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					Dendritic Polymer Adhesives for Corneal Wound Repair
Mark W. Grinstaff, PhD
Associate Professor of Biomedical Engineering and Chemistry
Metcalf Center for Science and Engineering

Great, thank you very much for the kind introduction. I’d like to thank Richard for
the invitation to speak today. I’d also like to thank the two previous speakers,
they very much set up my talk which I am gonna tell you about our recent work in
the lab on dendritic polymer adhesives for corneal wound repair. Great. So as
we talked about a little earlier, dendrimers have some very unique properties
compared to linear polymers and as Karen had mentioned these are globular
macromolecules and shown on the bottom left here, is a structure of one of these
macromolecules where you can see that there is a core and then from that core it
branches outward in a fractal like manner where you have layers and then at the
end you have in groups. And we have the ability to go in and make discrete
changes at the molecular level, either at the core, at the layers, or at the in
groups to optimize and to tune these types of macromolecules. So we can go
back and ask the question what would be the design requirements for a particular
application where interested and how to go back in to the lab and make those
changes at the molecular level to see what you can do with these types of
interesting macromolecules. Now, what we’ve done that’s unique is we’ve made
these macromolecules out of biocompatible building blocks such as glycerol,
which of course is found in your phospholipids, or succinic acid, which is part of
the Kreb’s cycle. And the idea is to have this well defined globular polymer
perform a function in vivo and then after it’s performed it’s function, it’s gonna
degrade into natural metabolites. The closest analogy here is a linear polymer
polylactic acid. This is what your absorbable sutures are made out of today.
This is a
linear polymer, okay and as such when you ever, when you work with a linear
polymer, you’re always working with the distribution of different molecular
weights. So in your polymer sample your gonna have some small molecular
weight, some medium molecular weight and some large molecular weight. And
therefore, any property that you see, be it a physical property, be it a mechanical
property, be it a biological response, you relate
that to that distribution of molecular weights. Alright. And what we can do with
dendrimers, which is quite unique, is that we can actually make single molecular
weight materials. And so we are able to make single molecular weight polymers,
okay, like polylactic acid that might have a lot of applications in the areas of
tissue engineering, wound closure and drug delivery. Here’s an example of a
recent one that we synthesized,
again composed of succinic acid shown in red and light blue is the glycerol. This
is a branch structure so it is not flat as shown there on the screen, and this
structure is about six to eight nanometers in diameter depending on how many
generations or layers we had. So our first generation would be GE 0 generation
1,2,3,4, and so forth. So again we are able to make these in such a way that we
have complete control over our composition.
Therefore any effect that we see, be it a physical property, a mechanical
property, or biological response, we can correlate to an exact structure. Now
how do we make these? It’s a very straight forward way in which we basically
start at the center and work our way out in a step-wise fashion, so in a very
controlled way, we can build this dendrimer up from the basic structure and as
such we have all these individual structures on the way to use in our analysis of
the physical properties. Alright. Now, what’s particularly nice is when we do the
synthetic reactions we get very high yields and so we are able to do this over
multiple steps and generate gram quantities in materials. These materials are
fully characterized, everything from NMR and mass spec, to elemental analysis.
And the take home message is that with these dendritic polymers we have the
control of structure and composition at the levels that you would normally see for
small molecules and pharmaceuticals. So now we're on the scales of five to
fifteen nanometers and yet we have the precision that we might have with small
molecules that are gonna be less than a couple nanometers in size. Let me tell
you about one particular polymer that we like and this is the tri-block system. We
have a dendritic block, a linear polyethylene glycol
block, and a dendritic block, and again we make this in the same way in which I
showed in the previous slide. We started at the beginning, and we just couple
out one step at a time, and we keep growing this dendrimer out from its core.
And again we can take this all the way out to a generation four system where we
have this dendritic wedge of polyethylene glycol linear spacer and then this
dendritic wedge. And now what we are
gonna do with this polymer is we’re gonna modify the outside of it with a group
that allows us to cross-link it and make a gel. So were gonna take this polymer,
were gonna put it in aqueous solution and then we’re gonna shine light on it and
when we shine light on it were gonna cross-link it and we’re gonna make gels.
Okay and I have some blue food coloring here. And these are hydrogels so they
have a high water content.
Anywhere from about fifty to ninety five percent water. Well we can mold or
shape them in any object that you would like. We can do it under physiological
conditions. So we can put a cell in here and trap a cell within this hydrogel
matrix. We can do it in situ. So let’s say you have a tissue defect site, I can inject
this fluid in there, come in with a fiber optic, cross link it, and form a well
integrated matrix. And finally by picking the right
polymer, the right dendrimer, I can alter the physical characteristics of this gel to
match the soft tissue I am interested in. So maybe you want something very stiff
or maybe you want something very soft, so the question comes back to the
clinician. What types of materials do you want and how do we think about those
materials at the basic level and then move towards the engineering side to
generate prototypes? So here’s an example,
here is a dendritic structure. We make a gel out of that material. It has a
modulus, around ten to the sixth. That’s a pretty hard plastic. If I take this
structure here, and make a gel out of it, its like Jello at home, if I make a gel out
of this material, it’s kind of like a goo. So the point is I can change my material
properties over a wide range by knowing exactly what my compositions of my
macromolecules are and what the
composition of these dendritic structures are. So we got interested in corneal
wounds and in particular to full thickness corneal wounds and as you all know
these can be caused by trauma, infection, and inflammation. And were gonna
look at 4.1 millimeter wounds here. If you think about it, when you have a wound
in your cornea, your cornea collapses. If you don’t repair it soon, you’re gonna
get scarring and that’s gonna disrupt
your vision. Standard care right now is to use sutures. Every time that you run
that needle through that cornea tissue, you’re inflicting more damage. Once
you’ve sewn it off, you need to make sure now all your stresses are equivalent
about that wound site, so you don’t have any asymmetry that would lead to
astigmatism. So what we are interested in doing is taking that wound, applying a
polymer to it, have it seal the wound. Have the
intraocular pressure increase, have that material be more elastic then the cornea
so that you can reduce the chance for astigmatism. Have it be the right refractive
index so you can see through it and then have it degrade as new corneal tissue
grows back into it, alright and we’ve been able to do that. It’s been a really fun
time in the lab. So here’s a nice linear wound. And the point is the number of
sutures that would go in to it. Of
course you know, sealing something like a perforation is much more difficult.
And again, we have to worry about all of these concerns that are out there with
using sutures, and at the end of the day, we take the sutures out. So at 4,6,8,12
months, you’re going to take the sutures out. You’ve inflicted more damage
when you’ve taken the sutures out and you’ve left another site for infection. So
again, can we come up with alternate ways
such as using a sealant? So what do we want? We want a material that will
adhere to moist corneal surfaces. We’d like something that can flow through
very very small needles. We can put it in place and have it stay there in place.
We’d like to seal a wound. We’d like to restore the intraocular pressure, of
course maintain the structure and integrity to the eye. Have it be elastic, match
the right refractive index, and of course we
would like it to promote the healing response there in the cornea. So in our first
experiment, we took some enucleated eyes, we made a full thickness four
millimeter wound, and then we either repaired it with sutures or with our sealant
and then we injected fluid into the eye at a constant rate to see at what point
failure would occur. So again, we go through and we make our wound, we apply
our polymer, and we can cross
link, and what we found is that sutures will hold up to about eighty millimeters of
mercury and then with our dendrimers going from a small dendrimer , the
medium size, large and extra large, we found that one of them was quite good.
So the question is why? What’s going on? Our working hypothesis is that we
have an interpenetrating network being formed. So you think about your protein
damage site, proteins are exposed, the
polymer comes down, and now when you cross-link that polymer you physically
trap the protein in this gel. And that’s where the adhesion comes from. If you
have a very small dendrimer, and you have very few groups, so you don’t get
efficient crosslinking so it doesn’t gel well. The dendrimer gets too large it cross
links fast, but it becomes a little bit hydrophobic and now after its gelled you can
pull it off and it delaminates. So the
question is, how do we design materials that will promote this inter penetration
and hydrogen bonding interaction with the gel. At the same time have a material
that will cross-link and fix it in place that we can get good adhesion. We did a
few more eyes and as you can see sutures are around eighty millimeters in
mercury, 4.1 millimeter wound, our sealant somewhere around a hundred, and of
course this is where the normal
intraocular pressure is and this is probably a realistic, you know upper limit. So
the point is we’re in a range that is very reasonable. You can do this for other
types of wounds for example, for , LASIK flap complications. Of course you
make the flap then you come in through with the laser and you oblate the tissue
here and you put the flap back. If that flap gets dislodged, or if you get an
infection that’s gonna be a problem, so you typically
clean it out, give antibiotics, and then you would suture it into place. And so can
we use this material to actually close a wound? such as a LASIK flap, and sure
enough we can go through the process and it works quite nice, and sure enough
at the end of the day we can make material that’s gonna secure that wound and
keep that LASIK flap in place. And we have a little extra dye there so you can
see it. So with that data we actually
moved on to an in-vivo experiment and we’re looking at we used the chicken.
Chick has very similar corneal structure to our own and we used thirty animals
received the adhesive, thirty animals received sutures. We did slit lamp
examinations in Seidel tests from about six hours out to day twenty-eight, and then
histology. And what we found is at day one we see the anterior chambers are
reformed, we don’t see any leakage so that’s
good, and by day seven out IOP’s are all back to normal. If we look at day seven
with the sutured case you can see, you can see some corneal scarring and some
hazing. Day seven with the sealant, you still see the scarring but you see less
hazing, so that was encouraging. And the histology at day seven, of course, you
see the sutures, and of course you see that the tissue is fairly irregular, and that
our epitheliums become
detached from the stromal. We think that’s because of the suture stresses put on
the wound there. With the sealant the tissue is a little bit more uniform and we
have a much better interface between our epithelial and our stromal tissue. We
go out to day twenty eight again you can still see where the line was from the
wound site, and in the sealant treated case, much more uniform stromal tissue
and a fairly good interface between our
epithelial and our stromal tissue. So in the midst of doing all this, we started
talking more with the clinicians and they said can’t you just make something we
mix together and it gels just like that? So we borrowed some nice chemistry from
the protein and biochemistry world and what we are going to do is take a
dendrimer that has N-terminal cysteine which is this group here, and have
pegged dialdehyde. If we mix this two
together it will gel and form these nice optically clear gels to pH 7 in a matter of
less than a minute. So we thought well, what's another procedure were you might
like to use a sealant and that would be in a clear corneal incision. You go in,
make your cut, come down and you would of course remove the lens put in the
intraocular lens, now at low pressure what happens is fluid from the outside
actually gets pulled in and that’s where
you can have an infection and MacDonald as well as others have shown that
there is really been more of an increase in ophthalmitis with this procedure over
time, so again can we close a wound such as this? Here is a three point
millimeter wounds again we did the self seal or the current treatment closes
somewhere or sealed somewhere around twenty five to thirty millimeters mercury
sutures up to around fifty and our sealant at
somewhere around one hundred. So the point is we can design materials that
have good tissue adhesion and good strength so that we can close wounds. So
to wrap this up I’d like to tell you we have some very nice ways to synthesizing
these polymers. We can build them out of a variety of building blocks, and we’ve
successfully sealed linear and stellate a corneal lacerations in-vitro and in-vivo,
we’ve done LASIK flaps, clear corneal
incisions and corneal autographs in-vitro. The sealant is about four to six time
faster you have lots of control with it and it’s a nice soft -rubbery texture. So why
a dendrimer? Why these types of materials at this level? Well, we can obtain high
cross-link densities at low polymer concentrations. In other words, we can have
materials that have high water content, and that promotes healing and gives us
the beneficial biological response.
We can make low viscous aqueous solutions for placement on the wound and we
can vary their physical properties. And I really believe these are some really
exciting and new macromolecules for ophthalmic applications. And I'm happy to
report this has actually been licensed and in the next year or two our hope and
plan is to be in the clinic with this type of material for corneal wounds. I need to
thank the students and my collaborators
they did all the work I just have the joy and pleasure to tell you about what
they’ve done, in particular Michael Carnahan, Michelle Worthier, and Lovorica
and Kim were instrumental in the very beginning of this project getting it off the
ground and my long term collaborator at Duke, Dr. Terry Kim in the cornea
department there. It’s been wonderful to work with him and his fellows, John Kim
Christianson and Al Stark. And
I'd like to thank the NIH for their funding. All of you for your kind attention and I
look forward to discussion later today. Thank you.

				
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