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Module 1: Sistemi di consegna rispondenti e mirati

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Video:

Hello everyone, welcome to another lecture of Drug Delivery Engineering and
Principles.
(Refer Slide Time: 00:33)

We have been discussing about how to go about and use responsive systems, so this is a
quick recap of what we learned in the last class. In the last class we started our
discussion on various responsive delivery systems particularly the temperature
responsive delivery system and so what that entails. So, in temperature we first looked at
LCST exhibiting polymers, so this is nothing but Lower Critical Solution Temperature.
And what this says is typically you will find that most things become more soluble as
you heat them up whether it is some protein, whether it is some polymers. But, there are
some unique type of polymers that actually show the reverse property where if you heat
them up they become more hydrophobic and they actually become less soluble. So, such
polymers are called LCST polymer and the temperature at which this switch between the
properties happen is called LCST.

And what we found here that , if you let us say make a hydrogel out of such polymers
which is let us say chemically cross linked. So, all of these places are fixed, but the
polymer itself will change property as a temperature changes. So, let us say if we now
heat it up above the LCST. So, for example, let us say the LCST for this particular
polymer system is about 30 degree celsius. So, at room temperature it is fairly well
soluble it is fairly hydrophilic. So, it will have a certain swelling ratio it will interact with
water molecules around it and will swell.
But once you let us say go to 37 degree celsius which is a body temperature what will
happen is this is going to become hydrophobic and it will not try to have interactions
with water. So, it will then try to have interaction with other chains which are also
hydrophobic. I n that case what will happen is these polymer chains will basically repel
water and try to come close to each other.
Now, well the since they are cross linked at this at these points and they cannot leave
those points, so basically there pore size is going to shrink. As a that is that is what the
LCST is and we discussed how this can be done. Similar very similar thing a variation of
this is sol-gel hydrogels which are nothing but instead of being chemically cross linked
they are physically cross linked and that means, that now these chains can move around.
But then if they are becoming hydrophobic they may not actually form any kind of gel ,
what will happen in these cases is if you have a polymer it is going to show a phasic
diagram which is something like this where you have concentration on the x axis and the
temperature on the y axis. So, only within this area will this be gel anything above or
below it will not gel. And so, that is because the chains will be too soluble at a higher
temperature, but then if the temperatures in this range they will have enough of
hydrophobicity. So, actually does a physical crosslinked gel
Then we also talked about pH sponsored gel which we have been talking about
throughout this course, but we looked at some more variations to that. So, how you can
vary the pH and have more swelling or less swelling and depending on that another
applications here we looked at an example where we had a colon specific delivery. So,
we had polymer chains that were cross linked within Azo bond and these polymer chains
themselves had lots of COOH group.

So, where this is the case what will happen is as it is travelling through let us say the
stomach these polymer chains are non-ionic, because they are already protonated there
carboxyl is not going to lose it is proton and they have a certain pore size. So, maybe
your drug is in here which is encapsulated as they go to a higher pH in colon. Now, what
is going to happen is these all are going to become charged, so all of these will become
charged as a result then started to electrostatic repel each other and the pore size will
increase.
So, this is basically going to become like this and not only that there is a lot of
microbiota in our colon which has Azo reductase as an enzyme that is being produced
and that is going to cleave these bonds. So, eventually you will have your drug release in
colon instead of your stomach or intestine. Then we also looked at glucose sensitive gels
and in these again there could be several variations, but one variation we looked at was a
pore that was covered by polymers that were fairly water soluble, so they will extend and
not let anything within this device.
(Refer Slide Time: 06:25)

So, let us say if your insulin is here the insulin cannot go there, because these polymers
are physically blocking these pores. But then lot of enzyme called GOD: glucose oxidase
was stacked to these polymers and whenever enough glucose is present in the
surrounding. So, let us say glucose comes this is going to react with this glucose oxidase
and produce H plus ions causing a local drop in the pH. These things will change

property with the local drop of pH and will shrink down and essentially become , can
find to the surface rather than spreading out, hence opening the pour for the insulin to
come out.
(Refer Slide Time: 07:23)

So, these are some of the systems and we discussed in the last class, and then towards the
very end we learned about some bio shielding strategies. And so, those were more that
let us say we have particles and we do not want our biological system our immune
system to detect them. Then what is a way that we can prevent that detection and there
are several things that we can do as we have already discussed throughout this course
and this is just sort of a repeat of that.

(Refer Slide Time: 07:57)

So, one thing that we can do is coat them with lipids. This is something very similar to
your liposomes which I have actually made out of a lipid, but even take your polymeric
particle. And then you can coat it with this lipid layer which could be made from these
lipids and derived from the human sources. And, that will mean that the body will only
see this and think that this is something of a self particle similarly you can coat it with
polymers.
So, this is few things we talked about those PEG is one of the good polymers that people
have been using although we also talked about there are some antibodies that are being
detected now against PEG. Similar to the PEG we can use carbohydrates, so this is again
similar to what normal cells are our cells have lots of proteins and these proteins are all
heavily glycosylated.
So, they also add to the shielding layer, so you can we can use something similar
strategy on a particles. And then finally, we can coat them with proteins that are also
present on the cell surface to sort of give a signal that this is a self protein or this is a self
material.

(Refer Slide Time: 09:09)

So, we will continue the discussion today in this class and talk about targeted drug
delivery system. So, the targeted delivery system can now a divided into two categories,
one is passive targeting another is active targeting. So, passive targeting is what we have
primarily talked about through this course now EPR effect was one that most targeting to
cancer as well as inflamed issues. And the major rationale behind that was that the blood
vessels that are present in this inflamed or cancerous tissue are much leakier than the
healthy tissues.
So, let us say this is a healthy tissue, and this is a tumor ,then in these regions the blood
vessels are leaky just because either they are immature or this lot of inflammation that is
causing this drop in the vascular network. So, what you can do is you can make particles
that are big enough to come out through these pores. And in that case they will keep on
circulating through the healthy vessels because they cannot go through, but as soon as
they reach cancerous vessels they can come out.
So, something like a particle which is 50 to 200 nanometer is targeted to the cancer;
however, this is a passive targeting because at this point you are not really forming any
active born it is not very tumor specific, it is any tissue that is leaky in it is blood vessel
will cause accumulation of these particles. So, this is what we call as specific targeting,
other things that tumor environment again. So, you can have let us say your drugs or pro
drugs which are targeted to let us say a local pH or something like that

So, that is again, let us say if I have a pro drug. Maybe I have this drug which
conjugated by some labile bond to this heavy molecule that is protecting this drug from it
is activity as well as it is degradation. But then this cleaves at a low pH and we know that
tumor environments also have a low pH just because they have a very high metabolic
rate. And so, this drug will accumulate everywhere, but in the tumor environment it may
break, it may also break in some of other environments. And so, this is how drug is
inherently passively targeted to these tumor regions or any region which is low in pH
T hese are all good, but these are again all passive targeting, there is no active targeting
involved and I am; obviously, one other method is to use a direct local delivery. So, if it
is for lung you directly give it to inhalation if it is directly you want to deliver it to the
tumor and you have a big tumor you can directly injected into the tumor itself. So, that is
also categorize in the passive targeting.
So, let us look at what is active targeting and as you can already see in the slide, so we
have this is a specific to a direct interaction. So, let us say there is a receptor ligand, so
we know may be a tumour cell because of it is high metabolic rate and because of
whatever mutation they might be expresses a protein x on the surface which is present
only in the tumor or at least in much higher amounts in tumor. Then if I then put my
particle with the ligand of x, then this is actively target to the tumor because now this is
going to go and bind to the tumor cells and the tumor microenvironments.
So, that is active targeting, so it could be on the basis of ligand and receptor. So, this
particular example , this could also be that a certain lectin or a carbohydrate is present in
the tumor. And, you conjugate your particle with the carbohydrate or the lectin to be able
to target it to that tumor cell or it could be any other cell, it does not have to be tumor,
but it could be other disease as well and then finally, it could be an antibody antigen
interaction.
So, let us say we know that there is an indigent this is very similar to the ligand receptor,
but more specific in terms of it is function and role. Let us say if they we know that there
is an antigen that is present on the tumor regions or that is present on let us say in a
bacterial cell, or in mammalian infected cell. We can then take our particle put these
antibodies onto that and these antibodies will then go and bind to this and that will be
more active targeting. So, that is the major difference between passive and active.

(Refer Slide Time: 14:01)

So, there are lots and lots of targeted drug delivering systems that have been tried. So,
this is just an example with liposome, but this could be true with any type of particle. So,
here is just a normal non targeted particle it is a liposome in this case it is speculated to
have an on circulation maybe it is diameter is about 100 nano meter. And, that is what
allows it to accumulate in some regions maybe you have certain charge on this as well
maybe this is negatively charged maybe this positively charged just depends on what
your application and target is.
And, but if you are looking at targeted nano particle what you will do is you can then
modify this targeted nano particle in such a way that the exterior surface whether it is the
peg chains or is just a particle with the exterior is modified with some target or some
ligand that is specific for the disease you are looking at. So, again there are several
targeting moieties that one can go through ,n there are antibodies you can use the full
antibody.
So, again antibody is also divided into an Fc region, and the variable region; Fc region is
basically the constant region. So, which means that this is fairly similar in most types of
antibodies, and then variable region is what allows it to bind to different targets. So, this
is fairly variable and triple (Refer Time: 15:30) different targets.
So, sometimes since mostly for most targeting specific applications you may only need
these regions. So, people are able to even separate out these segments to reduce the

antibody size and not have anything else around, so, you can use antibody fragments as
well you can use any small molecule.
So, maybe some cells are heavy in the requirement for glucose. So, you can even put
glucose on your particle and then because more and more receptors on this side do bind
to it, you can use a small peptide again very similar concept. So, why reduce it to this
why cannot we just reduce it to whatever the moiety of the peptide that is present on
these surfaces that are responsible for it is recognition. And then there is also another
thing called aptamer and we will talk about aptamer in next couple of slides.
So, all of these are targeting moieties that have fairly used. So, antibodies, proteins
which are again antibody is the type of protein, you have lipoproteins, hormones, you
can use charged molecules. And if you use charged molecule this is technically not an
active targeting this is a passive targeting, you can use poly specific polysaccharides
some low molecular weight ligands. And again as it is shown pictorially here you can
mix and match them you can see in particle again we put with 2 or 3 ligands to confirm
much more targeting to such particles.
(Refer Slide Time: 17:01)

So, as I said aptamer is another class that I will just describe it is not probably as widely
known as some of the others. And what does an aptamer? Aptamer is nothing, but is in
short oligonucleotide that can fold into a unique tertiary structure. So, we know what
oligo nucleotides are.

So, these are just sequences of your nucleotides ATGC and if I arrange them in a certain
order such that we have several of them and let us say if this sequence continues to 10
nucleotides. Then this structure itself can roll around and try to minimize the energy and
in that process it can form a 3 dimensional tertiary structure which could look like
something like this right. And then in many other shapes I mean this is just a random
shape that I have drawn.
Now, again this is fairly small and we are talking about 100 nucleotides only, so it is not
a huge protein like structure it is a small protein like structure and maybe there is a
protein that is complimentary do this. So, maybe I have a protein that looks like this and
if that is the case and the aptamer has the sequence complementary or the structure
complementary to this protein binding side then what will happen is this aptamer is
going to go ahead and bind to it.
So, here is your aptamer is maybe a protein and it may not be protein, it could also be
lipid it could also be something else carbohydrates it just depends on where you get a hit,
but something like that will then have affinity for your target or I should say protein
slash target. So, and this is what it relies on and now the field of DNA synthesis is
actually a progressed very rapidly and people have been able to bring the cause down
significantly as well as have the throughput very high.
So, if I want a sequence which is 100 nucleotide it is not a problem these days is fairly
cheap as well as you can have quite a lot of amount being produced in a very in a very
small time. So, it is fairly feasible to make lots of aptamers and lots of types of aptamer.
So, let us see how then these help.

(Refer Slide Time: 19:35)

So, as I said they will recognize a specific target ranging from small organic molecules
to proteins to even cells sometimes we do not even know what the target is. But, but we
know that it binds to certain cell where exactly it is bind to the certain cell may not be
known.
And so, on the basis of that people have now come up with a protocol and a method
which is called SELEX and this is nothing, but it is a systematic evolution of the ligands
by exponential enrichment. And it is an evolutional selection method and we will we will
describe it in a moment as to what it is. And one of the advantage with the aptamer
compared to an antibody, let us say we do find an aptamer and we will go into how we
find an aptamer. But let us say we do find an aptamer which is able to bind to a certain
target that you are interested in.
So, then it offers lots of advantages over antibody first is aptamer does not have any Fc
region. So, as I described that anti bodies and large molecules and that have an Fc region
in a variable region, the basic function of the antibody is to be like a tag for the immune
system. So, if an antibody is binding to a certain region or certain protein that acts as the
tag for the immune system to remove that because that is something that is considered as
foreign. And the way the body does that is through this Fc region, this Fc region has lots
of receptors on your immune cells and once these immune cells bind to this antibody
through the Fc region, and they will clear it off.

So, the last thing that you want is your therapy that let us say you put you are putting a
particle and you have put an antibody that you are delivering it with it to get detected by
the immune system and gets cleared off then you are losing lot of you drug lot of a
particle formulation. So, that is one advantage first, the second is actually it is quite a low
molecular weight. So, we are talking about 5 to 10 kDa, because of which that diffusion
limitations are much lower, so remember bigger the size lesser is the diffusion.
So, this can diffuse this can go through the small gaps it can find anywhere and it will
have a much larger range of a penetration into various types of organs and cells. And so,
in that regard this is much better compared to let us say a 100 kDa, 200 kDa protein
molecule like anti body and that will face this challenge. And I just want to point out that
this selex method is actually very similar to the phage display library which is used for
selection of some peptides.
So, in general when we are talking about aptamer you can actually compare them with
small peptides. So, small peptides can also have affinity against a particular target,
because of the same region they may acquire a tertiary structure or they may go at a
secondary structure that may have affinity either by the structure or just by the
interaction of the peptides with another amino acids on new target.
So both of them are similar in terms of their sizes as well as use for targeting. But;
obviously, their backbones are very different and aptamer do attain a lot of higher
tertiary restructure compared to peptide which usually are linear or secondly structure in
nature.

(Refer Slide Time: 23:17)

So, let us see how does the SELEX work, so here is a selex. So, what you can do is you
can have a library of lots and lots of these aptamers in here. So, let us say this is a library
of aptamers, and so you then put your target in that library and screen if anything is
binding to your target. So, let us say you started with 100,000 aptamers all different
maybe 40mer s or 50 nucleotide long in various permutation combination. And then
from there you end up getting maybe let us say 10 of them that that is showing some
binding to your target.
And you can test that through various methods you can run it through a phage gel, and if
you see a shift in the molecule and shift in where the protein is coming you can say that
that protein is now bigger because it is born to some other sequence in this case the
aptamer Or, you can stain for nuclear nucleotides on your targeted cell after washing and
that may show some affinity there then you do the in vitro optimization. So, you can take
this hundred or maybe this is your 10 you can do some binding affinity to figure out
which is binding in it a much higher affinity, and you can take them and do your in vitro
as says
So, you can see whether you cannot target your payload to yourself what is the
mechanism of action if it does target, if the target stimulation or emission whatever you
are trying to achieve is happening. Then you can basically take it to your in vivo stage or
if it does not happen you can go back to here you can try to screen with some other

library. Once that is the case or if it is happening, but if you are not happy with the
binding affinity you can then take that sequence that you know is working in some small
amount and you can try to then mutate it rationally one by one to get up much higher
affinity.
And then you can go in vivo you can see whether this works or not whether it is not
binding to any non-specifically to any other targets which may then cause toxicity and if
all of this works then you will essentially have a therapy that this will targeted. And I I
also want to point out that the DNA technology has progressed quite a bit and we have
now been able to made modifications in your nucleotides.
So, here we have LNA which is a locked nucleic acid or peptide nucleic acid and these
are just some variations to nucleotides and they are actually fairly stable in serum. So,
even if we inject this in serum you find that they do not degrade they are well tolerated in
the body So, this is one way the phage library is very similar also, where you are
screening for peptides, instead of selex library, you will have a phage library.
(Refer Slide Time: 26:27)

And, it is called phage library because we use bacteriophages to which are small viruses
of bacteria. So, let us say this is a bacteriophage and this will display peptides and you
see where these peptides are binding using this phage and a very similar method will
eventually give you a target through peptides also.

So, what are some of the shortcomings of aptamer, first is of course, that they are
negatively charged these are eventually nucleotides and, so the chances of them
interacting with your negatively charged targets. So, let us say my protein is negatively
charged, so if I have a protein that is bearing negative charge the chances of these
aptamer is going to bind to this protein is very low.
So, now, you are basically reduced your targets to half essentially, because if you assume
it is all random distribution then nearly half of your routine cannot be a target of this.
And then the other problem is that it is actually a foreign DNA because these are DNA
that is not what the body is used to. So, it can get recognized by the TLRs or like
receptors of the immune system and that may still cause some inflammation as well as
rapid clearance.
(Refer Slide Time: 27:53)

And then lots of aptamers have actually gone through the clinical trials this is this is a
2015 data some of them have actually been approved for certain treatments, lot of them
you see is actually in quite a advanced clinical trials. So, there is quite a lot of
enthusiasm with the use of aptamers is targeting moieties, and along with everything else
there are antibodies which are also in clinics it is a targeting moieties, peptides all of
these are fairly widely used. And we will talk about the processes to get clinical approval
in our nano toxicology in a part of this course.

(Refer Slide Time: 28:33)

And just very quickly tumour is again one thing that is widely used for targeting
basically most of the therapies in research are looking at tumor because it is a major
problem. And tumors do up regulate lots of receptors, because they need quite a bit of
nutrients for the fast growth and generation of new blood vessels. So, as a result there are
several molecules that are routinely up regulated and used for tumor targeting.
So, transferrin is one which is in protein that shuttles iron, you have EGF receptor which
is a growth factor receptor, similarly folic acid is another one that is required for growth,
VEGF is for blood vessel growth. So, all of these as you can see are quite obvious that
tumor may need these two proliferate and grow in size.
And so, because of this you can then design your particles with these targets displayed
that are going to go bind specifically to the tumor cell and get internalized, but not to
your healthy cells. So, that way you can then have more specific result compared to what
you will get without targeting.

(Refer Slide Time: 30:01)

However, there are some cons of targeted delivery systems and some of them are first of
all that it is important to note that very few targeted little systems are actually being used
in clinic. So, just an example of a target drug delivery we use and again there several of
them, but just an example is this particular antibody drug conjugate which is used for
breast cancer. But there are not many if you look in the research phage lot of people are
using these targeting moieties, but not all of them are being used in the clinics. And the
major reason it is still debatable whether adding these ligands actually results in any
therapeutic administration or therapeutic benefit when you are giving it systemically.
So, it is not entirely clear there are several issues such as once you add a targeting moiety
on your particles you have now increased the size. So, maybe earlier the size was about
100 nanometer, now the sizes become 150 nanometers. So, that causes change in
pharmacokinetics you can mask these peptides can get masked by protein adsorption. So,
as I said all foreign material that you are going to inject in the body will come in contact
with your proteins that are present and they may adsorb on the surface and eventually
none of these targeting moieties are actually available.
They may change the type of proteins are absorbing, they may even denature during their
journey throughout the body. And their accessibility is also a question mark, so that is
why there is lots of cons for targeted delivery systems.

(Refer Slide Time: 31:43)

And this is just a laundry list I just wanted to give it to you there are lots and lots of
clinically approved drug delivery systems, again these this is probably not the exhaustive
list it is from 2018. So, probably more and more have come out and have been approved,
but as you can see even after all this we have been able to as a whole field being able to
in actually influence quite a lot of peoples life. Because we have so many systems that
are coming into the market which are showing better replicated in the feed drug that is
why they are approved and, so this is just a laundry list I wanted to give it to you ok.
So, I think we will stop here and we will continue this further in the next class.
Thank you.