has proteins, the proteins will tend to aggregate on the surface and we will come to

the reason as to why they do that, but this is the property of only the surface. So, this is
why it is called adsorption.
(Refer Slide Time: 08:53)

So, let us talk about a surfactant as well which is going to be important in this discussion.
So, surfactants are usually compounds that are amphiphilic we basically talked about
surfactants and we talked about micelles; essentially nothing, but they have a polar head
group and a hydrophobic tail.
Some of the common examples of surfactants are detergents and soaps and what they are
is essentially hydrophobic tails with hydrophilic head groups as is also shown here and
they have a certain solubility in a aqueous solvent, but if you increase the amount that is
present in, let us say, water, eventually they will start to precipitate out and form these
micelle structure. So, that is why; that is why these soaps and detergents are very good in
terms of cleaning out the dirt from your clothes. Let us say if you have a cloth and which
has let us say some dirt which the dirt can be both hydrophobic or hydrophilic.
So, if you only use let us say water or molecules which are hydrophilic, it will only be
able to dissolve any sort of drug contaminant that is hydrophilic, but the hydrophobic
sort of impurities or the dust will still remain stuck to your clothes. But, if you have a
detergent which is both hydrophilic and hydrophobic, then it will go and solubilize both
parts of the dirt and that is why they clean the clothes lot better than the individual

components. So, essentially that is a surfactant that you use in your washing machines in
at the time of washing your clothes and all.
(Refer Slide Time: 10:40)

So, now we have that concept clear. Let us talk about proteins itself, since we are going
to talk about protein adsorption predominantly. So, proteins again composed of amino
acids they are nothing, but poly amino acids. So, as you can see here, several amino
acids are being conjugated to each other. They have an N-terminus which is basically
means the final protein which will have a primary amine at the end and a C-terminus
which is the final protein which will have a -COOH at the end. And so, this is a primary
structure which is basically all opened up.
You can have a secondary structure which means that these proteins or these amino acids
will self align into some sort of a complex structure which may not be linear, it could be
beta sheets, it could be alpha helix and these can then further self align into tertiary and
quaternary structure which becomes more and more complex.
So, we know that all amino acids are non polar,polar and anionic. There are all kinds of
amino acids as well as they are neutral chains as well and so, all of these properties are
present in proteins. So, the proteins have nonpolar areas, they have polar areas, they have
ionic and their neutral chains. So, a non-polar is essentially nothing, but somewhat
related here, and their structures can also be primary, secondary, tertiary and quaternary.

And again all of these can have multiple configurations depending on the environment
the protein is the quaternary structure can have infinite combinations of structure. So, all
of this adds to the complexity of these proteins.
(Refer Slide Time: 12:16)

So, when we say it will talk about protein folding we are saying that for the water soluble
protein, the folding is essentially driven by what is the medium in the surrounding. So, if
it is water, it wants to minimize the hydrophobic interaction with the water. So, what will
happen is let us say I have this protein. A long protein, in this case I am drawing a single
chain, spreading it out. Let us say this is my protein where this is the hydrophobic
domain and then these are all hydrophilic domains. So, this is hydrophobic, this is
hydrophobic, this is hydrophobic and then all of these are hydrophilic.
So, now if I put this protein in water what will happen is the water will love to interact
with the hydrophilic domain. So, it will go and start interacting with it will start
accumulating near and the hydrophilic domain, but then this green domain will not want
to interact with the water at all right because this is hydrophobic. So, it does not really
like water. So, what will happen is then this green body will start to self assemble.
So, eventually what will happen is you will have the structure where all the green
domains will tend to interact with each other because they do not really have anything
else to interact to, whereas, all the red domains which are hydrophilic domains will tend
to be away from the green domains as well as start to interact with water. So, I mean this

is one of the very simplest cases of protein folding that I have just described here. The

protein folding is much much more complex because as I said they have all kinds of non-
polar, polar, charged all kinds of moieties and hydrogen bonding being present.

So, all of this will play a role and will result in a structure which is very complex it is
typically a quaternary structure for any large protein and so, that is what defines the
protein folding. Of course, this is the protein folding in water, but if you change the
environment in the surrounding then the structure will change right. I mean if the same
folding was to happen in let us say an organic solvent. Let us say hexane- now hexane
wants to interact with these hydrophobic domains, but does not want to interact with the
hydrophilic domains.
So, what will essentially happen is again all the red regions will collapse inside and then
the green regions will be on the outside basically making sure that they are shielding all
the red domains which are hydrophilic and not liking hexane. So, now you can see that
the structure is completely changed. So, depending on the environment in which the
protein folding is going to happen you will see these effects where the protein structure
will change. So, it is fairly dynamic and it is actually very very sensitive to even just
small perturbation to the local environment.
You can change the amount of salt in the liquid and that will change the protein
structure. You can change the location of the protein from one part of the body to the
other and that will slightly change the protein structures all of this is very sensitive to its
environment.

(Refer Slide Time: 16:36)

So, as I said they are trying to minimize the hydrophobic interactions. Hydrophobic
domains folded into a core away from the water and they want to maximize the
hydrophilic interactions - all the polar and charged residues are on the outside.
(Refer Slide Time: 16:48)

So, because of this now what we are essentially saying is that proteins are both
containing a hydrophilic and hydrophobic domain and hence they are weak surfactants.
They do not have a strong hydrophilic and hydrophobic domains, but they have a small
small hydrophobicity and hydrophilicity in their individual amino acids and so, they are

weak surfactants and again because of this there is a relative difference in the
hydrophobicity, but not a very large difference. So, they can then easily change the
structure and adapt to whatever surface or whatever environment they are put in.
(Refer Slide Time: 17:34)

So, hence most bodily fluids which contain several proteins will result in protein
adsorption onto any foreign substance that the body sees. So, we have several types of
protein. For example, a blood contains almost about 400 different proteins at different
concentrations that is flowing through our blood circulation. And so, what will happen is
if I let us say put an implant for example- a pen in my body and then what will happen is
let us say this is an implant. For the purpose of this particular slide let us say that this
implant is non-porous, nothing can penetrate in - it is a solid pen. So, what will happen is
we will first interact with water. So, water is everywhere. So, water will come in contact
with this implant. Now, this water which in this case is serum contains several proteins
which are folded into a certain structure.
So, let us say this implant is hydrophobic maybe let us say a PLGA implant then what
will happen is when this protein comes in contact comes in contact with the surface these
outer domains these are hydrophilic right. So, they want to stay in the water, they do not
really want to interact with the surface and neither does the surface want to interact with
them. So, what will happen is the protein will open up and refold such that the
hydrophobic domains are more than contact with the surface. So, this protein is going to

reopen such that all these regions are hydrophilic and all the regions directly in contact
are hydrophobic.
So, all of this and because of these hydrophobic-hydrophobic interactions there is a
strong bond that is formed or the number of small weak bonds, but there is so many of
them that the whole interaction is very strong, so, this protein absorbs very strongly on
do these surfaces. And once these proteins adsorb, then the cells which are in much
lower quantity than the proteins will come and start sensing the surface and most of these
cells well actually also start to see these proteins that are absorbed. So, most of the time
the mediation of the cell attachment to the implant is being done through these proteins
that are adsorbing through either serum or some other body fluid wherever the implant is
put in.
So, the consequence is the adsorbed protein layer mediates the biological response. So, if
I say that the cell is the main unit that is governing whatever response we are going to
get then the proteins that are adsorbing onto it determines how the cells are going to
come and attach to it, what sort of signals the cells will get and hence essentially define
what sort of biological response the body will give to a certain biomaterial.
(Refer Slide Time: 20:35)

And as I said the proteins may denature on adsorption. So, the protein structure is not
very stable. So, once the environment is change the heating, the chemical agent and they
will all cause denaturation of the structure. So, when protein adsorbs, the interaction with

the solid there is a change in the surrounding chemical environment and this potential
change causes the protein confirmation change as well which you can call denaturation
which essentially means just change from the original structure. This does not
necessarily mean that they are going to completely open up. They just means that
whatever their natural state was, that got denatured.
So, they may take up either a completely open chain or they may have some other
conformation that is not typically found in the nature. And again there are different types
of proteins depending on the composition of the amino acids some structures are more
stable than the others. So, the magnitude of response that you are going to get on a
material for different proteins is also going to be different.
So, some proteins are very liable to denaturation. They will completely open up
everything in the structure, while some proteins are not really that liable in terms of
changing their structure. So, they may still maintain their activity, they may still
maintain the natural structure that was present originally.
(Refer Slide Time: 21:56)

So, how proteins denature on adsorption? We already covered a bit of it, but the
denaturation will depend on biomaterial surface chemistry and water wettability. So, how
much is hydrophilic; how much is hydrophobic; what sort of surface chemistry is there;
what bonds can form between the proteins and the surface all that we will determine it.
So, typically at hydrophilic biomaterial surface, which is rich in charged groups or

charged amino acids, these hydrophilic amino acid rich regions of proteins will
preferentially interact with the surface.
So, if I have let us say two surfaces - one is hydrophilic and another is a hydrophobic and
of course, I am doing this in a water environment which is hydrophilic and I have a
protein structure which is let us say like this. Then in when it comes in contact with the
hydrophilic surface, the protein structure may change a bit, maybe it is going to become
slightly elongated, but more or less the structure is going to be similar. Whereas, when it
comes in contact with the hydrophobic domain, it is going to become completely inside
out.
So, the structure will change quite a lot more compared to a hydrophilic surface just
because originally the protein was in a water environment which is fairly hydrophilic.
So, there is not much of a drastic change that is happening. However, this hydrophilic
surface can have a lot of functional groups that are reactive and that can further cause
changes. So, there is all magnitude and various degree of response that we will get.
(Refer Slide Time: 23:47)

So, typically there is a low denaturation as there are already hydrophilic domains present
outside when you are talking about a hydrophilic surface. That is why typically when
you talk about tissue engineering or talk about implants. The major emphasis is to make
the surface fairly hydrophilic so that you do not start denaturing lots of proteins that may
cause some toxicity.

At the hydrophobic biomaterial as we talked about which is rich in non-polar groups, the
hydrophobic amino acids will tend to preferentially interact with the surface. So, these
hydrophobic domains were initially buried inside the protein structure. So, they will have
to then come out and that is going to cause a lot more change to the protein structure than
let us say a hydrophilic surface.
So, in water-soluble globular proteins hydrophobic amino acids are in the protein core.
Thus, these will try to interact with the hydrophobic surface and change the structure
quite a bit.
(Refer Slide Time: 24:52)

So, again why is all this important? So, we again briefly already we have talked about
this, but if you have a solid substrate, the first thing that is going to interact is the
proteins and they will adsorb on the surface and then when the cell comes it will actually
not be able to see the solid substrate surface right. This surface is all hidden by this layer
of protein.
So, the cells will only be able to interact with whatever is present on the surface which is
in this case protein that is adsorbed and that is going to lead to any biological response it
is going to happen. Of course, the protein that is absorbing is dependent on the surface
itself. So, you can argue that you can sort of control it and anyways, but it is still
becomes very important to study the protein adsorption.

So, cell membrane has receptor proteins, including integrins which bind to several of
these proteins that are found in the serum and that is how they will bind to the surface,
that is how they will attach to it, that is how they will start functioning on that surface
and so, the cells that attach will recognize this biomaterial through these integrin
molecules.
So, in this case what we are talking about is these cells. When they attach to the surface
they have a special class of molecules which are called integrins and most attachment
and spreading of these cells on these surfaces will happen when these integrins bind to
their receptors or their ligands. So, these could be proteins like fibronectin, collagen,
laminin and several others.
So, when these proteins get adsorbed onto the surface only then the cells can go and bind
to the surface before that the cells will not be able to attach to those surfaces and actually
grow. So, as I said any kind of immune response will also be driven by these protein that
are adsorbed. So, everything is sort of controlled by the protein adsorption.
We will stop here and we will continue rest in the next class.
Thank you.