Video:

Hello everyone, welcome to another lecture for Drug Delivery Engineering and
Principles, let us talk about what we have been discussing so far. So, in this course we
have discussed quite a lot of things by now, more than half of the course is finished. We
have talked about normal pharmacokinetics of the drugs that are currently used in clinics.
So, how these things travel through the body, how do the clinicians decide what dose to
give and different sort of parameters for the free drug. Then we talked about that we
would actually not want the pharmacokinetics to be what it is currently, but to improve it
further.
So, that the patient compliance as well as the patient comfort is increased, because if you
look at the current drug delivery field, what we are doing we are basically giving tablets
every 6 hours - 12 hours which is not ideal for a patient and so we talked about why not
we have something that we give only once and hopefully that is going to help the patient
not take any more tablets for another week.
So, then we discuss several ways we can do this, some of them are actually being used in
clinics. So, we discussed about polymer drug conjugates we discussed about, various
encapsulation strategies in various kinds of matrices, various scaffolds and we talked
about why not we make them nano, so that we do not have to do a surgery. So, all of that
we discussed and then now we were discussing the tissue engineering, protein adsorption
part of it which we finally, finished in the last class. So, just quick recap what we
finished in the last class before we move on to the next module.

(Refer Slide Time: 01:59)

So, in the last class we talked about drug delivery in tissue engineering and the major
part of tissue engineering involves some kind of delivery this could be either small
molecules or this could be some biologic large molecule. So, this could be proteins and
DNA, could be lipids as well, and then it could also be even cells. So, proteins would be
growth factors, DNA could be a gene of interest that you are trying to deliver and all of
those things.
So, basically most of the tissue engineering is related and goes hand in hand with drug
delivery. And so again in this we talked about various strategies to release small
molecules or large molecules through matrices and one thing we talked about is particles
in scaffolds.
So, let us say if I want to deliver two proteins, protein A and protein B, but then I want to
make sure that there is certain kinetics of protein A which is different from the protein B
how do I do that, because otherwise if I just put it in my scaffold, both A and B, then
what will happen is as this matrix will degrade slowly and slowly each and every of
these molecules will start to come out, but then what if I want A to come out faster and
first and then followed by B?
So, this could be desirable in some of the cases because in some of the cases may be
growth factor A acts first and then only when the cells have reached the certain stage

then the growth factor B becomes useful. So, to do that what we talked about is we can
make a system in which you can essentially put A in the matrix of your scaffold.
(Refer Slide Time: 03:59)

And then you can take another polymer make particles out of it and put B in there and so
what will happen is as this outside matrices will degrade the A will release out and then
only when the water is able to access B, or maybe the B degrades much slower than the
polymer outside, only then the B will be able to come out.
So, this way you can tune, so that let us say if I have to plot release rate from the time of
implantation maybe the A will come out at a certain rate whereas, in this case the B will
be minimal at the start and only when a certain amount of polymer is degraded only then
it will come out.
So, essentially if I overlap with this, so this is for A and if I overlap with this, I will have
a more of a release of B closer to like this. So, that way I can get sequential delivery
because A is mostly coming out first, its doing whatever it needs to do maybe getting the
cells ready to a stage where B can start acting and its very efficient at that point and then
the B is starting to release slowly.
And this is again I talked about 2 proteins, but you can envision a system that can have
10 proteins, 20 proteins and you can just encapsulate different kinds of things in different
types of polymers that have different degradation rates and you can even, in this system,

this a does not need to be outside you can also have A in a second polymer. So, all of
that, so this gives a lots of power and tool to play around with to get what you want to
achieve.
And then another thing we talked about was major differences between natural and
synthetic hydrogels for tissue engineering, in this particular case we took example of a
collagen scaffold and a PEG hydrogel. And what we learn here that both of them have
their own advantages and disadvantages PEG gives you a lot more control in terms of the
properties, collagen is more mimicking of what the cell typically sees in the in vivo
environment and there are certain properties. It is already bioactive, because the cells
know how to deal with collagen.
It can manoeuvre, it can secrete more of it, can degrade it, so all of that is something that
the cell knows how to handle; whereas, with the PEG hydrogel that is fairly alien to the
cell, but then at the same time PEG hydrogel gives you a lot more control on let us say
the mechanical properties, getting large batches of the PEG which are fairly uniform and
all of this. So, there are certain advantages and disadvantages to both of these systems.
(Refer Slide Time: 06:53)

So, today we are going to discuss another topic called implant associated infections. So,
what essentially I mean by this is, you have now started putting foreign entities into the
body, but then you have to make sure that these scaffold these implants and you are
putting in the body are actually sterile and they do not start causing infections. So, this is

a big challenge the field is facing these days where quite a lot of time after something
that you have implanted, in a weeks time, you realize that this actually has some
contamination, has some infection with it. And what that means is, let us say, if I put a
rod for my bone fracture. But then this rod could end up having bacteria colonizing on
the surface and once that has happened the body is not going to heal because this is
something that the immune system is going to continuously keep on attacking. So,
immune system is going to keep on attacking, the healing process will not happen just
because the environment is not conducive to it and eventually the patient will have to go
back to the clinic, have to get this implant surgically removed and then get another
implant and make sure that the area is now completely cleared of all this bacterial
infection that has started at the site.
So, it is a very painful and a long process as well as extremely expensive and not to
mention it basically worsens the disease that the patient was suffering with. We will talk
about various aspects of these implant associated infection as we go along.
(Refer Slide Time: 08:35)

So, first few definitions, so let us start with biofouling and so what is biofouling? This is
a phenomena in which a protein or cellular or any kind of biological things attach to
medical surfaces and so as I said in protein adsorption we had talked about this quite a
lot.

So, if I have an implant and I put it in the body fluid what typically happens is the first
step? The first step is the protein starts to coat over it. And so this is essentially nothing,
but biofouling where this surface is now being fouled by the bio molecules. This could
be good, this could be bad, depends on their application, but this phenomena is
biofouling and then the cells can come in and that is also a part of biofouling as well.
And sometimes this could lead to loss of the device function.
So, if it is a thin layer usually it is not a big issue, but let us say if this layer of protein in
cells gets coated to a quite a thick layer around it, quite a thick as well as compact layer
around it, then what will happen? Let us say this was a glucose sensor.
So, now, that this glucose sensor is completely covered with a thick layer what will
happen is, it required glucose to come and get sensed at the tip of this implant, this
glucose now has a lot of diffusion problems because there is a thick layer of this protein
and cell that is fouling the surface. And the diffusion of the glucose is going to be very
different, so the parameters you optimized this glucose sensor to sense are going to be
very different. So, maybe the readings are going to get from this glucose sensor is going
to be completely wrong. So, this is the problem in such cases with biofouling.
Another term that is very widely used is biofilms. So, biofilms is one of the type of
material that fouls implant that you are putting in. So, what are biofilms? Biofilms are
colonies of some microorganisms typically bacteria, that are formed on the surfaces this
could be several micron thick it could be anywhere between 2 to 100 organism thick or
even larger for that matter. And this is composed of the live microorganism as well as
other material that these things secrete and essentially form a thick slime layer over it.
So, it is very similar to what I have drawn here, but now in this case this is essentially
formed by some kind of a microorganism which is also embedded in this. And its
essentially doing the same function, if it is something foreign that has established on this
implant, then our body is not going to accept it is going to continuously try to inflame the
area try to reject this particular organism that has shown up.
So, that is a big problem with biofilms these days it is one of the very dreaded topic in
terms of problems in clinics and patients and so lots of impedance has been given to try
to avoid formation of bacterial infections in biofilms.

(Refer Slide Time: 12:11)

So, having now discussed these two terms, let us discuss what is implant associated
infections. So, like all materials biomaterials can provide a conducive surface for
adhesion and colonization of microorganisms. Basically I mean you will find that these
microorganisms are very well adapted to grow on any kind of surfaces, you will even
find them on your walls, on your objects which are completely not relevant to materials.
So, these things are very conducive to grow in any extreme environment, on extreme
surfaces and the same thing also applies with biomaterials, where once they find that
surface, they are able to colonize it. So, typically the hydrophobic surfaces, the charged
surfaces, the functional groups that are present give a binding site for these bacteria.
So, I mean, if the surface is very inert, you will see less likelihood of that surface getting
colonized by the bacteria, but if you have any of these properties where they are either
hydrophobic or they are charged or they have some functional groups through which
these bacteria can attach to it, then those surfaces become a lot more conducive for this
implant associated infections.
And again we have discussed throughout this course that all of these are something that
we use for whatever we are trying to achieve in different sort of circumstances like
charged groups and functional groups are something that we deliberately put in. So, that
we can modify our material the way we want it, but then the bacteria also uses those
functionalities to colonize that surface.

This phenomena is actually even more pronounced with polymeric implants or metal
prosthesis less so in drug delivery and the reason for that is most the time when you are
trying to do drug delivery you are looking at, predominantly in major cases, you are
looking at something that is going to be only there in the body for about 3 to 5 days. Its
degrading it really is a very dynamic system for the bacteria to sort of establish itself.
Even though it does happen in that system as well, but it is not as major of a problem as
let us say, in tissue engineering where you are trying to put an implant for life or at least
for a few years and few months.
So, those things, if they get infected then the bacteria has enough time to sort of adapt
itself to its environment and colonize its surface and so you will find that quite a lot in
tissue engineering applications. But again of course, nevertheless there are some
sterilization and surface modifications that you can do to prevent this and to make sure
that this does not occur in any of the patients and so that is why it becomes very
important for any kind of implant related applications including drug delivery and we are
going to discuss them today.
(Refer Slide Time: 15:07)

So, let us talk about how does this infection happen, what is the road that leads to this
infection? So, the first thing is of course, you are putting in an implant, so there is some
tissue injury that happens due to that injury there is some inflammation and the implant
interacts with blood. So, if at the time of surgery let us say the implant was not clean or

the environment was not clean around it and bacteria are able to come and interact with
your body.
So, that is where it all starts at the time of implantation and then it essentially becomes a
race to surface. So, now, earlier I talked to you about when a new material is put in, first
protein comes to it then the mammalian cells come to that particular area, but now we
have another player here which is bacteria. So, now, these three components are sort of
racing to get to the surface first and colonize it. So, whoever wins that race will
essentially have major advantages in repelling anything else that is coming in afterwards.
So, let us say if the bacteria do win this race which happens in few cases then the
bacteria, then adheres to the material surface which is very similar to cell adhesion, I
mean whether its mammalian or bacterial; obviously, the receptors and the mechanisms
are a bit different, but it is essentially talking about a having some sort of bond formed
with the surface either through a protein layer or directly and that causes the bacterial to
adhere to that material surface. Once the bacteria is adhered it can then colonize it can
start to grow, it can aggregate, it can form this bacterial mass which we defined as
biofilm in the previous slide. So, all of that can start to happen, so that is the next step.
And then finally, a more mature biofilm is formed, so this is essentially causing
aggregation of bacteria as well as production of quite a lot of extracellular matrix from
the bacteria such as polysaccharides, agarose is one of those example. And once this
matrix layer and biofilm is formed the bacteria actually becomes quite robust and it is
able to even repel antibiotics.
So, essentially what has been shown in the literature if you have planktonic bacteria
which is basically meaning that the bacteria is free floating in a fluid and if you require a
concentration of X milligram let us say of an antibiotic to kill this bacteria.
Then let us say now this bacteria has colonized the surface, so let us say this bacteria is
here is colonized in an ECM matrix, then the amount of antibiotics you may require to
actually kill this bacteria and stop from growing is could be all the way up to 1000 times
of x.
So, that is how resistant they become and that is how difficult it is because once you start
giving 1000 times a dose you also have to then worry about the toxicity of the antibiotic

to the human body itself and not to mention we have several bacteria that are good to our
body such as gut microbiota, lung microbiota, skin microbiota and all of that suffers with
that heavy dose.
And then once this biofilm is formed, ultimately the device fails, this could be due to
several reasons maybe the device is there as a sensor then essentially the sensing
molecules are not able to reach to their target site. So, we gave an example already of
glucose.
So, in that case we saw that glucose is now having difficulty in permeating through this
layer, could be something else that is sensing maybe its calcium ions, maybe it is a
concentration of a drug, maybe its a concentration of a solute. So, all of that leads to
failure in sort of sensing scenarios and not only that I mean even if let us say this was a
scaffold that you put in to grow a part of your liver and if its infected, then the
environment there changes quite a lot because the body keeps on attacking that system
because the body is not going to accept a bacterial infection.
So, it keeps or attacking, but due to this biofilm formation it is not able to clear it very
well and we can talk about why that happens and eventually this is going to cause failure
of your device because if the liver tissue is not going to grow in that new area, then that
implant is useless. And in fact, not only that, because of the inflammation in all the
surrounding area what we will start to see is even the liver that has attached to your
implant which was healthy to start with will start to lose function.
So, at that point there is really no option, you can try some heavy dose of antibiotics to
see if it helps, but most of the time what you will find is that the patient has to go back in
and the device will have to be removed.

(Refer Slide Time: 20:25)

So, here is just some example of how grave this problem is, so we have biomaterials and
related infections. So, what you can see here, so they are just mentioning some of the
devices that are being used and this data is for the United States, but basically similar
proportion can be found everywhere and what you see is how much of these implants are
being used.
So, quite a bit as you can see some of the things like bladder catheters you are talking
about in excess of crores of implants and similarly all of them are required in quite high
number. And what you see is in some of the cases the infection is fairly high, I mean this
is after following all kinds of state of the art practices you are looking at bladder
catheters we are talking about 10 to 30 percent.
So, basically every third of the fourth patient will get infected and some of the cases I
mean it is all the way up to 50 percent. So, I mean almost half of the surgeries that we
will do you will have to come back and redo the surgery. So, that is quite a heavy toll on
both the medical community as well as on the patient.
Some of them are also fairly low, but then even then that is a risk that still very high and
then the worst part about all this is lot of this actually leads to the death of the patient.
So, the implant related infection is actually causing the death. So, the patient came in to
sort of get cured from a certain disease you try to cure that particular patient, but you
ultimately end up having to kill that patient or you end up and killing that patient just

because this infection was, so massive that the patient could not handle it. So, as you can
see especially with the heart assist devices you see that the mortality is very high.
Similarly, in anything related to a heart is extremely critical just because it is not feasible
to continue to manoeuvre around with it is a very sensitive organ if it stopped beating for
a few even few minutes the patient is essentially dead. And in some cases, the mortality
is again moderate which is again as you will see is related to some sort of blood and
something like that can be very serious.
(Refer Slide Time: 22:45)

Again this is just again for your reference, so here is a partial list of human infections
that involve biofilms. So, actually one thing that I want to point out in the previous slide
here, is if you notice here most of these implants these are some things that stay for life. I
mean you are talking about heart assist devices, so as long as the patient is alive they will
need heart assist devices, you are talking about bladder catheters, dental implants. So, all
of these are some things that I am going to stay in patient for life and typically what you
will find as these are non degradable implants.
So, the surface that the bacteria is presented with on which the bacteria started to grow is
going to remain. So, this particular surface is going to remain there and then the bacteria
will happily colonize it once it adapts to it, it is not a dynamic implant that the bacteria is
looking at which is typically seen in cases of drug delivery.

So, going back, here is a partial list of human infections that involves biofilms. So, the
previous case we were just talking about that there was infections, now you are talking
about infections that are involving biofilms and there is quite a long list here that you are
seeing and some of the bacteria that are quite often seen is streptococcus is one of them,
hemophilius is another one of them, you have E. coli, some fungal species also come in,
so all of these are there. And then they are few which are actually very common, so one
of them is Pseudomonas aeruginosa, another one is Staphylococcus aureus.
So, these are very rampant and actually you might have heard about them in newspaper
and various news around that these are also now becoming antibiotic resistant, so that
then compounds the problem. So, not only you have a problem that these bacteria are
coming in, forming biofilms which have more tolerability against a particular antibiotic
you also have bacteria that are forming powerful there are already antibiotic resistant.
So, once that happens there is really no way you can treat them because all the antibiotics
that we are currently using some of these bacteria are extremely resistant to all of those.
So, how do you kill that bacteria? So, there is only one way at that point, you’ll have to
remove the implant. And again there is quite a bit of them and pretty much anything that
you are putting in the body you find that there are all kinds of infections. So, you have
sutures even those little sutures that you put in, they get infected, create a lot of pus you
have to remove them, then you have these contact lenses they get infected with
pseudomonas.
So, forming a layer over your contact lens which is again not very good. you have
endotracheal tubes, vascular grafts we talked about in the previous slide, mechanical
heart valves again we talked about in the previous slide, orthopedic devices which are
quite heavily used for any kind of fracture and these implants are essentially metal
implants and S aureus is one of the major organism that sort of infects these bone
devices. So, this is a major problem and again we need to take precautions when we are
handling these things.

(Refer Slide Time: 26:11)

And again as I talked about, so there is a race to the surface. So, let us say here is a
biomaterial that you are looking to implant in the body and it essentially boils down to
who starts colonizing the surface first because it could be the mammalian cells that are
coming in or it could be the bacterial cells that are coming in and depending on what it
is, whoever comes in first, then gets an advantage because that particular cell can then
adhere to the surface and let us say now if a mammalian cell is trying to come, these cells
can essentially is repel it.
Because there is no conducive surface for this mammalian cell to come in attach to and
the same thing happens with the bacterial cell, once the mammalian cells are there if a
bacterial cell is trying to come in and attach to it and the tissue ECM, these tissue cells
they do not let this bacteria to come and colonize its surface, the body is well adapted to
take care of anything natural that is present so that it does not get infected with bacteria,
but then anything unnatural that is there the body really has no control.
So, let us say for example, if a bacteria does come in adhere to this multi cell layer
surface, but then what will happen the body can essentially just kill this particular cell
and along with that this bacteria, so that the bacteria is not able to do essentially stay in
the body. But then in the case where is residing on a surface the body has nearly no
option if this is, let us say a stainless steel rod or a titanium implant, and the body cannot
degrade it.

So, at that point the body is really helpless against such bacteria. So, as I said both
bacteria the infection causing agent as well as the host cell compete for this material
surface, typically the bacteria in the nature is well adapted to colonize inanimate
surfaces. So, you will you will see in your pipes may have layers of bacteria, your walls
have layers of fungus and bacteria.
So, the bacteria is actually very well adapted in terms of colonizing such inanimate
surfaces much more so than the mammalian cell, not to mention the bacteria divides
much rapidly in the mammalian cells the numbers can increase very rapidly and so that
way it has some advantages over colonizing a material. And so this bacteria has a
naturally high affinity towards material surfaces, so, which can then again lead to
implant infection and essentially fouling of that surface. So, we will stop here and we
will continue rest in the next class.