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Immuno-Isolated Implants

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Video:

Hello everyone. Welcome to another lecture for Drug Delivery Engineering and
principles. We are discussing immunology and immuno engineering part of this course
and looking at why the drug we have, is required for those aspects. So, let us just do a
quick recap of what we learned in the last class before we go further.
(Refer Slide Time: 00:47)

So, in the last class, we finished our topic on vaccines. So, we had discussed already
different types of vaccines based on timing and content. And then in the last class, we
looked at why we need to use particles as control delivery and sustained delivery to
deliver antigens to APCs. And the major reason we said is first of all, it causes
enhancement of the delivery of your antigens to the APC, the size ranges; size range for
uptake is much better when they are in between let us say 50 nanometer to 5 micron. It is
a big range and there is also ups and downs here, but it is much higher for anything
compared to let us say free protein which could only be about 1 to 5 nanometer.
So, first of all you can then now deliver quite a lot of things. The second thing we talked
about was you can do a co-delivery with antigens. So, you can deliver multiple types of

antigens as well as co-delivery of adjuvants. So, you can then ensure that since it is in the
same particle, the adjuvant and antigens will be in the vicinity or in the same cell and
that would make sure that the two-signal requirement that is present for activation of
leukocyte is met in a more sustained manner. And then we of course, also discussed there
are some challenges with these particles. It may denature your antigen sometimes that
could be critical and so those are some of the formulation issues that need to be taken
care of.
Then we started our discussion on immune-isolated cell therapy and as the name
suggests, this is to deliver cells. So, in this case your cells are your drugs and as the name
suggests here immuno isolated as we want to protect from the immune system. So, we do
not want immune system to attack these cells which could be from a foreign
source ,could be either from a different human or actually could be from a different
species altogether. It could be from pigs for example, and so that is what we were
discussing, and we discussed some of the concepts of first of all why that is required and
the secondly, how potentially you can do it.
So, in that we were saying that we can encapsulate in some kind of a semi permeable
membrane. So, your cells can just reside in this chamber in which the pores are small
enough to allow glucose, oxygen, your other nutrients to go in, but it does not allow your
antibodies and cells to go in. And that way you can have this isolated from the immune
system to some extent and we will see how this goes on in today’s class ok.

(Refer Slide Time: 04:05)

So, this is just a pictorial representation of what I mentioned. So, you have some kind of
a barrier, it could be polymeric, it could be something else. This is your this is a barrier
that will allow let us say dissolved oxygen, your glucose, some free radicals, nutrients
and complement factors to go through, but it will not allow your big molecules like
antibodies.
So, this is all you are seeing here is some molecular weight here so, but this will not
allow anything which is higher molecular weights to go through which could either be
big large protein such as antibody or could be macrophages and T cells, B cells; those
things cannot go through. So, that is the whole concept here of course, this has lots of
challenges and we will talk about that in today’s class.

(Refer Slide Time: 05:06)

So, let us look at various components of immune-isolated implants. So, there could be a
polymeric component, as I said this barrier could be a polymeric barrier. So, if this is the
case and if you want this to be for life, this has to be non erodible right. So, if this is the
barrier which is made out of polymer, it cannot erode. Because if it starts eroding, then
these pore sizes will start to increase and then you can have antibodies and even cells to
go in and out and that will not be good. This should be mechanically stable. So, of
course, it is not like you are putting in bones, it should be fairly strong, but it should be
strong enough so it does not crack.
Because what happens if let us say it cracks, then now you have created a big pore size
through which, first of all these cells that you have put in can come out as well as these
cells from the external and the antibody from the external host environment can go back
in. So, it cannot break and of course, it must be biocompatible.

(Refer Slide Time: 06:26)

And this of course, is fairly intuitive because these cells that are residing here. So, this is
the implant and this is your host. So, these cells that are residing here, they need to get a
continuous supply of your glucose and oxygen for them to survive. And if this is not
biocompatible, and if this polymer had that fibrous reaction that we had discussed
previously so, what will happen is there will be protein adsorption first followed by a cell
adsorption or cell attachment.
And it may even just fibrose out the whole implant and so in that case now your
diffusion for this glucose and oxygen have gone down. And that is going to be a big
problem because then the amount of glucose and oxygen these cells are getting is not
enough for them to survive. So, it should be fairly biocompatible for it to work.
They will not be polymeric; this could be let us see some membranes. So, if we are using
membranes and there are again some components and criteria that you need to take care
of. So, one being that let us say if it is a hemodialysis membrane made out of polysulfone
or something else, they need some support because these membranes are fairly thin and
so, they need some support. These membranes could also be made out of weak
polyelectrolytes.

(Refer Slide Time: 08:02)

So, this could be some charge interactions, alginate with poly lysine films can be formed,
but needs to be a careful with that because these charged interactions are fairly weak.
And they may come off in certain conditions maybe the salt concentration increases and
the dielectric changes and then these interactions may become weaker and may fall apart.
The other examples could be that they need to be conformal. So, what that means, is let
us say if this is going go on the site which might be not straight, but they are round. So,
these things can then surround that in a round shape as well. So, this should conform to
the surrounding. As well as this should be microporous which again goes back to what
we discussed earlier that it should allow permeation of some small proteins, so,
permeable to small proteins, but not cells.
Obviously, this is all we are talking about immune system, but if your cells inside your
implant or the membrane, they are producing a protein which is let us say 60 kDa, then
you need these membranes to be permeable enough. So, that they can allow 60 kDa
proteins to go through because, eventually the whole purpose of the implant is to get this
protein.
So, those are some of the criteria’s that you will have to consider while designing either
the polymeric components or these membranes. And then there could be matrices; these
could be hydrogel. So, again polymeric components is a wider term and these
membranes and matrices are coming under it. So, these could be hydrogels or scaffolds.

So, these could be alginate and collagen hydrogels could be polyurethane scaffolds,
coated with some hydrophilic polymer. So, again all of this is going back to what we
discussed in this part of this criterion components that it should satisfy all of these before
it can be used otherwise the therapy may fail ok.
(Refer Slide Time: 10:07)

So, let us look into further the next component is your cells. So, one was the shield
which in this case we discuss about polymers, but then what about the cells themselves.
So, these cells can be primary cells which means that they are isolated from a being and
their primary they are not being cultured in a lab or something. So, a classic example is
islets. So, this could be from an organ donor, maybe you are getting islets from
somebody who has just died for some other reason and their islets are still alive and
functional.
So, you can get islets. Those are primary islets. This could be hepatocytes. So, this is for
liver regeneration maybe liver had some fibrosis or maybe there some damage to the
liver due to some accident. So, in those cases you can use these hepatocytes from some
other source or there could be some other hormone producing cells that you are getting
from some other source, that is not self. So, these are primary cells.

Sometimes you can use cell lines. So, again in this case you are planning to immune-
isolate anyways. So, you can use cell lines, the problem is the cell lines is of course, that

these most cell lines are cancerous that all cell lines have been transformed. So, that they

continue to divide and in those cases you want to make sure that it does not end up
causing cancer. So, here some another example in this case, you are trying to get some
dopamine so using PC-12 cells, or this could be engineered. So, this could be engineered
with either cell lines or with the primary cells. So, that means, that maybe there is a cell
that you think can be used for this application, but it does not produce the protein that
you want. It might be suitable for other properties that may allow enhance survival in
harsh conditions for those cells.
So, in those cases, what you can do is you can take those cells, you can then genetically
engineer them to produce whatever protein that you want, let us say factor VIII in case of
hemophilia or EPO in case of anemia. And then once they have done so they can produce
now these proteins which the originally were not present. So, they have been engineered,
but then you can utilize some other property for these cells to integrate well with the
tissue and survive in harsh conditions, something like that.
And so, these engineered cells can then help in preventing any kind of immune response
and enhance survival. You can actually even have some chemical switch so, maybe not
always they are producing it, but only when the requirement is. So, maybe they are under
some feedback loop where when this ends high glucose only then the produces insulin or
it could be something else for some other protein. So, this should be some trigger that
can be put in as well. So, gives you a lot more control over these cells ok.
(Refer Slide Time: 13:04)

So, here are some model configuration that I will go over. These are a few of the
configurations that have been used in the past. So, let us go over this. So, first of all we
are looking at some sort of a capsule that has some pores, the cells are of course inside
you may even encapsulate some growth factors inside. So, in this case the refer to a cell
stimulant to make sure that these cells are happy initially. So, let us say that these were
islets or the pancreatic beta cells.
So, now, what you are doing is with this capsule you are preventing any kind of cellular
and humoral immune components. So, any cells cannot go through, any antibodies
cannot go through, there is also some extracellular matrix that is present. So, ideally most
cells would want to interact with some ECM proteins to be able to survive and have
normal signaling.
So, now we are adding some extracellular matrix within this polymer or this implant to
which the cells can attach to and be able to perform their normal signaling. Obviously, as
I already said you do not have antibodies going through this, then whatever this is
producing that is required for the body can actually also come out. So, that is also a big
criteria that along with the nutrients and oxygen being able to go in, it should be big
enough to allow let us say if is producing insulin which is fairly a small molecule, it
should be able to come out or if its dystrophin which is not So, small that should be able
to come out.
So, the self products should be able to come out and more than that the waste product
should also be able to come out. So, what will happen if you do not let the waste product
come out? Most of these waste products are acidic. So, if you do not let it come out, then
you will have a buildup of the acidic environment. The pH of this area may drop and that
may cause a death of these cells.
So, you want the waste product to also be able to have some sort of a circulation through
this membrane and then just to highlight this external matrix this is actually very
important. It is first needed to immobilize the cell and so, that they do not start clumping
together. The cells do not find any support to attach to they will start to just attach to
themselves and start to aggregate. Once they start aggregating, then instead of having
separate cells to which oxygen can diffuse through.

What you will have is, you will have, cells forming these aggregates which will cause
another diffusion barrier for the innermost cell to receive oxygen. So, to prevent that you
want to make sure that the cells have attachment sites around them of course, you want
improved oxygen transport.
Sometimes what is done is you can add some components such as perfluorocarbons
initially, to allow oxygen to be released within this environment over some period of
time. These are molecules that will release oxygen for an initial duration, but then later
on you then you rely then, these cells will induce some more blood vessels to grow in
and around this so, there is enough oxygen around. So, maybe initially you want this
oxygen shock not to be there. So, you give the oxygen in the implant which through
some chemistry and then you rely on the fact that these cells will induce blood vessels to
come in to this environment very similar to the way cancer cells do it.
Then there are other factors as well you want; obviously, immune protection. So, you
want the physical limitation of a cell volume within the chamber as well as immuno
protection from any antibodies. And again you want to ensure that the chemical
environment is acceptable. So, like for example, collagen gels have been induced shown
to induce epithelial cells to grow duct like structures. If they if you are trying to produce
some endocrine based islet cells so, those things should also be fairly comparable. So, it
is not necessary that you can put any ECM depending on the cell that you are choosing
you may want to choose a particular ECM protein over other proteins.

(Refer Slide Time: 17:29)

So, here is just a very crude example of micro encapsulation process. So, in this case
what you are looking at is and we have discussed this before and this process. You are
looking at an alginate-based solution. So, this is the hydrogel. So, you want something
like this that there is an alginate which is encapsulating several cells and the pore size of
the alginate is such that it will allow the oxygen and the glucose and the waste product to
move in and out, but it will not allow your antibodies and the immune cells.
So, what you have done is you have suspended these alginates at a certain concentration
in which the pore size will be such that it follows what I just described. You can put islet
sort of some other cells for that matter and then there is some pump which is then
pumping this alginate solution containing cells through a syringe with a certain diameter
and it is being pumped into a calcium chloride solution. So, what will happen is, if these
are alginate chains and again we have discussed this in a hydrogel class, these are
alginate chains which at this point are not cross linked. These calcium ions, which are
divalent will start to interact with the negative charges that are present throughout these
chains.
And so, it will go and start cross linking these chains using ionic cross link and whatever
cells are there gets entrapped in here. It has enough pore size to allow the movement of a
small proteins, but more cells cannot come in. So, that is one way you can make them.
Of course, in this case you have to be careful, alginate is not an ECM protein. So, if you

want the cells to be fairly happy in terms of cell signaling, it depends on the type of the
cell. You want to ensure that you put some ECM proteins also along with the solution
and they get incorporated and stay at the site. And then this process is very widely used
and the reason for that is it is a very mild process. So, if you look through the whole
process, there is no organic solvent, there is no high stress condition what the cell is
facing them. All its seen is some high concentration of calcium which is anyways a
physiological ion that is present. So, in most cases this process works very well with the
cells and the cells are fairly happy in the end product.
(Refer Slide Time: 20:25)

So, let us look at some of the implant designs now. So, the grafted cells must be less than
200 to 500 microns away from the host environment. And where does this 200 to 500
micron number comes? That is because you want the oxygen and nutrients to effectively
diffuse in the system. So, now, let us say if you put an implant which is let us say 5
centimeter by 3 centimeter just for example. Then what will happen? The cell that is
residing here is almost 1.5 centimeter at the very minimum is about 1.5 centimeter away
from this edge.
So, now the oxygen that is here or the glucose that is here not only has to diffuse through
this semi permeable membrane, but also has to go and find its way all the way to the cell.
So, what is seen is if you increase this size range to beyond 0.5 millimeter, it is greater
than 0.5 millimeter then these cells do not survive very well; they do not get enough

oxygen. So, and that is of course, relying if the blood vessel is passing right through
here. The blood vessel it could be further away; the blood vessel could be further away
from this implant edge by let us say 100 micron.
So, now that this distance is further increased so, that is a major limitation that comes in
the implant design. So, ideally you want the implant to be such where the cells are not
away from the host environment by more than 500 micron because, if we do that you
would find that the cells do not survive for very long. They need this oxygen and glucose
to come in fairly regularly in high amount.
(Refer Slide Time: 22:34)

So, that is why you go to microcapsules and the reason for the micro capsule is you can
make these capsules of the sizes that about 500 micron or lower instead, but at least
ensure that from the host environment, you are not more than 500 micron away.
So, in that gives your because its only 500 micron and let us say your cell is about 10
micron, then you can encapsulate quite a few cells not millions of cells, but at least each
capsule can have quite a bit of cells and then each of these cells is not more than 500
micron away. So, if you make it 1 millimeter, all the cells are within 500 micron of the
edge. This is also good because now what you are doing is instead of making a big
device like head earlier drawn in centimeter, size, skills, you have now used the same
amount of material, but instead you made several of these devices. So, the now surface
area to volume ratio is very high and that ensures that quite a lot of diffusion is

happening, and you can still in capsulate the same amount of cells or more; however, this
has a problem it is a difficult implant to retrieve.
So, let us say if something goes wrong with this particular implant, maybe the cells are
turning tumorigenic, they are forming tumors or if something is; something is
problematic with this implant. These are fairly small spheres and they can move around a
bit in the body and you it may be hard to then retrieve this. So, this is a sort of a
regulatory requirement that our regulatory agencies such as FDA put that if you are
putting something in the body; if its retrievable, the clearance is easier because of course,
if something goes wrong you can take it out.
What if these cells now started producing something else that is causing toxicity to the
body or what if they are producing too much of your insulin or too much of something
some other function that you wanted these cells to perform and that could also cause
toxicity. So, you want them to be fairly retrievable which is a problem with such design
nonetheless this is still very widely used.
And then you can have vascular grafts and microcapsules or macro capsules in this case.
So, macro capsules is incorporation of the whole dose. So, basically talking about full
organ or large cell mass to be implanted, it is easy to retrieve because it is a big device, it
is not going to go anywhere. So, it will remain in the body.
(Refer Slide Time: 25:14)

To allow more surface area, you may not want to use sphere, maybe you take a long
sheet which is let us say 1 millimeter wide and the these dimension could be in
centimeters. But that would ensure that all of your cells are not more than 500 micron
away and then this device can be also retrievable or you can have a hollow thing in the
middle going as well.
So, let us say you do not want this, you want it to be let us say about 2 millimeter, but
what you can do is you can have some hollow pores. So, all of this area can be exercised
by the host. This is what is protected from the host and that will still mean that you are
still less than 500 micron away from any of the edge of the host, but these are typically
difficult to scale up, it is not easy you are talking about manufacturing each of them
separately.
So, they are difficult to scale up and of course, they are harder to control the nutrient
diffusion through the flow device because they are big. So, it they may be non
uniformities throughout the whole implant because if its huge thing that you put in
maybe here the diffusion is slightly different than what is here. so you will have non
uniformities throughout the whole device and then there are vascular grafts.
(Refer Slide Time: 26:51)

So, that means, that let us say, I have an implant. What I can do is, I can graft a vessel
next to the implant or something like this and then connect this vessel to the host vessel.
So, this is host this is grafted and so, at that point you are directly grafting it. In this case,

these may not contain any cells because this is exposed to the environment. This is where
your cells are and then they can then act on whatever is required and still get lots of
nutrients. So, maybe this vessel is going right through a vascular graft.
(Refer Slide Time: 27:30)

So, here are some implant configurations. So, this is one of those things I talked about.
So, you have bigger device, but if you have the whole device filled with these cells and
immune-isolated and then what you will find is that the cells at the center are not getting
enough nutrients. So, one way you can get out of that is to have a blood stream going
through right through. So, now, you can then make it thicker. Other example is what I
discussed before.
So, you can make very small spheres such as 20 50 micron and put few cells in here
alternatively, you can have even more micro coatings in which you put yourselves and
very very thin layer. So, this is very nice in terms of the diffusion that can easily happen.
Then you can have a semi-permeable membrane and put in a polymeric matrix. This is
micron capsulation not preferred in this case and this is not going to last very long. You
can have big alginate spheres that we discussed similar to here; obviously, this is better
in terms of the cell survival.
However, in terms of retrievability, this is not good. Similarly this is better in terms of
retrieving, but still not as good as let us say this in this device and the cell survival is
lower than this particular device. And then you can as I said you can flatten it out. So,

that the dimensions on the width is in microns. So, that these cells can get any of
nutrients whereas, this device is long; so, they can still survive. So, these are just some of
the models that are out there that you can use to go forward; each has its own advantages
and disadvantages and it just depends on what application you are trying to target.
(Refer Slide Time: 29:28)

So, here is one of the devices that was used. So, this is used for insulin delivery and what
you are looking here is a very porous membrane and it is actually very defined
membrane. So, in this case you have pore sizes of about 24.5 nanometer and inside this
device, you can have cells. So, this pore will actually allow your glucose and oxygen to
go in and out whereas, it will prevent cells from going in and out; however, at that big of
a pore size, you may still have some antibodies going through.

(Refer Slide Time: 30:01)

So, this is what is done here. So, further analysis of what happens to the antibody at that
size. So, you have free islets of course, and you are looking at the diffusion of insulin out
from this device. So, you have free islets, you have different pore size devices and what
you find is, the diffusion of the islet is fairly equal for a longer duration.
So obviously, free islets do very well with the rest of them. So, this is free islets, this is
encapsulated, but eventually what we are seeing is what for all these size 18, 66 and
78nm; you get a fairly good diffusion of the insulin very comparable to free islets at a
period of 1 hour. But what happens to an antibody which is something that you want to
prevent. So, this is for something that you wanted to diffuse. This is something you want
to prevent.
So, now, let us see what happens. So, what you are seeing here is that as you are going
up. So, this is time again. So, for 78 nanometer you have quite a high diffusion of IgG.
So, this is not good at 66 nanometer you still get some IgG diffusion slightly lower than
78, but still fairly high, but at 18 nanometer you kind of eradicate it quite a bit although
some of it is still going in.
So, if you want to make a device, you would rather make a device with 18 nanometer
because in among these 3, this is better in terms of preventing IgG from going in, and in
terms of the functional itself, it does not really affect a whole lot. So, you do not really
see a significant difference here. So, but of course, it may be beneficial to even try

something smaller. So that this IgG diffusion gets completely eradicated, but of course,
we will have to make sure that the it is not something like the insulin goes down like this
because, then it is not good. We will stop here, and we will continue further in the next
class and discuss more about immunoisolated cell therapy and some of the various
factors that need to be taken into account and some will also give some examples to see
what people have done in the literature ok.
Thank you.