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Hello, everyone. Welcome to another lecture of Drug Delivery Engineering and
Principles. We are currently talking about Hydrogels and we are going to continue that in
this class like the last two classes.
(Refer Slide Time: 00:42)

Let us take a quick recap on what we have learned in the last class. So, we have talked
about hydrogels, we have talked about the physical hydrogels, which essentially we
talked about ionic in this case, then we talked about chemical hydrogels, we talked about
how these are formed. So, essentially what is the major difference between chemical and
physical. Physical are essentially molecular chain entanglements whereas, chemical may
have an actual bond that is present between these polymer chains.
And then we have talked about synthesis process. So, typically synthesis process for
physical hydrogels involves either heating or change in pH or any other trigger that
causes these chains to start interacting with each other and form these gels. Whereas, the
chemical hydrogels, all you have to do is just add the cross linker or somehow activate

the groups; it could be either through light or it could be maybe adding glutaraldehyde or
some other kind of cross linker that causes this trigger to happen.
Then we talked about some pore size calculations. So, very briefly we discussed this. We
are going to discuss this more in future classes and then we talked about drug diffusion
kinetics. So, how can we model, how the drug is going to diffuse. So, we found out that
we can just use the Fick’s law and all we have to really change is the diffusion
coefficient D, which will then be affected by whether the gel is micro porous or macro
porous.
So, this micro porous D does not really have much role to play here. Although there is
some factors that get added up; whereas, if it is micro porous then along with these
factors we add another partition coefficient Kr because these chain these drug molecules
will now start to interact with that as well.
(Refer Slide Time: 02:24)

So, let us talk about in situ cross linking hydrogels. This could be any of those previous
two hydrogels if we talked about, both physical or chemical, but what essentially in situ
means is this that these gels can cross link at the site. So, these are very desirable when
you talk about any kind of delivery because what you can do is you can have individual
components. You can mix those two components together and once you mix those two
components together the trigger is basically given for the polymerization to start and

these things polymerize within a certain amount of time. This could be over a period of
seconds or a period of minutes.
So, why it is desirable? Let us say if I am a clinician, I am a doctor and I am trying to
inject something through basically a syringe and want to have a hydrogel system to kind
of have this drug release over time. So, all I can do is I can mix the two component
together in the syringe and directly inject into the patient and what will happen is in that
time or in that trigger this thing will polymerize and wherever it was inject it will stay
there and sort of form a depot. So, these they call in situ because they form hydrogel
inside the body.
And the polymerization trigger could be several types. So, one trigger is time. So, you all
you have to do is just mix two components together and it may be like as I said few
seconds 10 seconds, 20 seconds or it could be minutes, 2 minutes, 3 minutes and within
that the polymerization will start. It may be temperature right, I mean I may not even
have to do anything in this syringe. So, in syringe is completely stable I do not even have
to hurry up, but I mean temperature let us say 25 degree Celsius, but when I injected into
the body our body temperature is 37 degree Celsius. So, because now there is this change
in temperature something got activated that causes the polymerization, the gelling to
happen and that 25 to 37 degree Celsius trigger is enough that it done polymerizes let us
say within a few seconds. Or it could be pH. Let us say and this could be again several
types. Let us say in my two tubes that I am mixing together, the combined pH of the
polymer here let us say is pH of 5 at which it does not polymerize.
The other tube basically just contains sodium hydroxide let us say with a pH of 8, but
when I mix them together that leads to a pH of 7 and at the pH of 7 this thing starts to
polymerize. So, this could happen it could be that I do not even worry about mixing with
NaOH. I just inject it into the body and a body pH is about 7 anyways and so when this
diffuses in, it can cause the polymerization to happen. So, any of these things can be
explored in if you are talking about in situ gelation.
There are some disadvantages to it. Obviously, there is lots and lots of advantages, but
there is some disadvantages. One is this will diffuse throughout the body right. I mean if
I let us say inject it into a area which is highly fluidic let us say if it is in my stomach or
in my peritoneal cavity or any place which has lots of diffusion and convection going

around then before it can polymerize it can just diffuse throughout the body. I do not
really have a control of shape.
So, that is a big problem because as you know most of these diffusion kinetics will
depend on what is the area through which it is coming out of the system. So, if the area is
compact and the drug is only coming out from this much surface area, you will have a
different release rate where whereas, in case of in situ cross linking what will happen is
once injected into the body, it can take whatever weird shape it flows in before it
polymerizes and now you have much larger surface area and much irregular surface area
through which the drug can start to diffuse out. In this case, it is very hard for me to
model how much drug is going to come out in a particular amount of time.
So, these are some of the challenges. Another advantage here is obviously, if I am
looking to fill a defect; let us say; let us say an injury and some part of my muscle is
gone and I want to fill it with a gel containing some cells, all I have to do is just put some
liquid over it. The liquid will automatically take the shape, let us say if this was my
muscle and there was some accident through which this is the shape that needs to be
filled. Now, I do not even need to worry about designing the shape. What I can do is I
can fill this with the polymer and once it gels it essentially just takes up the structure that
I want.
So, there are some advantages to it, but obviously, it has to be looked at what sort of
applications we are looking at before we can move forward with this. So, again this is
very widely used in research and there is lots of hope for this to get translated into the
clinic. We will talk about one major research paper on this to kind of understand this.
The paper is going to look at different things as well. So, let us get into it.

(Refer Slide Time: 07:34)

So, in this case this is a paper which is using an in situ cross linking, but then this is here
what I am trying to do is basically give you some ideas to how these hydrogels are being
used in the system.
So, in this particular paper what they are doing is they are using a maleimide based cross
linking of a PEG hydrogel to improve the reaction kinetics in the cross linking to happen
for both cell encapsulation as well as in situ delivery right. So, in this case the drug is
actually cells and they want to make a system that they can just inject into the body and
not have to worry about surgeries and all.

(Refer Slide Time: 08:13)

So, in this case the objective of the study is to use a maleimide as an alternative cross
linking moiety to polyethylene glycol and when I say alternative there is try different
types of cross linking methods and see which is very widely used.
So, before this paper came out people typically were using acrylate or let us say some
EDC NHS or some other use some other form of coupling, but now this paper is
proposing using a maleimide base cross linking. So, what they have done is they have
compared the cross linking moieties which is again as I acrylates, diacrylates and vinyl
sulfone which is another functional group.

(Refer Slide Time: 08:57)

So, maleimide as we have discussed in the past is a structure like this in presence of
thiols that this reacts with the sulfhydryl group. So, thiols all will have a sulfhydryl. So,
something like cysteine or any other thiol group might be present due to some other
molecule and this will directly react with this. So, this is again very extensively used in
peptide bioconjugate reactions, but then here they are now proposing it to use it with
hydrogels.
The reaction kinetics is very fast as you will see in the rest of the data in this paper. This
has very high specificity for thiols. So, at physiological pH this is again for most of the
biological applications we are looking at physiological pH. So, this maleimide group has
a very high affinity and specificity for thiols. So, it would not have too many reactions
going around.

(Refer Slide Time: 09:50)

So, let us see what they have done in this paper. So, they used hydrogels. These are again
as I said these are hydrated cross linked polymer networks. They are kind of a synthetic
analog to the extracellular matrices in which the cells typically reside. So, this is a very
similar structure as I told in the last two classes and there is ease of modification and you
can modify them. They will result in some bioactive properties which are independent of
what you are encapsulating in them.
And in this case they have use PEG hydrogel. Again as I said PEG is very widely used.
So, and somewhere reasons we already know that it is very low protein adsorption. It is
actually used in different areas to prevent the protein adsorption from happening. It
minimally causes any inflammation. Although we have talked about some PEG
antibodies weak form, but in general if there is no PEG antibodies it causes very low
inflammation.
Safety of use in-vivo; again it has been used very widely for last few decades and there
lots of commercially available products that have used different functionalities to
incorporate into them. So, all of these are a plus to this.

(Refer Slide Time: 11:08)

So, here are some of the methods that they have used. As I said these maleimides will
actually react with thiols to form this thioether bond and what they have done is they
have purchased the PEG commercially available which has the four acrylate groups, it is
a 4-arm PEG. So, here is the cross link between the four arms of the PEG. So, this is arm
1, arm 2, arm 3, arm 4 and each of these arms contains a maleimide group at the end. So,
and then instead of maleimide they also have done similar modifications and I have got
an acrylate and vinyl sulfone which are two of the groups that were used quite a bit at the
time when this paper was published.
And then again this reaction happens at a pH of 7.4. TEA is kind of a catalyst to this and
so for acrylate the polymerization happens on the basis of a UV cross linking and the
vinyl sulfone also reacts with thiols to form bonds.

(Refer Slide Time: 12:09)

And so, that is what they have done they have done reactions. The reaction that they
have done is they have a PEG macromer, they put an adhesive ligand. Why is this
required? What do you mean by adhesive ligand? This is a ligand that reacts or that
actually interacts with cells and causes the cell to adhere to it. As we said that PEG does
not allow any adherence. So, they have put some adhesive ligands on here that will result
in the PEG to be cell compatible. The cells can now bind to it.
And so, once they do that, they get a structure like this, where let us say at whatever ratio
they are making let us say they are putting this in a ratio of 1 is to 1, then each of these
PEG macromer will result in on an average will have one of this adhesive ligand and
then they have another peptide cross linker. So, this is kind of a cross linker which
contains thiols. So, this one also contains thiol, but it is only a single thiol. So, it can
react once, but it would not form a gel, but in this case now they are talking about a
peptide which contains thiol at both ends.
So, now it can essentially cross link in a whole network like this. And they have used
different conditions they have used the adhesive peptide at 4 milli molar or 400
millimolar, they have used for different times up to an hour and then the cross linking is
done by a peptide which is also cleavable which is a protease cleavable, so, that means,
the cells can secrete proteases. So, let us say the cell wants to move around and it is
entrapped in this giant mesh, so, if a cell is here and it wants to move out from here it can

just secrete some protease, degrade this and this will open up and then the cell can move
out.
And, so, all of this resulted in this hydrogel structure as you can see here it is they have
formed it in a disk shape. So, it just took the shape of the disk that they formed in and it
resulted in hydrogel.
(Refer Slide Time: 14:10)

Let us talk about some of the cross linking chemistry that they have used. So, as I said
the acrylate is radical polymerization. So, if you have some UV based methods, some
photo initiator is used, that will result in polymerization to happen. Remember the
drawback here is of course, the UV itself is toxic to cells and the photo initiator is also
toxic to cell. So, you can actually have reduced cell viability there. So, that is a problem
that was identified with acrylate so, quite a bit of time.
And it is not really very conducive to do an in situ delivery and we will talk about that it
does not really happen very fast. The reaction is very slow and in the reaction is slow and
if you inject it, let us say it is if it takes 30 minutes and then 30 minutes all your hydrogel
components will get just diffused out into your body and it really would not form a depot
at the site you are injecting it at.

And then the other reaction is the Michael type addition and so, this is basically a
Michael reaction where a nucleophilic addition happens on another nucleophile. So, this
is what they have used to react the maleimide to the thiols.
(Refer Slide Time: 15:27)

So, again, as we said they have incorporated cell adhesive ligand so, one of the ligands
have been incorporated is this RGD molecule. This is a molecule that is very widely
found in fibronectin or vitronectin and other ECM molecules through which these cells
have ligands and when they find RGD in a polymer chain they can just bind to it and
kind of tether themselves from there. So, this is what they have used.
They have also modified this to also contain a thiol so that there is one sulfydryl group
present through which it can react and then the RGD group is still available for the cells
to bind to. So, that is what they have done. So, they used 20 kDa 4-arm PEG with thiol
containing adhesive peptide. And then in a step two, what they have done is the
degradable peptide is this. In this case this is the whole peptide and this VPM site is
where the cells will cleave it.
So, when the protease are in the solution they will cleave this VPM peptide, once you see
it and then again they modified it with two cysteines each containing a thiol and so, again
what that does is that allows you to sort of have a control on the system where now you
can have polymerization happening from this as well as its degradable. So, I hope this is
clear.

So, now, you are talking about let us say this is our 4-arm chain one is containing RGD
that is capable of binding to a cell and the other arms are then cross linked through this
VPM. So, if I use another color. So, now, you have a chain containing VPM from the
rest of them which will then be bonded to another 4-arm PEG. So, that is how the whole
structure is formed. So, this is zoomed in to one of these and that is how it is. So, now,
the cells can actually degrade this VPM moieties to move around as well as bind to this
RGD structure.
(Refer Slide Time: 18:01)

So, here you go, it is just the same thing that I just draw. So, you first react it at a 1 is to
1 ratio. So, you essentially get right functionalized with RGD and then you mix this with
the ratio let us say 1 is to 3. So, now, you have three sites on which all the VPM gets
bonded as well it starts cross linking throughout the network.

(Refer Slide Time: 18:29)

So, now let us look at what the data they achieve from this. So, first of all, the PEG-
4MAL shows higher RGD and VPM incorporation. So, this reaction is more efficient

that is what they are trying to propose. So, let us look at that. So, in cases of PEG-4MAL
what they see is even if they have low amount of the TEA which is a catalyst here, even
in 10 minutes you almost get all the reactions. So, on the y-axis you have the unreacted
thiol. So, they measure how much of the unreacted thiol is present. So, if they were
stoichiometrically mixed, so assuming that all the thiol should have been finished, that is
what they see that almost they get 100 percent thiol conversion and the same thing
happens at 60 minutes. Whereas, if you look at look at PEG vinyl sulfhone or PEG
acrylate at least for the concentrations of TEA for 10 and 60 minutes you only see very
little incorporation. So, I mean you are talking about only 40 percent or in this case case
very little.
If now you increase the TEA concentration which is again a catalyst here, you get to 100
times. So, I mean this is essentially we are talking about 100 times increasing the catalyst
concentration even then you are seeing not a very good incorporation only after quite a
bit of time you start seeing almost 100 percent reaction and the same thing applies with a
PEG-4-acrylate. So, clearly shows that this reaction chemistry is with much more
efficient to continue with this particular system.

(Refer Slide Time: 20:07)

Then, what is the different polymer concentrations we can use to form this hydrogel. So,
what is the lowest possible weight? So, and if you continue to reduce the polymer chains

you may not get a hydrogel at a certain point of time. So, they found that with the PEG-
4MAL even a 3 percent gel can be formed whereas, with acrylate you can only get a gel

at a minimum of 7.5 percentage of this polymer and with PEG-vinyl-sulfate you get at 4
percent and then the PEG-DA is also forming at least at 7.5 percent.
And the gelation times are also very different. So, the PEG-4MAL forms within 1 to 5
minutes whereas, the other takes up to 30 minutes and longer durations. So as I said, they
are not very conducive for in-situ gelation because if it is taking 60 minutes to form a
gel, then by the time it is injected in 60 minutes all the PEG polymer will be all dispersed
throughout the body. So, and not only that if you are encapsulating cells and if you let us
say doing it in a cell culture dish what will happen you took gravity the cells are now
settling.
And, so, if let us say if this is your gel 3D structure. Let us say this is your gel 3D
structure. All your cells will be kind of populated at the base and the rest of the gel will
have a very sparse population because of the gravity these cells are settling. So, that is
why you will not have a very good distribution of the cells either if the gelation time is
not fast.

(Refer Slide Time: 21:51)

Then they went ahead and looked at the hydrogel swelling ratio. So, what they found is
that the PEG-4MAL had a very high swelling at lower percentage. So, this was the 3
percentage which is the minimum they can get and as you increase the polymer
concentration the swelling decreases. Whereas, in other cases, you do not really have
much swelling happening because you are requiring quite a high percentages.
(Refer Slide Time: 22:24)

And so, then they went ahead and encapsulated some cells for 3 days and here is the data
for that. So, what are we looking at now is on this row direction, you are seeing each

condition. And so, if you can see, so, the bottommost is a PEG-4MAL and on this
column you are seeing essentially what percentage gel was used. So, this goes from 10 to
4 percent and what do you see here is that the PEG-DA does not really form gel at the 5
and the 4 percentage as was shown before like the same case with the PEG-4A.
And the PEG vinyl sulfone does form gels, but if you look at all these cells, they all look
fairly rounded. The only time you are seeing a little bit of an elongation is on this one,
but all the rest of them they look fairly rounded; that means, when the cells are out, they
mean they cannot really tether to something. So, when the cells are tethering they start
stretching, they start elongating and spreading onto the surface or onto a gel, but in this
case you do not see that.
Whereas, if you look at PEG-4MAL at higher concentrations, yes, the cells are sort of
rounded, but as you are decreasing the concentration of the polymer at 4 and 5 percent
you see them very well elongated. This is this is how a typical ECM matrix will look
like. This is a collagen gel which is the natural ECM matrix that we find in our body and
so, this is how the cells typically look. And then the only two conditions that are able to
mimic that are these two. So, the rest of them are not even able to do any kind of
mimicking like collagen.
So, the stains here used are essentially Calcein AM, which is a dye that is permeable
through the cell membrane and it goes in. And once it is there it is reduced inside the
system to a byproduct which then has a fluorescence. So, only the live cells fluoresce.
And then you have a non viable stain which is a propidium iodide, which is a dye that is
unable to diffuse into the cells are alive, but the membrane is compromised. It diffuses in
and binds to the DNA and then gives the fluorescent red. So, you can see which one are
live and dead. So, all of these are most of them are showing green so, which means that
they are live cells.

(Refer Slide Time: 24:42)

Then, they further went ahead and did some metabolic activity. So, they found that
metabolic activity is high in maleimides. So, here you have maleimides in the red and all
of them you see that compared to a 2D control, so, in these are all compared to a 2D
control in which there is no gel, you find that they are fairly well metabolically active
whereas, the metabolic activity decreases in other conditions and this is again just a
collagen control. Collagen of course, is a higher metabolic activity for these cells.
These are C2C12 which are essentially some muscle myoblast cells and you see that
even though the activity is not as good as collagen, but compared to the other synthetic
polymer chemistries, they have a much better metabolic activity.

(Refer Slide Time: 25:31)

And then let us see what happens when you apply this in-vivo. So, what these authors
have done is they then put it on a mouse heart; so, on a live animal. So, this is what
happens. So, when you just dispense the little bit of liquid onto these mouse heart the
beating heart. What you see is then the liquid was then spread for a little bit, but then it
started polymerizing. So, you get a polymerized gel taking the shape of the external
surface of the cardiac wall.
And not only that what the authors have done they have encapsulated some FITC in here
so, which fluorescence green. So, this is all gel, this is all tissue, the cardiac tissue in this
case and what do you see is there is some sort of an interface. So, the gel is was actually
able to diffuse in and polymerize and then the cells were also able to move in here. So,
what do you kind of see is an overlapping region. So, you do have this overlapping
region in which these cells are interacting with the tissue and this is a very compatible
surface.
So, that is basically all on the in situ hydrogels for this class. We will continue further
with a hydrogel discussion and some more computation of the network pore size and all
in the future class.
Thank you.