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>> And so the day I'd like to talk about some of the work that that I I did. So Ph D. student and also some of the work. That I'm doing. As opposed to Bob Langer sabot MIT and the title here is engineering biomaterials based inspired by biology and challenges and opportunities.

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So this is how this talk is going to go. First of all I like to identify some of the challenges that I think is facing the. Part of me. Sorry. Well. Identify some of the challenges that's facing this field of materials engineering and and then I'll describe two approaches to that we think might be.

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Promising to tackle these challenges and I'll give specific examples for each of those two approaches and then in the end. I'll spend some time talking about what the future. I think will be like in this field and also my own research. So over the last number of years biomaterials has have really experienced a lot of tremendous successes.

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In terms of making functional devices and use them in in the in to change people's lives. So here I just like to give a few examples. So here you see a official at total artificial hip. So it's based on it's made of a variety of different materials including such as metals metal album or is a ceramic some polymers.

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And here in such a way that. It supports the skeleton and it functions like a joint. And here is an example of a total artificial heart. Which is really an engineer. Marvel. It's made of again number of different types of materials including metals and polymers and it's engineering in such a way so that it can perform the very important function of a hard that is to pump blood.

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And here is see a very different kinds of material which is first of a polymer. Polymers and its soft material is always a lot of water and you can see that it's very flexible very soft and this is actually the contact lens. It's made of the material is called a hydrogen because it's a lot of water.

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So the function of that of this material is to correct vision and here another example is polymers containing drugs and in in the in the Matrix and slowly release them to model the disease processes so so far the the success of these all of these devices have really centered on two issues the so-called compatibility and performance.

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And these are these two are interrelated. So by a compound of the ability. Knowledge they refers to that but serial and once it's inside the body. It doesn't do harm to your body doesn't create any inflammation doesn't create a new response and performance of course these different devices have their own desired.

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Being a replacement or enhancement of physiological functions. But again. Where do we go from here. So again I think there are there are tremendous drawbacks as well even with these successful examples. Again in the in those two aspects by compatibility and performance for example. This total hip here.

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It's only good for about ten maybe fifteen years and there's there could be a lot of problems associated with it. You can get loose and you can get the generation of Bones because of your face. Specialist shooting a lot of stress and. And it could be there can be very slow chronic inflammation and in this case artificial heart.

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It's in contact with blood and it's pumping blood in such a high flow rate. There's absolutely there's going to be damage in the in the of the blood and bones so. So these are the challenges. I think that is really facing the biomaterials community is. These are two things I identified of course.

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First of all how do we make biomaterials with well defined structures of functions. Right now we're not really very good at doing that. So for example if you're making a polymer and you're making a polymer with using the traditional chemical services and this is what you would get in terms of the size of the product you're getting.

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I call this a molecular head coach in the N.T. which essentially is a Gaussian distribution of a different challenge and the species here involved can be a huge number. Like millions or hundreds thousands of So You Think You're making one polymer like polyethylene but actually you're really dealing with.

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Many many different kinds of polymers each of them with a very distinct structure of property of course that's going to influence any biological applications and on another different scale on the microscopic scale. Also there is tremendous heterogeneity what you see here is an example of pollen lactic acid.

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Actually it's. It's one of the F.D.A. approved material and in this case is a tissue engineering scaffold. And you have a liver cells sitting all those and you see those fibers. They really go. All kinds of directions and there's very little control there that there's space between the fibers is very heterogeneous.

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So this of course. It's going to have a tremendous influence on. The performance of this material. To to engineer. You know that scaffold so and the second challenge I see is really that we're making biomaterials we're not just making any materials. So how do we make those materials that can be sort of interactive with the biological environment not just really sitting there passively so so I see this we need really a update of the definition of compatibility from the current point of view seeing a name plan as a passive inert nontoxic genic material rigor to upgrade to something that's active and really can can be smart in some ways to communicate with the biological environment and modulating the biological environment.

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So all right so how these are the two approaches that we are using to deal with those two challenges the first approach really is based on. Understanding the biological molecular molecules and how they are put together and how they self assemble and how they interact with each other to form these architectures so.

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From here is the example you start to have the D.N.A. which in code is all the genetic information and then you have a protein and the proteins. Well self assemble to form these super molecular structure in this case is active filament and these super structures further form. Structure in this case the skeleton.

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So by contemplating on on the properties of structures of these biological molecules architectures. We decided that we're actually going to use these molecules such as proteins as building blocks to make materials and the way we pretty. These materials is using the well developed the recombinant D.N.A. technology. So by this approach ideal Lee or in principle one should be able to produce a material with exactly defined.

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Size and composition and structure. This is in. Exactly. The large contracts to to the more traditional polymer technology but of course the chemical the traditional polymer chemistry. Has been has a long tradition a lot of the basic questions that has been answered. So we're going to actually take a cup.

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Use a combination of both of these two. To combine these two technologies to make materials and the driving force of the materials can be either chemistry or more of biological oriented processes such as self assembly and the second approach is really based on the understanding of biological processes the systems.

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So in this case we start from a clinical problem for example if you're let's say you're treating cancer. Or you want to engineer a tissue. So you go back and understand really on the molecular basis cell or bases or tissue organ bases and ultimately this established system spaces to of all these processes involved in those diseases.

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That you want to treat and then go back to see what elements you need to put to be put into the material so that you can achieve. Your desired goal. So now move on to give some examples. So the first approach. I will give two examples one is about using engineer protein domains to drive microscopic.

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Structural change of polymers hydrogels the other example I would go through very briefly is to use by a lot. Template ing and processes to form an animal nanomaterials for the second approach. I will use a an example using polymer the variable polymer microspheres for the control delivery of D.N.A. vaccines.

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So the first example here's some background about hydrogels so Hydra gels are really polymer chains. That are tied together through these so called crossings and these are all very hydrophilic chains they absorb a lot of water. So instead of dissolving a salute in in buffer they would swell and maintain a certain shape.

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So there is in the end there is an equilibrium stay where you have achieve a thermodynamic between the hydration of the polymer. And the surrounding solvent and this this type of material has already been used extensively in the biomedical devices. All kinds of applications shown here and a very interesting and intrinsic property of all hydrogen is called phase transition so this is similar to the role of the physical phase transitions that we see for example water freezing or water evaporation.

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But it's done in a at different scale with a different identity. So here for example here you have a hydrogen which is an equilibrium with a solvent and then by changing the environment such as these different factors. In this case you change how the polymer chain interacts with the solvent and you can actually drive the solvent away from the polymer you get sort of a localized precipitation.

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And that's called a phase transition of Hydra job and usually as a confident by a change in volume a change in physical properties such as optical properties. And these face transition process is is very specific to a specific stimuli for example something that's responsive. Something that occurs at a.

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Temperature would not occur at different temperatures very specific and the process is also highly co-operative and in many cases reversible. So the currently available. Phase transition based polymer materials is really doesn't leave much room for engineering. So we came up with a new approach where we call this a hybrid hybrid system where we have essentially the system will contain two different components one is based on synthetic materials.

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So we're intent to use this as a mechanical support and to absorb water to keep everything. Smalling in water and to minimize nonspecific interactions involved in this second opponent component which is based on proteins and the proteins here is actually the vital component where we're trying to take advantage of the stimuli responsive behaviors of protein conformational change to drive a macroscopic property change of these hydrogels so.

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We started with a relatively simple protein folding motif called the coil coil. So what is required coil. So here is a whole bunch of native proteins that have very diverse functions and and structures but all of them contain part of it a quote Cuomo T V So it's basically the two or more off the hilla Cs winding around each other to form a super helix and the beauty of this motif is that a lot of the biology has been done so that we know a great deal about how the amino acid sequence correlates with the structure of this of this.

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Motif. So for example here in this case you're looking down from two of the heel loss' and you will see the first and the four positions of the amino acids are often hydrophobic residues so they serve sort of a service a zipper to glue these to ketosis to gether to make the make it stable.

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So then we moved on to produce. Two proteins we call one and two and when we made these using the recombinant technology we constructed the recombinant genes and put them in eco live expressed and and purified them from bacterial culture and then we looked at the secondary structure of these proteins so we're verified that they do Photoshop very correctly into our for healer cities and we found that one of the proteins.

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Actually has a thermal temperature related conformational change at about thirty seven degrees. Whereas the other one doesn't really have any change. So then we moved on to construct this this material this hundred thirty National material where we have a synthetic polymer backbone. That is able to conflicts with a metal ion and then we've engineered histed in residues on the ends of these proteins so that they can bind to these ions and form this crossing so interesting when when we increase the temperature.

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We saw that in the case of the hydrogen which contains the first. Protein which. Is a capable of undergoing confirmation a change. We saw this dramatic collapse of jobs from the state to almost ten percent of this original volume and and in common at the same time we observed also a dramatic change in optical properties.

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So actually the other gel which contains the other protein Rola didn't have any change. So what's happening here. So this is what we think this is our hypothesis so we'll start with the really lot extended are for helixes structures and if you unfold these things and they're they form these random structures and they these run of structures occupy less amount of volume and that's going to that's the driving force.

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Which brings the hydrogen all to a collapse. And to illustrate a little bit better here. So let's say you have. Have the same number of neo asses in a protein and depending on the different conformations occupied different space different amount of space and if you move from one confirmation to a different confirmation that change in volume.

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Would bring about the change in the hydrogels So then we moved on to constructs are more common complex structures in this case. These are blocked proteins so we're taking many a protein second of a native protein called nice in which is one of the motor proteins. In charge of interstellar transport and we clone that this part of the protein sequence.

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Consists of part of the helix. And part of a Rand. And we make we made one copy of the gene. And then we also duplicate it to put two copies side by side with each other and also then we move on to make a three three repeats as well and we also we were successful.

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To be able to express these proteins in bacteria and purify them. So we did a number of characterizations of these proteins looking at secondary structures and how the Associated with each other in solution and how much space they take up in this in this water based solution. And we came up with this model here.

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So remember the we call this chaos one which contains one copy of that that gene that we taken from kind nice and and compared that with two and three copies the really interesting here is that we think that that these rather than forming a linear structure they actually within each other to form these more compact structures so and also very interesting that.

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They display sort of the hierarchy structure where the tribe or is actually can be viewed as a combination of a diver which is shown here. Plus a monomer scent. On top of it and in the the behavior or the thermal transition behavior of the traveler is a combination is the sum of the monomer and the dimer And so we used this model that was hypothetical but we were able to explain how the proteins behave in the in the context of a three dimensional hydrogen.

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So this is really good and then then then we I'd like to move onto to a different kind of material which we which is sensitive to calcium what you're looking at here is a very primitive single cell organism whole called Boortz a cello. So this is a cell body and that lives under nice water and it fastens itself through this long organ now it's called spasm in the.

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Two way solid support such as Aleve and then if you in the presence of trace amount of calcium mine it contracts dramatically and if you take away the calcium relaxes and it relaxes back and interesting thing about this motel of it is that it's not dependent on A.T.P. like for example muscle contraction and basically the organ now which consists of a lot of fibers protein based fibers and hydrophilic sheath wrapped around it.

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So the mechanism of this contraction is really not known right now but there's the working hypothesis is that you have these well lined fibers and in the presence of calcium somehow. This organizes the fibers and causing the fiber to contract. So here I can I say show you a movie.

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Of the contraction this is real time and the contraction process is completed in one sixty S. of a second. It's one of the most violent biological in in biology and the relaxation is kind of a little bit slower it takes about a couple seconds or so. Later on only in the recent years people found isolate and also later cloned a gene which enclose a protein called spasming.

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And really the specimen is found throughout these organ now it's all over the place and. Has a very strong sequence on the homology with calcium binding protein. And the way it works is that it's a dumbbell shape and it binds with four calcium lines and in the presence of a target Pattaya which is sort of perpendicular to the slide it wraps around it to form this very compact structure.

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This is also one of the most the largest protein conformational changes which has ever been observed in biology. So the question now remains is does this vast man. Work similar similar ways. Similarly to or does it have a distinct mechanism and more relevant to what is is that we want to develop materials so what kind of engineering principles can we learn from from this these structures so we can make better materials.

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So we produced a recombinant in a lab technician and also constructed fusion proteins of G.S.T. with these with these spasming and we compared. Spasming fusion protein with the first fusion protein using a technology surface based technology called course Crystal micro balance basically you have of course Crystal and Co the crystal with a layer of proteins and if you have any protein structural change.

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It's going to change the the properties of the crystalline and this very sensitive. So the bottom line is that we found that these. Spasming is behaves very very differently and. It has a very dramatic conformational change in the presence of one of calcium mine. So this is really very interesting and then.

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Just by chance we sort of observe that when we start with the G.S.T. spasming fusion protein and we add calcium to it and if we shine U.V. light to it for just a few minutes we observed the formation of these type of structures these are a very extensive large.

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River like structures and some of them are really large in terms of size and some of them are really thin but these are highly irregular structures there are sort of along these fibers and the formation of these structures is highly dependent on the presence of calcium you can treat this with E.D.T.A. you remove the calcium.

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This is what you end up. You have all these globules locked you don't have these fibers structures anymore and the dimension of these fibers is actually roughly on the same sort of same scale as the spasm of a cello so. So what we think every. So far this is all has been really interesting.

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We have a working hypothesis in terms of how this works. Based on these data. So we think that that really that this protein. Very different from. The uses of the calcium binding to change conformation change and use that to do some of the motel of. So this is also some work ongoing that we're using this principles and and sharing variations of these proteins to to make calcium sensitive materials.

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So now I'm done with the first example here to illustrate this approach and then I go to this. Nanotechnology related approach this sort of. We feel that that the biology is fired engineering approach is not only applied to the terrorism biological molecules. But also it'll be interesting to use take advantage of these methodology.

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To Engineer materials. So these are just a number of examples of the. Different structures that people have been able to make so far and. Almost a lot of these structures were are made using the traditional chemical synthesis where we're often involving. High temperature high pressure the use of Callas.

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Which is really not very compatible with bile. With biomolecules bio processes so we decided to take. An approach that's based on protein assembly and templating to make these materials. So we decided that we're going to focus on a very common. Protein called actin which is very important side of Sokal protein.

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And this is a monomer called G. acting and in the present physiological salt itself assembles binds with it. Assembles into these extensive fibers. Shown here. And so we have developed ways to. To use this process and this process is very reversible you take with a solve it goes back to the monomer So we've been able to construct.

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A metal clusters along these fibers and in this case it's really a few metres long which is really really long and and in some cases. The alignment of the arrangement of these. Metal clusters are very are really straight so. And we're trying to improve on the methods of these to hopefully we can engineer something like a like a wire that's really capable of conducting current.

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So of course you can think about using. Using these materials in microelectronics but also. Where we're interested in more interest in is to a combination of these. These nanostructures with entities such as biomolecules or even cells. All right so now I'm going to move on to the second approach.

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Let's see. All right. Talking about D.N.A. vaccines. So what is a D.N.A. vaccine the traditional vaccine is based on a ten year viruses pathogen antigen such as protests and peptides and the any vaccine is really starts with a with a piece of D.N.A. and the D.N.A. encodes the engine that you want to immunize against and you put it into a plasma and you replicate that.

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That and then you inject the plasmid into an animal and hopefully the gene will get expressed the protein will be produced and presented. So that you will get a response so. In terms of the immune system how the immune system works inside the cells this is just a typical classic go to pathways.

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What is really involving that. This second this half of the graph here is the so-called exogamous pathway where this is more of the how the traditional vaccination works. So you have an antigen that's coming from outside of the of the body being taken out by cells and being processed chopped.

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Noted onto these molecules called a cloud class to molecules and then come back comes back to the cell membrane which elicits a mere response and. So D.N.A. vaccine works in a different way. Takes advantage of the other pathway which is where you have the antigen the protein produced inside the cells and that is process and chopped and loaded onto the image the class one molecules and then goes back to the brain and created a new response.

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So it is known that plasterers class two pathway is really important in eliciting and about are related immune response where you were in the class one pathway to elicit both the ends about immediate as well. The cell based. Cell with her. Response. So this is how the system works.

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You have these two. Different response and seller response but really the important part of these two pathways is how the antigen is being made and present it. So this is where we think that that which we can we should be able to contribute to is that this is is after contemplating on these immune knowledgeable processes.

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That we the hypothesis is that if we can achieve a way to selectively target certain cells inside the body that are professional cells that are made to present and process antigens then. We should be able to get a better near response. So the way we're trying to do this is by using a polymer microspheres encapsulating these D.N.A. vaccines and make these microspheres into a certain size for example one to ten microns in diameter.

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So that they're too big for. Normal cells to be eaten up but they're perfect for. For example dendritic cells or macrophages to be taken up and processed. So let's imagine if these microbes your internalized by this cell. The enable somehow can somehow escape these vessels and then eventually reach to the nucleus and gets expressed and processed displayed on the cell membrane and then can be and elicited so the polymers that we have been using is really based on this new polymer called Paul also Esther's and we are trying to as I am or using this other polymer.

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Poly lactic acid as a comparison. So this polymer here is. Probably the most often used by the girl polymer. So far because first of all it's very compatible and it's approved by the F.D.A. for in the VO use but the problem with this polymer is that you make it implant out of it.

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The degradation of this of this polymer really is. Throughout the entire matrix so. And in that process. You generate a very ass acidic Diggnation product which has the potential of damaging the D.N.A. inside these particles. So this new polymer that we've been working on in collaboration with with with other people is that this really doesn't degrade like that in this case is a bulk erosion in this case the polymer really resembles a bar of soap where only the surface which is in contact with the solvent gets the grated and gets falls apart and wears the interiors of the polymer right here remains intact and by doing so there will not be any acidic.

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Decoration product cumulation so that the D.N.A. confirmation will be maintained throughout the entire process. And also by manipulating the chemistry of this polymer one can incorporate different trigger molecules so that you can have you can sort of these processes that they're predation by changing the environment so. These are these are the number of different monomers that we have incorporated into these polymers so for the first two categories are really the basics of the of the forming the bond the polymer backbone.

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And these different variations of the same hollar it gives you a very good. Flexibility in terms of controlling the header for. Felicity and rigidity of the polymer chains and this polymer is we call agent acid polymer So why this jury investigation this polymer is crude is generated and it's a it's an acid so and then and then that's sort of like a service or callus So you're speeding up the polymer degradation.

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As their production process goes last it was a self. Accelerated. Nation and we decided to incorporate another monomer which contains a tertiary group which is. Charge at neutral Ph. So what we're hoping is that because the end is D.N.A. is our large molecules that are positive and negative charge.

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So by incorporating this positive charge there will be create some. Interactions between the polymer and the D.N.A. So I have to have you to focus on two polymers that we made one has all these components. Except this last. And P two has. Certain fraction of the I mean contained.

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So we encapsulate the D.N.A. into polymer microspheres by using a very well defined. Double emotion technique and we were able this is just to show that we're able to make these nice a spherical particles with very smooth surfaces and we can have very good control over the size of these particles in this case it's centered the average size is about five microns.

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And the distribution is fairly narrow. OK So then we looked at. How the D.N.A. of behaves inside the particles and we extract the D.N.A. out of the particles at different time points to see if these the C.N.A. is remain active in terms of the conformation we run run gels to see if they maintain active confirmation.

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Because that's really important in terms of protein expression. So the bottom line is that we saw that. A large portion of the D.N.A. remains as the most active form and even up to thirty five days of decorative release and we looked at quantify the amount of D.N.A. released in terms of.

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With time and we started different ph buffers and how the from PH affects the release kinetics and we found this really interesting phenomenon that both of these two polymers release very slowly very little mound of D.N.A. A neutral. Ph within of course certain pure. TIME But if you change the PH to.

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Slightly acidic for P one. There's a dramatic very fast release burst and it reaches releases all of its D.N.A. where speed to has a cerebral lag time and then it starts to take off. So this difference here has to do with the presence of these those I mean contending polymers in P two polymer.

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And the reason why we're doing we're interested in these two different ph is because remember the one of the figures I show the hypothesis where you have the spheres go through all the cells and there's as they first go into the cell that go into these vessels called Fadl so that are kind of slightly acidic.

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So so we're trying to mimic really the biological processes where the microspheres when they're inside the cells what kind of environment that they're going to be in and to see. To try to assimilate that in vitro and then we looked at how these polymer microspheres degrade inside cells this is a macrophage cell line after and with polymers for four and five seven days.

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This is using force and prosperity. So the conclusion there is that we see with time we see. A progressive degradation of both of these two polymers and P P one actually degrades. Slices faster than P two which is in agreement to what we observed in vitro and currently we're doing on animal testing with using model antigens.

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Looking at the production of antibodies and also the the ability to elicit cell responses. Basically we're looking at the ability of activating T. cells using again the model antigen. And the preliminary data show that. Compared with controlled experiments where you don't in mice that there was no treatment or you treat it.

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Immunized with D.N.A. and they could be in a without in the Palmers of these they don't really have any activation so P. Using these polymers we got fairly good activation. But all of both of these P one M.P. to do. Because they were better than by itself. So this is really encouraging and we're.

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This work is ongoing. We're also going to be doing a show in the process of doing a tumor challenge experiment where we immunize the mice first and then you. Knock one of them was tumor cells and see if it can really prevent tumor from. From growing right now I'd like to thank a number of people here and my team my advisor been really extremely supportive and.

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Providing a wonderful environment for people in the lab to to pursue their sort of their own interest and then a number of wonderful collaborators at MIT this is in the Langer lab and also some other neighboring labs in biology and also in Whitehead Institute that helped us on there's aspects of these projects and also advisor at the University of Utah and a number of people fact the member there which helped me.

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Throughout the years when I was. Student there so and these different people. Contributed and collaborate with all different asset aspects of the project and acknowledge the support for my postdoc. Study by the an age now I'd like to move on to talk a little bit about what I see the future of this field is and how my research is going to contribute to.

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So. This slide pretty much summarizes the approach that I'm going to be taking So the really the important point here is that I will be this is a bio inspired So again really understanding how. Biology. Is made up on the molecular level and higher level. Interactions between different biological components and the process each involved in the bio in biology.

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And this is one approach this is one starting point. Another starting point is based on African nations. So really understand what is what the problems are have very. Potential have critically important. Impact and then go back to design rational of these these materials to modular these processes and the approach in terms of engineering is a unique combination of a bio office is and the polymer chemistry.

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So. This is what I see is really unique in this approach because by incorporating biology into the more traditional. Polymer materials. It really opens up a lot of opportunities for for making. Interactive material so I talked about in the beginning. So I. These are just briefly about the three different things.

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I thought about that I may be interested in pursuing is the first one is really based on the design of smart materials based on by a molecular structures and interactions. So if you look at these three different things. Proteins side looks like skeletons and the extrasolar matrix. You will find that that the common feature of these three very different identities on different scales.

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Have really. Very common features is that they are structures. So these are the character is destruction properties of typical hydrogen all. And you can find all of these properties. In these three different materials have the proteins have the south side of class and skeletons and then you have these cells sitting in in.

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In the middle of a three dimensional actual cell the matrix. So after looking at these. I'm thinking it would be really interesting to. Make materials based on these properties. And to really mimic the structure and function of these. That identities. So. Expanding on some of the earlier work that we did.

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Really to expand these and to incorporate a lot of large different variety of molecular structures and interactions. So for example in this case if you create a polymer Hydra gel based on a protein conformational change you change conformation you change a property of the gel and in this this particular type you start with an aggregated protein and somehow trigger.

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Put in the trigger and dissociate these proteins you will get a property change in the gel and in this case you have a protein and a lot again. And if you destroy the light in certain ways you would change the property of the child is also a similar case and more complex and I have some examples where there are certain.

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Actually there are many many different examples in biology. That one can take advantage of creating these materials. So these are the some of the long term things that could be a political. By this technology such as tissue under a scaffold drug delivery devices actually. Incorporate these structures in microfluidic devices is going to be also very interesting.

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Because of the presence of biomolecules and that's conformational change like an admission that these could be useful as file sensors and those these are more practical practical applications and from the more basic science point of view first was just really interesting to make new materials and secondly. I believe that this could be really useful model system to look at how proteins on the molecular bases the that.

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They would change their structure and how to respond to. To a stress on the nanometer scale and also more importantly more relevant to the tissue engineering is the process of trying to. This could be also another system to study that right. So now I'll move on to a second.

[00:42:11]
The second proposed topic is for tissue engineer or general medicine. So all of you know that there are several key components in tissue and you're in generating a tissue organ. So you have the source which is interesting. You can use primer cells or stem cells or other cells and have to be soluble factors such as growth factors you have to give the different kinds of growth factor to cells at different time cores and different patterns different dozes and very competent complex and then there are another component is mechanical stimuli.

[00:42:47]
You have different kinds of patterns and different shear and different stress so and this is where I say the polymer scaffold. And what I hope to do is to engineer polymer scaffolds so that they're able to integrate all these components. To achieve this final goal of engineering tissue.

[00:43:08]
So this is a hypothesis what I call a design or three dimensional scaffold whereby you really using the protein and sharing technology as I described the four one is able to be able to incorporate. Diverse different functionalities into a single scaffold. For example in this case the vision that for the chemical properties you would should be able to incorporate different.

[00:43:30]
At different modules there are some of them are some of the room hard and in combination of these two different things you would get a desirable mechanical properties of the scaffold. And of course you can think about if you want to interact with cells you can put different kinds of molecules into these into these into the chain and also you want to be able to have the scaffold to be able to be remodeled so to put in as magically degradation sides.

[00:43:57]
Israel is going to be important and also. It should be also possible to incorporate growth factors for example in this case as a tether growth factor as part of the scaffold to rather than adding a solid growth factor which is going to be really easily diffused album being consumed so and if necessary.

[00:44:19]
A synthetic polymer can be incorporated just to maintain again the mechanical. Strength and add in water solubility water a small ability of the of the scaffold through these very highly. Well defined crossings. So these are certain things that we're looking at these scaffolds and eventually to move hopefully to move from the IN VIVO to in vivo and I would like to really to build a platform technology.

[00:44:49]
So that depending on different target tissues one wants to generate regenerate to go back and tailor different composition of the materials so that there are individual two for particular tissue. All right so the last topic will be last field would be gene therapy. So this entire field of gene therapy is really bothered by one issue that is the delivery problem.

[00:45:18]
That's really the bottom of the field. So you can imagine that if you want to get your gene the gene to where you want before you even reach the cells. You have to deal with all these different problems this is more like a more assists system related problems in terms of Smith's ability and distribution where you have the genes located at let's say even if you can achieve all these goals and you can sort of target your D.N.A. or Gene to or particular cell and they can go into the cells.

[00:45:47]
There are also and numerous barriers and processes these genes have to go through so so that they can be sort of successfully expressed so how do you overcome all these different issues and. All these different hurdles. At different scales. So that's that's the challenge right now. So again a summary of the state of the art of the actors a lot of the viruses have been used for actors.

[00:46:14]
Of course they're extremely efficient and. Many times they can be permanently integrated into the genome and the expression level is very high but very serious drawback is that it's a safety issue. Many of these viruses really. Can create problems. So on the other hand. Other non viral doctors such as based on these molecules look it's polymers have tons of proteins They're in general very safe to use but they're also very low in the fish unsee and the Djinn expression is often very transit.

[00:46:49]
So and also. Their structure is really not very well defined especially compared to a virus and these polymer these materials are really very heterogeneous like had to strike in the beginning of the talk and it's also very challenging to incorporate functionalities to these systems. So again learning from biology.

[00:47:11]
Here you see a virus and this is how virus looks like and these are proteins that are on the viral membranes and that that are in charge of either targeting or getting help the virus get inside the cells and get trafficking moving inside the cells and get the.

[00:47:27]
Expressed so. And here's another example of a naturally occurring protein family that. That are. Built to bind with the N.S.A. These are transcription factors you have these modular type of organization have a D.N.A. binding domain. You have another functional domain connected by a link her. So our have all this is is what if we can make a structurally mimic of these materials.

[00:47:54]
Hopefully we can also get the functions as well. So here's a hypothesis. I imagine that you know one can make a modular molecule based on different functionalities for example you can have a domain that combines the D.N.A. and you can have all the other domains that are can be in charge of targeting to specific cells or trafficking within the cells.

[00:48:18]
And you have these linkers to attach them through self-assembly you would end up with this nanometer sized complex. So that all these functionalities are exposed and the D.N.A. is really protected in the interior of the complex. So this this sort of mimics a real viral virus structure. Except it's made of synthetic materials and are a few things that one should consider and eventually I hope that.

[00:48:46]
It would be really nice to move this to the clinic so that it can be really useful and of course the living out the very tremendous medical applications and hopefully this will have a very clinical written all of an impact. So that's the end of the talk and thanks very much on ready to to go to questions if you have that thank you.

[00:49:11]
Yes. In terms of the the organism. Yes it is it's very versatile. In our society system we have a looked at directly the calcium sensitivity of the material. What was so far observed was that the formation or the self-assembly of that excessive material is very much dependent on the presence of calcium and that formation of the material is very risk is reversible sort of you have formation and you take away the calcium it doesn't exist anymore.

[00:49:51]
So the logical the next step that we're doing is can we make a material that really mimics that contraction process that we observed in the. Biological organism. So that's worked on. Right. That is not reversible in those two examples I presented to you and there are reasons for this.

[00:50:15]
I think. First of all protein protein. There's a very large chance that it will may not fold back it's just a kinetic factor that's probably limiting it. Secondly if. And we're not just looking at a protein. That's sending a solution by itself. You have all these how to fill it.

[00:50:34]
Polymers around it and that the present of these polymer chains will restrict the three dimensional. Flexibility of these of these probes instructor so they may not be able to rotate in full back to its proper state. And lastly. Because of the unfolding process. Some of the hydrophobic residues on the protein.

[00:50:56]
Are exposed and they can interact with each other and not specifically so that they would actually form something really compact and more like a spherical sphere structures and they may even come out of the solution. So that process of course is very hard to be reversible. And in terms of applications in certain after it has a real nice to have reversible process but in certain applications if you just want to have a wide shot type of use then it may necessarily have to be the case.

[00:51:27]
She's so. Yeah. It's. Crazy. Forms of structure. That's right. It's explains very little. That's right. This structure was that's right. Of course ultimately. If one can do crystallography and really solve the structures that that's going to basically ask your question or thought about this and it's just sort of out of Already John where really the material is so so yeah.

[00:52:24]
It's plays. Well based on the data we have we think this is the most reasonable model. There there might be some other models but. I think as far as we're concerned this is the most possible model. Now. So the question is how how how. Stable are these forces are OK.

[00:53:00]
Of course that depend depends on what kind of protein you have some of the proteins are stable and some of them are not so it depends on get it depends on what type of YOU WANT TO YOU WANT TO YOU want to use for example in terms of tissue and you're a scaffold.

[00:53:16]
Eventually you want the scaffold to go away. So then the protein you don't want to be to be there forever. So you want to to go away to be degraded and actually you may want to deliberately put into some elements so that they can be that they can be controlled and trigger.

[00:53:34]
In terms of other applications. Again depending on the exact configuration of the material. The stability issue is is not too much of a following. Hey. Facts. The first question is so how you want to use the calcium responsive material in the file of course you have a lot of calcium mines present inside the cells is that going to be a problem.

[00:54:47]
So my answer is in that particular case is not most probably not going to be using the evo. And I see that as an opportunity rather than a challenge is that this approach really allows you to incorporate. By using different proteins so that you can have really. Biological specificity.

[00:55:05]
Which a synthetic materials are really not capable of doing so for example if you want to really use something inside the body and you've got to really understand what is special about the the where you want to put your implant or material so that the response will be only specific to that particular part of the body or any particular cell type you know for example if you want to really want to do something with tumor.

[00:55:28]
For example targeting a drug to a tumor one of something want to have some of the targeting mechanism to be very specific to that particular tumor. So second question is the immunogenicity potential immunogenicity of these proteins. First of all. Depending on the application if you are not going to eventually implant these materials inside the body.

[00:55:49]
That's not an issue. Secondly if you do. Incorporate these inside the body. There can be a number of different approaches to do. First of all you can use humanized proteins you can use proto. Really you know part of the human protein and then of course we settle on a particular application.

[00:56:07]
We're ready to really mass produce that material and the very first thing we should be doing is really to test that protein. With an animal model see if you have any genetically there. So that's a very good question.