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.