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>> I've jumped on the nanotechnology bandwagon and since getting invited me today I'm going to be talking more about our work in the bio tech area I think in nanotech area then biomaterials in tissue engineering. I'm going to save that talk largely for Hilton Head and my golf clubs.

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So I want to give you two examples of how we've been trying to merge the capabilities of an area of material science that is focused on intelligent materials with applications in biomedicine. And in material science there's actually a pretty well developed wing of that field. That's been concern for many years with the development of a class of materials called intelligent materials and I know when I first came to bioengineering from my background more in bio chemistry and biology.

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Now I was a little skeptical about how smart these materials really were. But I've become convinced that they're pretty darn smart and the basic property of a material that is termed intelligent is that it can sense and respond and it can do so. Reversible E. and so upon an input or some sort of stimulus in active or intelligent material and then undergoes a change in its structure function properties to provide an output a response type of signal or change in properties.

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And a great example is a strain sensor where the input is mechanical defamation that leads to some output in signal called voltage in the degree of strain. Modulator the degree of voltage and so you have this coupling between a signal and a strain and these materials. Ideally are fully reversible so it can sort of be a reporter of string.

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Our own interest has been to couple these kinds of active materials that change their properties in response to environmental signals or changes with the control of biomolecular activity such as protein binding enzyme activity D.N.A. hybridisation And so what we're really talking about in this case is a set of materials that are polymer based.

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Rather than a kind of book material. And so the biomolecular materials that I'd like to describe to you are hybrids between smart polymers in protein or D.N.A. I'll focus on protein today. And what we're interested in doing is coupling the changes in the smart materials such as changes in conformation shape volume their mechanics changes in signal output chemical properties and use that.

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For applications such as switching bio activity controlling in Xyrem reactions to couple to buy a sensor or signal detection. Process that we call bio matchmaking where a complex mix yours of bio molecules in for example diagnostic applications you can control when a particular component is turned on or off when and where two particular components such as D.N.A. and protein come together and and to do that with a time controlled element so that you can do it sequentially from a complex mixture and and this really isn't possible to date with current technologies.

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When you put molecules in there. There aren't any really good methods for controlling which ones are on which ones are off and so things usually have to be sort of control. Temporally in terms of which components are there you can't just start out with everything you have to add them sequentially to control that time axis.

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So in the first part of my talk like to address this development of what we call smart biomolecular components or materials that control when and where molecules come together and devices and when and where their recognition properties are turned on or off that sort of the functional part of it and the polymers as you'll see serve as both an antenna to allow us to couple in this is sort of the talking part from our viewpoint and it's the listening element from the biomolecular material component so this provides a listening capability that we can send signals into but because they're active materials and change their properties that change in properties represents an actuator kind of function and so they both sense and then they can talk in so they kind of listen and talk and this change and in say as I've just drawn the cartoon this change in confirmation from more extended higher volume confirmation to a smaller conformational change can be used to for example in the simple cartoon block the association of two proteins in the collapsed form then staring at the two proteins you come together in this represents an on state.

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Whereas this is an off state and we've really become particularly interested in biomimicry microfluidic applications of these kind of capabilities where you could for example in a microfluidic stream. Again stream. Again inject complex mixture is of proteins and control where in a stream by controlling where this signal that converts this polymer into the collapse state.

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Kurz and so may occur at a given spatial location compared to where they're injected. So a little more on these smart polymers they exist in one state as an expanded coil that's very hydrated and depending on their composition then with small changes in ph temperature as I'll show you with specific wavelengths of light you can derive an intra P.D..

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Directed collapse. That's driven again by the entropy of water release and so this collapse state can be generally considered as a more hydrophobic state. In this hydrophobic state they can actually also aggregate and this is also very interesting for microfluidic applications because you're diffusive properties of these aggregates change and they can do so.

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Reversible a so you can bring molecules together if you hook them onto these polymers or drive them back this way. Or in some applications you're just interested in using this confirmation to control activity and another property of these smart materials though is how sharply. They enter convert between these two states.

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This is one of the best studied the smart polymers poly in US approach will occur. This is the chemical structure here and it's this poising of water between this our Kilmore hydrophobic group and where a can hydrogen bond. To these positions that drives this sharp intra be change as a function of temperature in so you can see over just two degrees Celsius you go from a clear solution where these polymers are expanded in isolated polymers to this collapse and subsequent aggregation that then scared.

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There's light and becomes target and this is fully reversible over this temperature range with in us probably acrylic. And you can see how sharp that transition is so it's a what's called a critical of it. Now what happens. It's a very sharp switch. Now if you cope Plimer eyes Nipe am with more hydrophilic monomers for example such as acrylic acid you can shift at what temperature and control it.

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What temperature this transition occurs and that will also a factor and sometimes broaden it out a little bit so that you can also rather than just a pure switch think about intermediate confirmations between this and that. So our first idea in this illustrates the concept was to couple these smart polymers in this is a cartoon of a protein here near the active side of a protein so this may be a ligand it might be a substrate if this was an enzyme and that we would position.

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This response of polymer at a special location that was fairly near the active site. And when we then triggered this reversible change to the collapse state as we initially initially drew this up. We pictured that you might affect the off rate so that you could object to leg and or you might block the re Association of the leg and what the protein just Eric Lee and what we've demonstrated is that this general concept is valid and by controlling again the composition of this polymer you can do this with changes in ph that are one to two ph units so very friendly to bio molecules with light or with temperature in again concept is that the polymer is the listening device the antenna and the actuator to control this active site to turn proteins on and off.

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Now. In a little more detail. We've done a lot of work initial work with a protein called strapped ABBOTT And that's one of the most used in the biotechnology field in diagnostics and in laboratory assays and this is a molecular model of struct Abbott and shown here in purple and what stripped out and does is bind a small molecule called biotin his chemical structure shown here and from the X. ray crystal structure you can sort of see the biotin and here.

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It's a tetrameter So there are four subunits here each binding one molecule biotin what we found is this is a model of in a surprise that approximate molecular weight that we've been working with and this is sort of a schematic to show you that if you hook it up here near the binding site you can sort of picture how it can serve as a gate to the Association of a molecule into this active site.

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And so the polymer we've found can serve as a very effective gate to prevent the association of biotin on to the protein. What we've also found on the other hand is that we're not very good at once. Biotin is found at a jacked in the biotin So getting biotin back out and if you really look at the structure and you can sort of see the difficulty here.

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This is a very big buried binding pocket. You know if I really showed you all the atoms on the protein. You wouldn't see biotin at all. You just see this very end this car box in the group which is out on the surface and so the polymer can't really access this more buried binding pocket and so it's been very difficult for us to come up with the way of coupling this change of the polymer to the ejection of biotin Because this and isn't really attached and so you don't really exert any force.

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On this kind of scheme where we have been able to manipulate. The binding in this buried pocket is by you. Using a polymer that is not only just interactive but it has reactive chemistry is chemical moiety is scattered along the backbone and in this case we engineered a unique cysteine residue at this position shown here relative to the binding pocket and it is directly adjacent to one of the most important binding contacts trip of fan at position one twenty that we knew from other studies was very important in this binding a biotin and so we positioned this attachment site here.

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But because this. Polymer can be grafted at multiple points walk and happen is this is Diski magically the polymer can be attached at multiple points such that when it collapses it can actually generate force. And what we've seen is that this is the only way by using this pendant polymer where we are cross-linking at multiple points that we can get release and so this is a control where we just look at biotin the amount of bytes and bound on the Y. axis as a function of temperature so you're going between the expanded and collapse state of this polymer and there's that control just tells you that there is no temperature dependence of biotin binding and that's a problem.

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There's no reversibility to this binding on the other hand with this pendant polymer we can at least begin to see some release when we collapse the polymer we lose about fifty percent of the bound by it and this can be cycled. So that eventually if you go through many cycles you can get rid of all the bound by it and so it shows you the capability that you have in controlling how the polymer is attached we control where it's attached that's another important design parameter and sort of generate generates this model that got us going OK what I want to give you are some.

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Unpublished study. Where we've looked at a sort of a different class that I think is much more amenable to this idea of proteins that have exposed pockets and so the problem here is that the Palmer doesn't have any access to this buried binding site but if we look at an enzyme then I want to talk in more detail now about Inzamam.

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Here many enzymes and many other types of protein such as antibodies have a much more exposed pocket and these are three residues in this particular ins I'm that are the important catalytic site residues in this entire group it defines a large open groove on the exterior of the enzyme that's excessive all to polymer and this is a very different setting and we've turned to spit the results saw a lot better.

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Switching activity. So in this particular example what we're after is generating our first photo response of switch in our first application with in zines the idea again is similar. Now this becomes an enzyme but it becomes a bigger more open cleft for the substrate to come in but it has the same idea with the photo response of polymers what we're after is switching in different directions with different wavelengths of light.

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So for example you could go to this off state collapse of polymer with far U.V. a radiation at three hundred sixty nanometers and then go back the other way with a distinct wavelength of light of say four hundred twenty animators. And after a lot of work a very talented graduate student.

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He developed such a polymer and this is the chemical composition it's a CO polymer of dimethyl acrylic. In an age so been seen moiety shown down here and he actually developed an all sort of be giving results to different. Classes within this general family of CO polymers where the.

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Been Xen. Group is attached either through an ester linkage call that the D.M.A. had to really remember that or an amateur linkage D.M. am and. The important thing about this. I'll show you in a second is that for reasons we still don't understand that subtle change in astro vs Amad linkage give this these two polymers opposite photo responses so that you can either go in this direction with U.V. and back the other direction with four hundred twenty nanometer light or vice versa you can go in this direction with visible light and go back the other way with far U.V..

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This is driven just really quickly by the trans this photo I summarization of the a's are benzene moiety. And why we think that this is coupled to this. More macroscopic change in the polymer going from expanded to collapse is the fact that in the trance state. There really isn't a very significant dipole moment which affects water ordering again whereas in the cis state you have a much larger dipole moment.

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And that's coupled again to driving this water release that underlies the change in the polymer confirmation and it's fully reversible by these different wavelengths of light. So this is what that sort of temperature and photo responses of these polymers are it's a little complex because they're both temperature and photo responsive which is very useful from an engineering standpoint.

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So again we're looking at her ability of solutions of these polymers so that when it's clear you're in this expanded hydrated isolated state but up here you're in the collapse state where they aggregate and scatter light. In so you can see first of all that we've selected the CO polymer composition.

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So that this transition occurs at around forty degree cells. Yes and that was chosen very carefully because that's where this particular in Zion Target has its maximal activity. But the interesting thing is that if you study where this transition occurs under U.V. versus visible light you can see that with this particular polymer this is the Ester linkage the U.V. light increases E L C S T.

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And so down here somewhere in this range of a slightly above forty degrees. If you look vertically at a single temperature you have this maximal difference where the polymer is more collapsed up here and less collapsed. Here under U.V. versus visible light and so at that isothermal temperature by shifting the polymer between these two photo radiation States the polymer exists either more collapsed or expanded and the interesting thing that we still don't understand very well is that the AM Amad linked.

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Polymer goes in just the opposite direction so you the lower Z L C S T So you can just go in the opposite direction which may be important for some applications but not so important there radically. So we've been working with a model in Zion called Indo glutinous twelve a clever Asian region in core International which is a big producer of in zines and maybe I certainly didn't appreciate what drives gin and cores business they're the second largest in design by you know what drives their business Balkans Imes.

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A couple of things one is laundry detergent. If you if you've seen the commercials where it has some special component that degrades protein stains food stains they put in zines in there and it's mainly a marketing thing the other big use for glue going to ace in zines.

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What these do is degrade cellulose and so what these are you. For is in making your jeans look like somebody else wore them before you by degrading to a fine degree of control how much fraying how much sort of stone washing you know how what kind of look that they have and so the ends of magically degrade the Cellulose Fibers and that's what these in zines do.

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And these are the catalytic site residues that are involved in this just a model show you. Roughly kind of what size we would expect for these stimuli response of polymers relative to this particular in Zion and what we do is going to genetically engineer handles here by putting in Sistine residues that define positions and then hooking the polymer up and but by deciding where you put this hook you can move the polymer sort of closer to the enzyme active site or farther away and this is a bigger better cartoon this would be a model of the substrate.

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It's a really cool enzyme it kind of looks like a hand in the hand just grips around the cotton fibers. And that allows it to degrade along the fiber length and so you can see relative to the substrate there's a very exposed pocket. The idea is in the expanded state the polymer would not interfere with that this would be the on state.

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It was say a change in temperature or show you photo radiation you would collapse the polymer and it would sort of block off this substrate from being able to get in there and that would be the off state. We found that there are two key parameters of conjugation side in the polymer molecular weight in terms of the molecular bioengineering that's part of this.

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This shows you a little better side view so that you can see that the fire would be pointing into the plane of the screen here and it's kind of a hand that wraps around that and then I'm going to show you some results. By conjugation a Palmer at this position which is very near and located sort of down around this axis so that.

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We picked this position when I designed these so that the fiber couldn't get into that fit all the way through that groove so well. Whereas this one. Position was moved a little bit away and then we also have a control where we've conjugated to this in terminal position which is the farthest away from the binding site.

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We use pretty standard chemistry these polymers have been modified with a so fired roll. Reactive group of vinyl so a foam group so that the Sistine that we engineer into the enzyme can react and just with the end of the polymer So this is an example of the polymers just in grafted onto the enzyme.

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There are two Application formats in two that are commonly used in all these devices and some of these you want to use an immobilised conjugate and there's a number of different ways that we hook these polymer protein conjugates on to say magnetic beads a chemistry isn't so important but we can also then view this as a switch for free proteins that might be in a microfluidic device for example or just in a laboratory assaye such as P.C.R. something like that where the conjugates are brought together into aggregates and this aggregate what we've seen for example is in the off state.

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But again it's reversible to send it back out to this state where the polymer is more expanded and this would correspond to the on stage just free and solution. Well how well did these work we were surprised because we work so hard with strep Davenant for so many years and I really think it's a function of this exposure of the binding site but these work really is spectacular switches.

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So this is with a small molecule substrate called O N P C. At forty five degrees Celsius like I showed you at the sort of optimal ice of thermal condition. Sorry this is the wrong. This is. Super chair this should not be isothermal show you that in a second.

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This is the temperature response at thirty two degrees where the polymers expanded and you get about eighty percent of the activity this is a real drop I mean just having a polymer around there does not down the activity of about twenty percent but that isn't so important. Really what's really important is that when you collapse the polymer.

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You get a nearly complete turn off of the activity of the enzyme and this is that position. This is jargon by us protein engineers showing that we've changed a naturally occurring asparagus in it I mean no acid position fifty five to Sistine And so this is attached very near to the active site.

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And that's what is going on there. This S twenty five C. is moved a little bit away from the active site but still pretty close. And you can see the general trend that as you move it away in the off state here when you're collapsed there is significantly higher activity about twenty percent activity and then when we move it to predominately to the end terminus this is an ideal control I would really are trying to do is make a control where we're attach back here but you can see the general trend when we move it a little further away yet then you begin to get significant activity at the higher temperature where the polymers collapsed and so the conjugation position sort of makes sense.

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Just from a simple Sterrett model. The molecular weight also shows a trend. Although it didn't turn out to be so important. These are our with this is what the Astir polymer this is what the Amad polymer is a jargon here but again with polymers of ten thousand molecular weight or greater as we've seen you get this nearly complete turn off.

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When the polymers collapsed as we go down to a six thousand molecular weight polymer you begin to see more activity in that off state and at three thousand a little higher. Yet in when you go all the way to. Just physical mixtures you see no difference in activity between thirty two and fifty two degrees and so it does require conjugation and there is a molecular weight effect although even the three thousand molecular weight polymer at these positions works pretty well.

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What we're really interested though is the photo response of and so I have splices in incorrectly Now this is the isothermal condition so we're at forty five degrees where there is maximal difference in the polymer being expanded versus collapsed under visible versus the U.V. photo radiation and so what the Astir linkage.

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I'll just remind you under U.V. conditions the polymer was expanded and what we and indeed see is that that's in the active state. Now what's interesting is that here we have lost about forty percent of the activity which still actually isn't from an application standpoint. Really all that significant.

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It's still active enough but what's important is that when you collapse under visible photo radiation go this state. It's a pretty darn good switch in the same with the Amad link you go between the on state and the OSCE date but in this case the Austrade is U.V. versus visible and this is a control interesting control where we just put that a Zo been seen monomer So it's not the polymer just a small molecule on to the same position and you can see that the small molecule.

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Photo I summarization does not act as a switch so it has to be a larger polymer to get this kind of switching activity. And in terms of the reverse ability then in the cycle ability these are also really beautiful and much better than anything we've ever saw a stripped out in and so if we look at thermal cycling first what we're plotting here is total product accumulation as a function of time and so you're just sitting there with a Q. vet with.

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You're conjugate in there and you switch it between thirty two degrees and fifty two degrees and this is kind of like real time here on the axis and you just switch it back and forth and measure product accumulation. And so both of these polymers have the same temperature response both the Amad in the aster so you'd expect them to be the same.

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So at thirty two degrees where the Palmers expanded you accumulate product the enzyme is sitting there working away but when you shift it to fifty two degrees just in the time that it takes as you know it. Fastest we've looked at a sort of seconds here of manually.

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I'm shifting this to a fifty two degree water bath. It's turned off and it's turned off. Indefinitely for as long as we looked at it but you shift it back to thirty two degrees and now you're at switches back to the on state and so you can accumulate product you can turn it off and you can keep cycling it like this.

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So it's a real time switch and with the photo cycling we've seen similar properties but the two polymers now have opposite responses so under visible this particular D.M.A. M. polymer is in the on state the expanded state shifted to U.V. again in real time it's off on and off and the other one just has the opposite.

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Photo response. So these conjugates really show the properties of what we were after initially in terms of being able to turn things on and off in a reversible fashion. So in summary where we're really heading with this are in applications such as microfluidics streams and it holds not only for proteins but we think also have some very early results with D.N.A. that are interesting but again you can control.

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You know sort of which components come together such as the green versus the purple and this represents a time axis because at this point if you're shining the light at. Disposition of microfluidic stream. Then the purples may be in the on state and working whereas the Greens are turned off and this is totally reversible so that you can capture D.N.A. molecules release them at different times points in a more complex device environment or maybe just a laboratory assaye system where there isn't necessarily have to be a microfluidic stream.

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You can turn and signs on and off or just activate them. And so it's sort of like a platform capability that we see lots of applications for. It's an experiment that we're trying. And so were initially trying it by a micro injection. Because there are many enzymes. For example kinases where you could have it turned off but then turn the kindness on it had to find time with a light stimulus for example where that in there could function for a given set of time where you might turn on a signaling pathway for example or turn off the pathway.

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But we don't or we're far I guess from having a result we disorder of. Dried some initial experiments. The conjugate in that case so it's one of these with an in Zion. Where the polymer would be positioned more appear around the active site where it be in an off state at thirty seven degrees.

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Because that's another reason why we were interested in this forty degree range but then with light. You could switch it. On or Off can go in either direction. All right a second example that's a little less biotech E. and maybe a little more drug delivery. Is a different set of polymers in here what we're after is a very different goal but it shares this property where the polymer has.

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Percival in Vironment only specific changes in properties in this particular application the what we're after is the more effective intracellular delivery of bio therapeutics and so these are sort of the new biotech drugs that are based on D.N.A. It might be plasma D.N.A. for gene therapy. And what we're interested as is trying to make non viral even remotely competitive with viral delivery efficiencies or one that we've developed quite a bit is with antisense nucleotides you are talking about small linear pieces of D.N.A. or protein drugs that function inside the cell.

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To date. All of the protein drugs that are out there function on the extracellular face of the cells. None of them really work inside the cell directly but there's a whole class of proteins that people are interested in and shown by my current junction have interesting firm suitable properties such as transcription factors or neutralizing antibodies that if you could effectively get them across this barrier.

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You could have a wholly new classes of protein drugs and also R.N.A. therapeutics and the core problem is really simple these macromolecules are not transported across a cell membrane effectively and when they are taken up they're taken up into of a secure trafficking pathway where they're taken up into the end as OMH in the real problem isn't the end his own per se but the fact that this is rapidly traffic to the degradation.

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Degradation compartmentalizes And these are all degraded in that compartment. So our technology that we've been trying to vel gets things out of the end is ome into the side of Plasm where they can then diffuse into the nucleus or reach their target. From the side of Plasm. So when we started thinking about this what we were after.

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Is this. And that a delivery system that could could address this need. And if you. It's informative to consider how biology has solved the same challenge viruses of C. solve the same challenge because they have to get all their D.N.A. efficiently inside the cell and to the nucleus not just into the secure trafficking pathway pathogenic organisms such as diptheria have have the same problem in that case it's a protein drug equivalent that they have to deliver to the side of Plasm and they share some important properties.

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You can think of these is really like a little drug nanoparticles drug delivery systems and there's proteins on the outside of these viruses are retrovirus example that direct the initial uptake targeted uptake and so that's one part of their drug delivery system ness pretty well appreciated I think what's less well appreciated as if they have a very separate component but a very efficient component that solves this problem of getting out of the into zone in it's coupled to a unique gradient.

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That's generated in the end as they have an A.T.P. dependent proton pump that lowers the PH all the way down to five in late into zones. So this is a large chemical gradient and this is what these organisms evolve to take advantage of out here. The proteins that destabilize membranes biological membranes or inactive because you're at seven point four as a PH drops they become activated in destabilize the membrane so that you get enhanced transport of the virus out of the end as ome and from there.

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Then another series of miracles happens to where it diffuses or is actively transported into the nucleus where you can then get final gene expression. If you look at this a little higher degree of resolution there's a fair amount known this delivery system component has a. PONY called him a gluten that represents this smart component that can sense the ph ph seven point four The structure of this protein has been pretty well characterized here it is it's inactive and so is this is we're looking inside out.

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This is inside the end is ome ph some point for initially it's an active and it's inactive as this still fully goes through your body. But as it starts to sense this drop in Ph. It undergoes a big confirmation change in the exposure of side chains that can then destabilize into zonal membrane and that enhances the transport of the cargo the D.N.A. or R.N.A. across that membrane and the way this works.

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A little more specifically is that there are amino acid side chains that are proto native there car box like groups and so there P.K. matches biology and as their protein needed that simple change going from A C O minus to C.O.H. triggers this change and it's been well characterized as well.

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Characterize also in depth theory a toxin There's a cue card box a little residue that is the PH sensor that triggers this same. Membrane destabilisation So we got interested in this because we were interested in whether you could sort of learn from how these viruses was we're doing this but design you get into synthetic polymers.

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I mean polymers that have an intelligence in that they can sense and change their properties reversible. In regard to PH and the advantage of polymers is that they're not viral proteins which are extremely immunogenic they're also extremely expensive and while they've shown promise in the laboratory they're not practical from a real life engineering standpoint and polymers have a lot.

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Of advantages and are much more realistic to think about than these viral proteins. So I want to just close by showing you one example of a family new family of Palmers that sort of are mimicking this viral. System in the key is it has different parts as we've designed at the backbone shown here an orange is going to have this capability of it low ph is being membrane destabilizing but it ph some point for being inactive and then we've grafted onto that that.

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Membrane disruptive element element through a Ph degradable winker the actual cargo and targeting element so a targeting log and or protein or nucleotide can be attached through really simple chemistries in a peg group through this ph to degradable linkage to such a backbone and the key here is that we're going to generate this ph degradable this ph sensitive sensitive element that decouples the cargo and targeting ally against from this membrane disruptive element.

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And the concept then thinking of the application is that this is a poly Merrick delivery system that can target receptor mediated into psychosis it doesn't have to have targeting but it can but the key new thing is that this is a functional polymer it's not just a carrier and so at low Ph.

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This falls apart in this backbone that has this ph dependent membrane disruptive activity can partition and destabilize this membrane and enhanced the transport of these elements that then sort of break away like the lunar lander module haven't Hance transport to the side as also these are functional polymers a direct into zonal escape of these bio Molecular Therapeutics.

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Here's a first generation polymer does show you what the chemical composition is. And this was sort of based on some preliminary work that we had done it didn't come out of the blue but this is a Turco polymer of butyl mouth accolade and this is the membrane disruptive element.

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And this is a key part of it here. This is a stiring bins out to hide that has an acid tell link or linkage here and these are the PH sensitive chemistries and the nice thing is that by tuning the specifics of the star in bins out to hide for example putting oxygen versus nitrogen it affects the electron withdrawing capabilities and you can tune these kinetics from minutes to days hydrolysis rates as a function of Ph.

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So when you lower the PH these bonds are cleaved and these peg groups and whatever go with them are liberated and so this is sort of the separation and then this is the membrane disruptive element. The design again was really crucial here to couple it to the biology and so that normal trafficking kinetics and going from the in his own to the lysozyme or fall on the timescale of say zero to sixty minutes.

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OK And so this is a time frame that you have to beat. OK And this really a big challenge because it's seven point four seven point four It needs to be stable but then within you know thirty to sixty minutes as this is acidifying that has a time aspect to it that needs to fall apart.

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OK so it has to fall apart fairly rapidly and yet be stable and again lots of great work by a graduate student or a mercy to care of this and so that the hydrolysis if you look at hydrolysis sort of the breaking apart the PH dependency. It's a very slow background of Ph seven point four as individual groups.

[00:42:00]
Hydrolyzed So these are stable on the time frame of a date at two days at seven point four before they become member in active but very quickly on the right time frame of zero to sixty minutes at PH five point four C. This rapid hydrolysis and that it turns out is just directly a function of the proton concentration.

[00:42:23]
Well how about the membrane disruptive properties. Well we use an inside out assaye that allows you to calibrate versus other agents that a bit peptides and proteins for example have been developed where you add the polymer and you ask as a function of PH How well does it liberate hemoglobin from inside the red blood cells so it's kind of a model membrane now mass a tick to see how active are these polymers and well after a twenty minute incubation and this is really again out today is it Ph seven point four.

[00:42:56]
You don't see enough hydrolysis to give you membrane disruption. But at PH five after just twenty minutes. This thing. HUME allies is all the red blood cells under the standard conditions at of are sort of a gold standard in the field and so what this means is that this polymer will liberate.

[00:43:19]
All the hemoglobin from ten to the eight red blood cells in one mill and P.B.S. How does that rank. It's actually or three to four orders of magnitude better than the best viral protein and we've seen this with a whole set of these polymers is that they're actually more active membrane disruption than the biological proteins are it's kind of surprising that you have a sample of amplification effect because there are large polymers versus small much smaller proteins.

[00:43:52]
Just to give you one example to show you that these really sort of do work and we've done plasma D.N.A. we've published that. Also published just recently I think it's just coming out with a protein but I want to show you a biomaterials application. One of our big interests and I'm sure you heard about this last week with Buddy.

[00:44:10]
Is a control of the macro phenotype and control. Of activation and one class of drugs that we've become very interested in our antisense all of the nucleotides that knock out specific. Gene product or the messenger R.N.A.. That are tied to particular gene products and so what we've done is just modify the peg groups here with man knows that targets Man us receptor is on the macro.

[00:44:40]
And in this case we just put on. Lysine groups to Lechter statically complex on the negatively charged antisense So this is the full carrier. This is a group that is then designed to be wants to takes up these polymers to hydrolyzed and liberate the cargo and I'm in this with an enhanced transport out and it turns out that the macro fires been a really tough model and I was the other reason why we want to try this because while macro fires are very efficient at taking things up.

[00:45:12]
They have an unusually high concentration of degradation in zines So it's been very difficult to get antisense in America if I was really hasn't been very possible practically to do that and also the same is sort of true of proteins in D.N.A.. In our particular target it was done in collaboration with Ron Mayer is a surgeon at the Big One of the big hospitals in Seattle at Herbert view and one of his residents joked to Sherry and they identified Interleukin one receptor associated kinases a good target for antisense therapy and it plays a key role in this.

[00:45:58]
Believe me as an abbreviated signal. Pathway representation but the key thing that's tied to downstream that we're going to ask for is tumor necrosis factor and we wanted to knock this down by knocking debt and by knocking out. This Iraq transcript and Iraq is a good candidate because while it's tied to phosphorylation a vent that regulates its activity.

[00:46:20]
It is ubiquitous aided and so it's degraded. In the Proteas Nomen So if you knock out the transcript. You can knock down the levels unlike a lot of other intracellular kinases who are sort of constituent of Lee produced and present. So there's a lot of data here but I mainly want to point out that what we're looking out for each of these samples is a non L.P.'s stimulated versus L.P.'s stimulated production of sorry that should be T.N.F..

[00:46:53]
Here on the Y. axis. So when you stimulate. Normally you get a big operate elation of T.N.F. and want to ask. Can this anti Iraq. Nucleotide knock that back down and the polymer in we've seen this in a lot of different toxicity assays and it's also been sent through some very initial In Vivo screens and has very low toxicity and the reason is because it's seven point four It's not active.

[00:47:23]
OK And so this was a good sign a polymer did not sort of super stimulate further T.N.F. production which these cells are very sensitive to another control is the sense story and so this is not an anti science and it can hybridize and lead to degradation of the target M.R. in a so it's up there.

[00:47:42]
The anti-science alone may have a slight effect but this is the problem when you just put in antisense Olive goes. They're not efficiently delivered and so you don't see really knock down into enough production but finally if you look over here with the antisense plus polymer delivery system we get very sick.

[00:48:00]
If it can knock down T.N.F. production. And so we've seen this with several different types of antisense also in the macro and other types of cells and so. What we can say at this point is that in cell culture. These seem to have usually high activity getting antisense And we've also in different cell types like I said done proteins in D.N.A. The concept is sort of general for all these classes.

[00:48:28]
So in conclusion then we've developed I didn't have time to really show you. But there are really a whole different families of chemistries of these polymers that have the same property of coupling high membrane destabilizing activity with controllable ph profiles and if they're very interesting because you can control does this happen it.

[00:48:48]
PH six point five or does it happen at five point five and that's important. Biologically Because that this is a really a gross oversimplification the into zome is evolving at all times toward the license. And there are different biologically and it may be better to disrupt earlier maybe better disrupt later for all we know and so you can investigate that by controlling at what PH these become active and we've seen that it enhances gene and protein delivery with low toxicity.

[00:49:20]
This was a PALMER I didn't have time to talk about but the encrypted polymers say the same. So just in conclusion then I'd like to acknowledge certainly my good colleague Alan Hoffman We have a joint group working in these areas as a person who did all really of the work that I showed you the second I'm giving his name to Bob because I think he's looking to get ready to look out for some academic positions being dead and say sheesh I'm a bogey did the smart polymer protein work that I discussed and we have many nice collaborators.

[00:50:00]
Ron Mayer and Joker Sherry in particular on that antisense example were very important and this work was funded primarily by the end I H. and also by us. B R C Where we're interested show here of delivering these kind of drugs from scaffolds and bio material coatings so that you could enable a much broader class of potential therapeutics to modulating say macrophage after activation.

[00:50:32]
So that's it. Thanks very much. You know last question why. Well that's sort of the computing technology in a sense I think or that's what's always asked of me is how does this comparative to the tap peptides and these peptides that apparently have some ability to move macromolecules across directly across to the side of Plasm.

[00:51:15]
And those are exciting and you know what the way I look at it is that we can easily use those on our polymers number one because we have a modular system where we can put targeting like Ganz or a tap peptide onto our polymer Now the problem with those peptides on the other hand is that they're nonspecific And so for in vivo use you really have to worry about those peptides because the first cell that they get to there.

[00:51:45]
They will try if if they're working as advertised which I have some questions about they will go across that membrane. And so in vivo you have to have some way of sort of introducing specificity to your delivery system now in vitro. They seem very attractive and probably very useful and so but for in vivo you have to worry about that.

[00:52:16]
Well yeah that I think that's an interesting question. Going I think. The question is which dominates though. And so even though you have a targeting ally again it will depend on the kinetics of the bio availability by availability of distribution as to how quickly do you partition to the target site versus along the way the thing is bouncing into other cells and if the tap peptide is there then there's no reason why it couldn't transport Still even if it has a targeting like going for a different cell types so that all that's really interesting I think is just so emerging right now they'll be interesting to see plays.

[00:52:52]
Well we've been in the in the in vivo studies that we've just started are in two areas one is with plasma D.N.A. So it's a true non viral delivery system in this case the polymers are packaged together with a viral system that has a condensing agent and the plasmid And so the the polymer that we have is an additive that sort of again equivalent to human gluten and.

[00:53:47]
Well we hope it's the way the formulation turns out to be very important and we actually think that's important a formulated and lasso that is available and coating the surface. But what we've seen is that it does not significantly affect bio distributions because it's just mainly it's the part of the condensing agent in the D.N.A. And so what this is an interesting case where it may actually help not have so such great in vitro.

[00:54:16]
Properties because in vitro you don't have to worry about bio distribution what people have found is that you cannot use these left as that look great in vitro in vivo because they get taken up by they are yes system for example and rapidly cleared to get around that you Pega late.

[00:54:34]
But then the peg a lation. Really kills you on uptake at your target. And there again then this is skate becomes a very important parameter and what we've seen is that the polymer in these cases where the system has been optimized for bio distribution but exhibits poor uptake the polymer then becomes very important in vivo OK And one study that we've kind of been doing.

[00:54:57]
And so it's it's a very complex. From an engineering standpoint because all these different parameters play off of each other and it's still yet to be seen how well our Palmer really enhances but it's for sure. Needed in the non viral. Too because it's a real barrier getting inside this to the side of Plasm now in the anti sense.

[00:55:21]
Case it's kind of interesting because there when you attach it to a polymer has completely different bio distribution and then the antisense alone. And this can be either an advantage or a disadvantage depending on your application but you've got to play to your strengths and for example polymeric drugs accumulate at tumors because of the leaky vasculature And and so you just have to take that into account and they have completely different bio distributions and they antisense alone and you need to that that will kill you for some applications it will open up possibilities and other applications.

[00:56:17]
Will see I think the that's where the river from a design stand where we've always said it has to be reversible because that if you have much membrane destabilizing activity and these have potent D.C. able to PH some point for your trouble. You know. And so the key thing about these polymers is that as a real cooperate back say it in the side of Plasm once the into zone is disrupted or it's not we don't even know if it's really disrupted it's destabilizes why choose that term carefully that at some point for their back to being very pegged like they're hydrophilic and we haven't seen any toxicity we haven't been able to get a toxicity profile because at seven point four.

[00:57:02]
They're just there's no toxicity and I think that's a key thing that inside the into zone but only at that low Ph. Are they active. Now there are additional more complex questions as you accumulate these and you destabilize into zones too much then there are there are into zonal storage diseases where that that may evolve over time and if these things are accumulated and so I think the bigger question at this point is clearance how are these cleared out so they don't accumulate inside of the cells and that we have not gotten into yet.

[00:57:38]
And of course it's a new Ph D. thesis topic to make these bio degradable right. But still maintain these properties can you do that while it's fun. Academically to think about but I think that my own feeling is that they're not going to be too toxic in their many applications where you don't have to worry about just continuous big.

[00:57:57]
Delivery problems that you can go in with a relatively safe amount and they will get clear just like Peg does pegs approved less with. Now is this for the first part. I mean with for the second part. Well they are there random Co polymers and so they do carry multiple.

[00:58:39]
Doses of whatever the antisense is and I maybe I'm not understanding your question correctly. No it's better. It's cooperate of and in it. Yeah absolutely. Well it's a huge effect if you want to go in that direction. Now we've tried to optimize our polymer So it's just the disruptive element but it's kind of interesting you can couple it with membrane disruptive peptides that are based on these viral proteins and see that at concentrations where those peptides have no activity whatsoever that if you conjugate an equivalent.

[00:59:33]
Concentration of the peptides they're completely membrane disruptive and that's because you're Yeah you're saving the system entropy because they're already joined together and there's no interplay loss and in associating those peptides in such a way when they go into the membrane that they're there are disruptive and so it's very much a co-operative idiot effect in that case.

[00:59:57]
Well it's. It's there's an analogy but it's I don't think it's multivalent see it in this case it's not actually multivalent see it's because what these peptides have to do is assemble into a membrane disruptive. Assembly and in that case what it is a question of is the assembly favored walk concentration Do you have to have to drive that assembly of that so that those peptides to associate an aggregate together to become disruptive.

[01:00:33]
They're isolated there's an intra be cost to associating them. But when the polymer. Is brought into play. Then there are there. It's interim molecular assembly. And so it's not multivalent see but it's more of this interest co-operative eighty is you know effect rather than multivalent statement. I understand your analogy to multivalent see going alone together you can talk about gravity.

[01:00:59]
And then we need to bring the one with them when they are all. That. Thank you very much. Thanks. Always fun to be here. Everyone.