Nanoparticles - Chemistry, Structure and Function

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Nanoparticles - Chemistry, Structure and Function Karen L. Wooley, PhD Professor Washington University in Saint Louis Department of Chemistry Well, good afternoon, and, thank you, Richard, for the opportunity to talk today. I’m a Synthetic Organic Chemist, and a Polymer Chemist, and therefore, the emphasis of the presentation will be on the control of chemistry, and how controlling chemistry can allow for the construction of materials having well defined structures and compositions, and how that then can lead to control over the function, as it relates to materials being translated to medicine. I was very interesting that Mark Ratner’s conclusion slide, in his discussion, tends to agree with my introductory slide here. And that, we’ve spent many years over in the lefthand box, on fundamental research, and now we’re to the point where nanoscience is emerging in applications that involve the nanotechnology. Of course, this involves a great deal of research that is of fundamental nature, and that will be the bulk of the presentation, that I will make today. However, I will highlight several examples. For example, those listed on the right side of the screen here. Beginning with, metabolite sequestration and how metabolite sequestration can be performed by microparticles, and perhaps, nanoparticles, as a means to lower cholesterol. And then, continue later in the presentation with diagnostic screening, using nanoparticles, ultimately, drug delivery, and I’ll try to highlight some attempts to mediate and manipulate biological functions. Before I begin those examples, however, I want to spend a moment going over some basic terminology and some basic fundamental polymer chemistry. Since you’re in the medical field, you have probably had some exposure to chemistry, and this slide is a rather, in some sense, detailed example of three important terms that I’ll be discussing throughout the presentation today. The first of those involves polymerization, and polymerization is simply the connecting together of individual monomeric subunits to create a polymer chain structure. So, poly means many, and mer is the subunit. Now, the polymerization process can involve many different types of chemical structures, and I don’t know how familiar you are with the drawing of organic molecules, but let me just point out, that on this slide, that each of these lines shows a carbon-carbon bond. When there’s no atom label incorporated into the structure, you can just assume that it’s a carbon. And so, we have in this structure of allylamine, two carbons here that are doubly bonded, a carbon-carbon single bond, and then a carbon-nitrogen single bond, and the nitrogen has two terminal hydrogens bound to it. Ok, so this kind of structure. So this carbon-carbon double bond is reactive, the polymerization that involves opening up that double bond and establishing now, single bonds between the carbons, between individual monomeric subunits. So, that’s an example of polymerization. The second term that I’ll be discussing throughout the presentation is, crosslinking, and crosslinking involves the linking between individual polymer chains. So, as that is shown here, these green balls are now crosslinks, that link together this polymer chain with that polymer chain, and also

with that polymer chain. So that, if we bring small molecules together into a polymer and then the large polymer molecules, or macromolecules, together into a crosslink network, ultimately, we can have a matrix that is of varying size, from the nanoscopic regime to macroscopic objects. For example, a bowling ball is essentially one large molecule that is a bunch of crosslinked polymers, linked together. Now, another term that I’d like to highlight is, chemical modification, and this can be done at any stage here. Monomers can be chemically modified, polymers can be chemically modified, and crosslinked networks can be chemically modified. And, I’m showing here the chemical modification of this matrix, with these red units now, which are meant to represent cationic functional groups. And, the reason I’m showing you this particular chemistry, is because this crosslinked network structure of this chemical composition, that bears these cationic moieties, is one that’s used in a product called, WelChol, and WelChol is used as a material to lower cholesterol. And, the way that it operates, is that the WelChol is ingested, and the intestinal tract, where the bile acids are secreted in order to digest fats, the WelChol then will sequester the bile acids, capturing them and carrying them out of the body. That’s important because that then requires that the body convert cholesterol to generate new bile acids, because normally the bile acids would not be passed through the intestinal tract, rather they’d be reabsorbed after they had performed their digestive function. So, WelChol works very well, and it’s produced by the chemistry I showed you on the previous slide and then those crosslinked chemically modified materials are ground up into microparticle. Now, the question then, would be, what is the performance of a nanoscopic particle relative to a microscopic particle? Now, we don’t know the answer to that, in the case of WelChol, but there is an example, again, on the market, from Elon Pharmaceuticals, that involves this general concept, that if we have a particular mass of material and that’s broken down into subunits then the surface area increases, while the mass remains the same. So, we can say that surface to mass, or the surface area to volume, increases. And, if these subunits are broken down into further smaller pieces, then of course the surface area increases even further. And, this concept is utilized in the Elan Pharmaceutical’s development of nanocrystal formulations, in order to make more bile available. Any type of drug molecule that is part of this nanocrystal and framework, and in this particular case, again, it’s a milling process, and a grinding process, that gets down to the nanoscopic size scale. But, if we want to create something that’s nanoscopic in size, that has this very large surface area, relative to the volume or mass, and also, presents interesting chemistry on the surface, in order to mediate biological interactions, then we probably don’t want to go through a process of grinding and milling. But rather, through building up a structure that has all of the components placed within a molecular framework, that’s well defined. One of the very interesting molecular frameworks that being explored by many people in academia, and in industry, is what’s called a, dendritic macromolecule. The term dendritic means tree-like and it’s shown schematically here. Rather than each of the monomers being linked together through the polymerization process to generate, a linear polymer chain that acts a lot like cooked spaghetti when it’s in a solution environment. Rather, in this

case, the dendritic macromolecule has its monomeric repeat units branching, so that their linked together in a branching fashion, in order to generate a highly branched and globular structure. Now, in the presentation that follows mine, Mark Renstoff, will talk with you about the application of dendritic macromolecules and ophthalmological areas, and I’m going to go through a few slides that detail the structure and properties of these very interesting tree-like molecules. One particular example of an application that is being developed for the dendritic acromolecules, aside from the ophthalmologic applications, is in treating viral infections. And, this is being developed by Starpharma, a company from Australia, or in Australia, in collaboration with Donald Tomalia. And, Donald Tomalia is really one of the fathers of dendritic macromolecules. He began synthesizing these structures back in the early 1980’s, while he was a Dow Chemical Company. Dow had no interest in dendritic macromolecules back at that time, and Don left Dow, and continued working on dendritic macromolecules, he’s started up several companies along the way. And, it’s been Don’s firm belief in the unique properties of dendritic macromolecules that has lead to many, many people joining in this effort, and now you can find hundreds of examples of various dendritic macromolecules, from fundamental studies, to development work toward applications. Now, the applications that are being pursued most heavily for dendritic macromolecules relies upon their overall framework, as I’d indicated, where if we can control the chemistry and the structure, then we can control their function. And, in dendritic macromolecules, there are three main things that we need to highlight. The first is the core domain, the core unit here, and then the monomeric repeat units that are used to build up the framework, and ultimately, the dangling chain ends that are the termini for each of the growing arms of the dendritic macromolecules. Now, these structures are typically drawn like this, where they have all of the chain ends emanating from the central core unit, and all the monomeric repeat units being intermediary layers, but there’s a high degree of flexibility in these molecules, that allows for them to adopt multiple confirmations. Most applications would expect that the chain ends would be on the surface of the structure and available for binding in a polyvalent fashion to, for example, biological receptor. But, I’ll show you that it’s actually possible for the chain ends to be tucked inside, or presented on the surface, and it’s this dynamic process that’s actually the most exciting toward development of these macromolecules for controlled applications. So, I’m not going to go through a lot of examples of the solution state properties, except to highlight, that again, because of their small size, and their structure, and the high degree of the number of chain ends, they have enhanced solubility, relative to linear polymers. They don’t tangle-up like linear polymers do; rather, they act like globular molecular ball bearings. They have high dynamic radii that increase with generation number, so increase as are grown larger and larger, and ultimately, the viscosity of the structures and solution goes through a maximum and then decreases, and so their of interest as viscosity modifiers, for example. The core environment is very interesting because the core environment allows for the sequestration, for example, guest molecules, either for capture, as we saw on the example of WelChol, or for temporary packaging, and for example, a

therapeutic agent that’s to be taken to a particular tissue and then delivered or released. The solid state properties of the dendritic macromolecules is also very interesting, and I will highlight one example, that was a study, performed in my laboratory in collaboration with Jacob Shaffer, and Department of Chemistry at Washington University, to determine the actual confirmation of the dendritic macromolecules in the solid state. Their thermal and rheological properties are of importance, as well, as is, their interfacial behaviors. So, when we started this work back in the late 1990’s, again, the dendritic macromolecules were drawn, either three dimensionally, or more commonly, two dimensionally, with all of the chain ends presented on the surface of the molecule, and as I had indicated, many people were interested in using those chain end moieties as ligands to find a particular receptor. So, what we did was, in the study, was we labeled the chain termini with a carbon thirteen label and then put a flourine nineteen label near the center of the structure. And, then in another molecule we labeled an internal intermediate monomeric repeat unit with a carbon thirteen label and then put the flourine inside. We then used rotational-echo double-resonance NMR as a molecular ruler, in order to measure the distance between this carbon-13 and the flourine, or all of these carbon-13 actually, with the flourine, which is all the carbon-13 labels at this radial distance to the flourine. And, it was found that the distances from here to here, and from here to here, were about the same. And, in order for that to happen, as you can see based on this structure, there has to be the inward folding of the chain ends. We were also able to perform dilution studies, in order to measure the internuclear distances between carbon13 and flourine-19 labels. And, that indicated that there was interpenetration and interdigitation of the arms of these dendritic macromolecules, just as you would expect, for say, burs and next to one another stacking, and interpenetrating and tangling up, to some extent, but not to same extent as would occur for a linear polymer. Now, some very interesting recent work that’s utilized this inward folding of the chain ends, or pulling them out through favorable interactions with the external solvent, has been reported from Thayumanavan's lab at the University of Massachusetts Amherst, where he has incorporated into a single dendritic macromolecules, both hydrophobic and hydrophilic functionalities, so carboxylic acids that are water-soluble, and decyl chains which are, organic-soluble. By placing both of these hydrophilic and hydrophobic at each of the chain ends of the dendritic macromolecules, he has both of those poised to, either, to inside if the external environment is dis-favorable for solvation , or come outside of the structure, if the interactions are favorable. And therefore, if he places this molecule in a polar solvent like water, the hydrophilic groups are presented on the surface, where’s if he places it into an organicsolvent, and then solvate those decyl chains, they can extend from the surface of the structure and tuck the polar groups inside. So, he can go from a structure, which is a typical micellar structure, to one that is an inverse micellar structure. This is very, very interesting, because what he was able to then show, as well, is that a hydrophobic dye molecule will pack within this hydrophobic core domain, and aqueous solution, and then, it’s expelled when it’s placed into organic solvent. Where as, a water-soluble dye will pack inside this polar core

environment, when it’s in a fatty material, for example. So, in the ophthalmology field, you can imagine, this would be a very interesting as a delivery device, where the reversibility of the hydrophilic and hydrophobic functionalities within the molecule could allow for transport. For example, through membranes, and also, releasing of therapeutics at various stages during the transport process. Now, I don’t know if that work is ongoing, but it is a very interesting potential application. Now, the dendritic macromolecules structure does not necessarily have to be uniform throughout the entire framework. Rather, there’s an ability to create unsymmetrical structures. And, Jean Fréchet’s laboratory at the University of California Berkeley, has reported several reports in this regard, where I am showing here, some of the schematic representation for what are called, bowtie structures, where a dendritic fragment would be one side, that’s not the same size as the dendritic fragment on the other. And, this dendritic fragments might bare some linear polymer chains extending from it, for example. And then, if this dendritic fragment is grown larger, so there are a larger number of chain end groups for attachment of those linear polymers, then we can have a higher number of linear polymers extending from the dendritic structure, which is again, non-symmetrical, and then that can be continued again. So, you can see there’s a lot of variability here, in the way that the structures and components can be put together, and it’s again, the chemistry that allows for control over this. Now, in Jean Fréchet’s case, they’re working with polyesters. And, those are very interesting because they are degradable polymers, and on one side, so this a dendritic macromolecules that has two sides, a branch structure here, and a branch structure here, coupled together in the center. So, you can see that these dendritic moieties are of different generation levels, or different sizes, and on one side polyethylene glycol or actually this polyethylene oxide, chains are conjugated to chain termini. If you see the terms polyethylene glycol and polyethylene oxide in the literature, the way to distinguish them, is the polyethylene glycol is a glycol, it has a hydroxyl group at each end. Where as, polyethylene oxide, is a polyethylene oxide chain that has only one hydroxyl chain end. It's a sideline. In the case of this structure then, after conjugation of the polyethylene oxides onto one side, there are hydroxyl groups that remain on the other side of the structure that can be utilized for the attachment, for the example, of therapeutic agents, and those could be attached, for example, through a labile linkage. And so, there is a large degree of variability here, in terms of the number of therapeutic agents that could be attached to the structure, the number of polyethylene oxides to provide of water solubility, and also, the length of the polyethylene oxides, and whether those can come back and wrap around the therapeutic, and protect it or not. So, there’s really a lot of potential for these types of structures. The types of structures that can be synthesized are really as large and as varied as you imagination allows. Where, the chemistry now is developed to the stage where it’s a plug-and-play kind of system. Where you can see that these are several schematic representation, that are really cartoons of the types of chemistry that can be employed, but you can see that are various kinds of star and dendritic structures that use pieces of linear polymer chains, and branching dendritic units, coupled together in different ways, and of different compositions, that’s the different

coloring here, in order to create very complex macromolecules. But if we want to extend to the preparation of nanostructures that are say, larger than the dendritic macromolecules, but not synthesized through iterative, repetitive chemistries that rely upon coupling together individual components in a repetitive fashion, then want to combine the covalent chemistry, for example, formation of strong chemical bonds, with supramolecular chemistry, which is the self assemble that Mark had indicated in his presentation. And, my laboratory has been focused upon the structure that’s on the far right side of the slide, which are called, shell crosslinked linked knedel-like nanoparticles, and Knedel is a dumpling, so they have a core shell morphology, and those are derived from blocked copolymer micelles that are assembled, as Mark had indicated, through supramolecular chemistry, through noncovalent interactions, weak interaction, for example, hydrophobic interactions. An intermediate between the dendrimer, of which, typically, is of about, one to ten nanometers in diameter, and the micelles and their shell crosslinked products that are typically 10 to 100 nanometers in diameter… there’s an interesting area where single linear polymer chains can be collapsed upon themselves and covalently linked together, in order to perform crosslinking chemistry, not between the polymer chains, as I had shown on the second … third slide of the presentation, but rather, intramolecularly. And there’s a nice example of that that was reported from Carig Holicker’s laboratory at IBM research center. He’s since moved to the University of California, Santa Barbara, wherefore, a polystyrene chain, which is like the styrofoam that you might find in a coffee cup, the incorporation of some chemically reactive groups within the structure, just a few of them are all that are required, allows for this linear polymer chain to bare reactive chemical functionalities dangling from the side chain, so as side chain functional group. And therefore, when this chain is placed into an environment, a solution, where it collapses upon itself, if those reactive moieties are allowed to form covalent bonds then a collapsed globular structure can be prepared in one-step. It is similar to a dendrimer and having a globular shape, and very different properties from the linear polymer, from which it originated, but is produced in a very simple fashion. Now, if we extend this from this one-step intramolecular chain collapse, into the formation of multimolecular micelles and crosslinking, there are also interesting structures that can be produced, of larger sizes then would be present here for the single chain collapse. I want to spend just a quick moment indicating that it’s very interesting to be able prepared each of these nanostructured materials, but it’s another thing, to be able to characterize them, and I think Mark had highlighted that in his presentation, and he also gave you some very nice examples of what it means to be nanoscopic, in terms of the perspective. And, I like to use this example, because what it does, it indicates that if we take ourselves and compare the height of a person relative to the diameter of the earth, then the relative height of an average one meter person, compared to the diameter of the earth, is about 10 million times smaller, and for a particle that has 100 nanometer diameter, it’s about 10 million times smaller than we are. So, we’re really looking at very small objects, and as Mark had pointed out, looking at these structures is quit difficult. And so, I like to show this cartoon, where this researcher approaches his

colleague and says he’s really made this magnificent discovery. He’s created a molecular computer, and he goes on about all the things that it can do and all calculations, the real calculations it can perform, and you know, it’s got great speed and everything, and he say’s that at the moment he’s having only one small problem with is molecular computer, and she asks, “well what is that?” and he said, he can’t find it. And, that’s really one of the key challenges in the laboratory is the identification of the nanostructures and really not only not being able to find them, but to quantify their size and also their properties. So, as Mark had told you, we often use what is called an atomic force microscope, which is abbreviated, AFM, and, I like to relate that to an old record player, where it essentially scans across the surface and reads out the surface topography, as well as, the surface properties, in terms of the modulus of the material, whether flexible or a hard surface. And, we also, employ transmissional electron microscopy, which rather, than using light, a visible wavelength that we can see with our eyes, uses the electrons, to pass through the material, and measures the contrast of passing through an object, verses, just the gird on which the object sits. And, another type of visualization for nanostructures and one of their key applications that’s being developed is in the area of in vivo imaging, and in vivo imaging involves labeling the nanomaterials with, maybe, radioactive elements, or magnetic residents, active elements, in order to track the positions of the nanostructures in vivo, by MRI imaging, or by, for example, PET imaging. And, the example that I’m showing here, is a real technology from Alza, that uses liposomes, and these liposomes are assemblies of surfactant molecules, into vesicles that are, typically, about 100 nanometers in diameter. And, it’s been found that the size of the liposomes is critical to their blood circulation times. Where, if the structures are less that 100 nanometers in diameter, then their not taken up by the RAS system as quickly. Where as, if they are larger than 10 nanometers as well, there allowed to circulate because they are not excreted through the kidneys. So, something smaller that 10 nanometers, it’s passed very quickly through the kidneys, and if it’s larger than 100 nanometers, of course, it’s taken up by the liver and the lung and things, the spleen, as well. So, the idea is that we’ve got to be between about 10 and 100 nanometers for utilization of nanoparticles, and biomedical applications that are going to rely upon their blood circulation. And, the images shown here, are illustrating that when these liposomes are of appropriate size and also, decorated with polyethylene oxide chains, that they're able to circulate for long period of time and allow for imaging over large periods of time. Now, if we then go from these structures, to not assemblies of small surfactant molecules, into vesicles, but rather, assemblies of again, of polymer chains, and to micelles, then it allows us to very accurately tone the size of the structures, and get well below the 100 nanometer size of the liposomes, and I’m showing in this example again from Jean Frechet’s laboratory, UC, Berkeley, that the assembly of the block copolymers into micelles, and in this particular example were quit large, about 90 nanometers in diameter can be a reversible process. In fact, there’s a dynamic disassembly process that occurs, but there can be some degree of stability to the block copolymer micelles, that has allowed them to be studied as a drug delivery

device. For example, there is a significant amount of work out of Kataoka’s laboratory, and I can provide references for anyone who is interested. As Jean Frechet’s laboratory had conducted recently, they wanted to allow for the assembly of block copolymer micelle, to use the core domain to use, as say, a host for the uptake of guest molecules, and they are using in this case nile red as a label, in order to demonstrate that this nile red can be packaged within the core domain of they hydrophobic chain segments, here, based upon these acetal groups dangling as side chains off of what was polyaspartic acid. So, this block copolymer has a polyethylene oxide chain, and a polyaspartic acid, so this is watersoluble. Once these hydrophobic groups are attached along the backbone of the polyaspartic acid, this generates a hydrophobic segment, this allows for the polymers to assemble into multimolecular aggregates, which are block copolymer micelles, sequestering the Nile red within the core domain. And, then upon the reduction of the pH, or upon the introduction of acid, then that catalyzes the cleavage of this acetal moiety to release the aldyhide product and also the diol. This then reestablishes hydrophilicity to this block segment, and allows for dissolution of what was micellar aggregate. And, each of these processes was monitored, where the release of the benzaldehyde was monitored as a function of time, and at pH 5 the release was much greater than at pH 7, or 7.4, and at the same time, the release of the nile red, upon the breakdown of the micelles, was much faster at pH 5 than at pH 7.4, and the release of the nile red, coincided with the release of the aldyhide, to indicate that it was a pH triggered hydrolysis that caused the destruction of the micellar assemblies. Now, as I had indicated, my laboratory is interested in the shell crosslinked knedel-like nanoparticles, and our interest is based upon the desire to create very robust assemblies, and this is again, conducted by comparing, as Jean Frechet had done, an amphophilic block polymer that has a hydrophilic and hydrophobic chain segment, and placing that into water and allowing the supramolecular assembly to take place, to generate nanoscale assemblies. We then follow that process by crosslinking, so linking together the chains, and we do the crosslinking, either in the shell layer, by reaction along this chain segment, or in the core domain by reaction along this chain segment, so we can do chemical modification selectively here, or here, to get very different structures. Here, these are nodular structures that have hairs of polymer chains extending from their surface, where as in this case, this is a crosslinked network, that is a shell layer, and the hairs are extending inside the structure. So, structurally these are very different, and their properties and extremely different. For example, if we now degrade the hydrophilic hairs off of the surface of the structure, we end up with a particle, whereas, if degrade the hydrophobic hairs from within these structures, we end up with hollowed cavities within a shell. So, again very different structures resolve. Unfortunately, I’m not going to have much time to go through examples of the chemistry, but let me show you that overall it is a very simple process, where we spend a lot of time making these amphophilic block copolymers to be very uniformed, and well defined structures, so that they undergo an assemble in a uniform way. And again, this assemble and disassemble is reversible process, and by then, performing chemical modification that involves covalent crosslinking throughout

the shell of the structure. The robust shell crosslinked nanoparticle can result here, and then that can be further manipulated, and chemist tend to have a very strong desire to, not only understand matter, but also, to manipulate matter on the molecular level. Our manipulations have involved, for example, physical manipulations, where the addition of the solvent now for this core domain, allows for extraction of those chains from the material in a process where we, essentially, do a threading of the polymer chain from the inside to outside, but because it is covalently attached inside, it can’t be removed from the shell, and rather, this is just an inversion of the structure. If we then cut that bond between the core chains and the shell, and then extract the chains, now we can remove them from the system, so long as their small enough to escape the shell. And, as I am showing here, sometimes those polymers are so large they can’t get out. So they can be trapped inside. If we instead cut these chains into small molecules, and extract them out, then that leads to the hollowed structure. And, my laboratory now is spending a great deal of time, taking this hollowed structure, which looks a little bit boring maybe, because it’s simply just a cage, and we’re decorating the outside of the cage with ligans that combine to particular cellular receptors and decorating the inside of the cage with functionalities, that then, enhance the uptake of guest molecules, to generate, essentially, an intelligent packaging material, that can package, for example, imaging agents or therapeutic agents, within the core of this structure, and then having targeting moieties on the outside, that allow for transport and delivery of the package. Ok I can see that I’m out of time, so I’m going to skip through the details of a lot of this work, and I’ll be here all day if you want to talk about it. Sorry, I’m skipping a lot. Let me show you quickly, that we’re interested, not only, in spherical structures that lead to these cages, but also, more recently, by manipulation during the assembly process, we’ve demonstrated that we can make donuts, that are nanoscopic in size, and is shown schematically here, where we make a more sophisticated polymer, and control the conditions under which the assemble is conducted, and these donuts are really interesting. We’ve only found these recently and now were looking at tuning the chemistry inside the edge of the donut, to use that as a capture device, as well, as inside the core domain, and then we’ve got the surface. So, there are many different regions in order to make very sophisticated materials. One sophisticated material that’s not from my laboratory, but is from Quantum Dot corporation, involves the use the quantum dots for in vivo imaging agents, and the beauty of quantum dots, as Mark had pointed out, is that the wavelength at which they absorb and emit, can be tuned, and also, they are very bright. And so, there…and there not susceptible to quenching as our florescent chromophores And, in one of the key problems with using quantum dots, is that their inorganic crystallin materials, which are in some cases, toxic and in other cases, not dispersible in water, and also, maybe, don’t have sophisticated surface chemistry, again, to target them to particular tissues. So, Quantum Dot has developed some polymer chemistry that’s based on polyacrylic acid, that has a degree of amidation to incorporate some hydrophobic chains that can stick to the surface of the quantum dot, and these carboxylic acids are used for the crosslinking reaction, in order to first coat

the dot with this polymer, and then, to stabilize it by tying it all together to make a robust coating on the outside of the quantum dot. And, then they’ve performed in vivo imaging, in this case no using any targeting moieties, but simply illustrating at the same depth of tissue here, their able to get much better resolution with the quantum dots, than for a florescent dye. So, let me conclude, by saying that organic materials of nanoscale dimensions, whether the entire structure is organic or whether simply a surface covering to inorganic package in the center, have a significant potential and a wide range of biomedical applications. And, the key is, that chemist have to able to control the methodologies, by which these materials are synthesized, in order to achieve well-defined composition structure and properties. This has to be performed coincidentally with rigorous physical chemical characterizations, and this is all the fundamental work and then additionally, fundamental work, in order to evaluate the performance in biological systems, for which their designed to perform a particular function. And, this process takes many years, but overall it allows for the translation of nanoscience, to nanotechnology. I’d like to thank you very much for your attention and I look forward to the discussion later this afternoon.