 OK, thank you. Professor Gao received his specialist degree from Xi'an Jiao Dong University of China in 1982. And his master in PhD degrees in engineering science from Harvard University in 1984 and 1988, respectively. He served on a faculty of Stanford University between 1988 and 2002, when he was promoted to full professor with tenure in 1994 and full professor in 2000. He served as the director of the Max Plan Institute for Metals Research between 2001 and 2006. And before joining the faculty of Brown University in 2006, at present, he is the water and number professor of engineering at Brown. Professor Gao's interest focused on understanding of basic principles that control mechanic properties and behaviors of materials in those engineering and biological systems. He has been elected to National Academy of Engineering, German National Academy of Science, and Chinese Academy of Sciences. And he is the editor-in-chief of the Journal of Mechanisms, Francis Solis, the leading journal of his field. He has received numerous academic awards from John Simon and Guggenheim Fellowship in 1995 and to recent to the Rodney Heel Prize in Solid Mechanics from the International Union of Theoretical and Applied Mechanics in 2012, Prager Medal from Society of Engineering Science in 2015, Nada Medal from American Society of Mechanical Engineers in 2015, and the Theodor von Kamen Medal from American Society of Civil Engineering in 2017. So let's welcome Professor Gao. Thank you. Thank you. OK, can you hear me? This is working. Thank you, Ting, for your kind introduction. And thank you all for having me here, giving this prestigious lecture. It's my great honor to share with some of you some of the work in my group on nanomaterial cell interactions. So let me first acknowledge two graduate students, one postdoc in my group, who did actually the work. And I also benefit with a lot of faculty collaborators. So Bob Hurd, he's a chemical engineer. I've been working for over a decade. Professor Agnes King from Brown Medical School and Professor Manu Nakis from Brown Medical School, Professor Pada Flahovska. She actually used to be a Brown. She moved to a Northwestern University this year, and also two faculty from Harvard Medical School. So I've been working in this field for the last decade. Basic idea was looking at the mechanical aspect of how nanomaterial interact with cell. Of course, there's a huge chemical aspect as well. They redox the reactions. But those aspects, I'm not addressing today. I'm addressing from a mechanical engineering point of view today. So the kind of problem I'm interested in, so-called mechanics of cell nanomaterial interactions. Here's a couple of review papers we wrote in the last couple of years. Mostly we're interested in how a special cell membrane interacts with nanoparticles of various geometries, such as a sphere, or a cube, or a tube, fiber, or a soft or stiff pad. These are called one-dimensional nanomaterial, or two-dimensional nanomaterial, or sometimes this is called zero dimension. Also the surface chemistry sometimes come in, but not always. So also we're interested if this nanomaterial get into cell, what are the destiny? How does the material interact with intracellular vesicles, such as lysosome? What are the biological consequences, their biological effects? So here's some summary. Cell-optic pathways of nanomaterial with different geometrical properties, such as size, shape, orientation. Mechanical properties, such as stiffness. Or chemical properties, such as surface functionalization. So how these different properties influence the cell-optic pathways of the nanomaterials? Second of all, the intracellular packaging of nanomaterials, so after the nanomaterial get into cell, they have a package of issues. So how are they going to go into the cell? What are the consequences? Toxicity damage of nanomaterial to cells and membranes. So these are the issues we're interested in. Let me show you a few backgrounds again. What is the driving force? What is the background motivation for this type of work? First background I mentioned is so-called nanotoxicology. Essentially, this is an emerging field, especially in the last decade or so, since the National Nanotechnology Initiative. President Clinton initiated this almost 20 years ago now. So essentially addressing the biological environmental impact of nanomaterials, huge field. People from different fields can all contribute understanding. But there's a need because the engineer nanomaterials. So these are not a result of natural evolution, but because we made them in the laboratory, or we fabricated them, there have become a significant fraction of material flowing in a global economy. It is estimated about the majority of them. This is like an estimate a few years ago. 100,000 tons of this nanomaterial, most of them will end up in landfill. They will end up in environment. No one knows their long-term consequences. So there's a need to know how to influence the biological world, the environmental ecology. Two specific examples, carbon nanotube production exceeded several thousand tons per year in 2013. These are really man-made nanomaterials who are chemically very stable, ultra stable. They're not easily degraded by biological enzymes. So they will be there for a long time. And there have been many applications, or have been used in many applications, such as energy storage, automotive parts, boat hose, sporting goods, water filter, thin film electronics, coatings, actuator, lots of applications of this material. This is a booming nanotechnology. And so a lot of this material will eventually end up in environment. So what are the effects on our life, on our species, not so clear? And graphene is a newer, even newer nanomaterial. Graphene was around 15 times annual production, 2009. And five years later, it's grown by a factor of 10. Today it's even much higher. So all this just shows a rapid growth of nanotechnology, nanofabricated material. Their environmental biological impacts are unclear at this moment. OK. Another motivation for this type of study is called nanomedicine. A lot of people are using nanoparticles, nanotechnology, for diagnosing cancers and treating cancers. So a lot of people. So I'm giving an example of antibiotic. How this nanomaterial can also be used for the benefit of addressing bacteria issues. As we know, since 1940s, since they emerged in the penicillin and the various antibiotics, this has made a huge difference in the quality of life. A lot of today's population, a large fraction, exist because of the antibiotic. This is a big advance in medicine. However, bacteria also been evolving. So there's a war between the human development of various antibiotics and the evolution in the biological world. They keep getting new species evolving, mutations, and things. And currently, the looming crisis means the human pace of innovation has not been able to keep up the pace with the emergence of new bacteria. In fact, the World Health Organization issued the crisis, the warning. The most recent release was a year ago, February 27, 2017. They identified a couple of bacteria in a critical stage, wider critical. This antibiotic called copper panium. Copper panium is the last resort. It's not prescribed generally for antibiotics. It's only for the last resort if everything else failed. So these emerging couple of bacteria become resistant to copper panium. That means we have no cure for them if they break out. So the challenges here in this field is the development of a new antibiotic has not been able to keep up with the pace of antibiotic resistance. Most existing antibiotic targets the protein, or cell wall, DNA synthesis pathways. They do not work on non-growing bacteria. If the bacteria, they're called persisters, some of the bacteria stop synthesizing, they become sleeping, they go in the sleep. So the current antibiotic have no effect on them. So the potential solution to this is two questions. Can we use modeling simulation to speed up the pace of drug innovation discovery? First, we need to speed up the innovation pace. Second, can we seek membrane active antibiotic that kill persisters? Because these persisters, they're not synthesizing so traditional antibiotic on the market, they target some synthesis pathway, they do not work. Can we develop such things? So one of my topic today is I'll give examples of this application. Another background is the rapid advance in bioimaging. Traditionally, optical microscopy is the prime tool in using medical school. Well, material science, we use electron microscopy. But electron microscopy do not work on live cells. You have to kill the cells. You have to stay dead materials, which is a big disadvantage. So optical microscopy, the resolution is limited to optical wavelengths. So in the last 20 years, the last two decades, there are various near-field scanning microscopy and other fluorescence techniques emerged. So to bring the lens dimension into nanoscale. In fact, 2014 Nobel Prize in Chemistry was awarded to three scientists, Eric Betzig, Warner and Stephen Hale for developing super-resolved fluorescence microscopy, which brings optical macro into nanodimension. So many of the things we can only speculate, maybe a decade ago, can today be visualized. This is one example. Showing the phyllo-podia is a structure on a cell membrane. Phyllo-podia, it's called pseudo-legs of cells, play an important role in the cell migration. You can see these structures are typically 100 nanometer or even thinner. But this movie actually is not an electron microscopy, it's optical microscopy, it's a nanostructure evolution. This is only possible, this is a paper from a paper published in Science in 2014. So not even possible five years ago, but today, using these highly-resolved optical microscopy, you can begin to study the mechanical aspect of nanostructures in the drug with the cell. In a limited time, I thought today, this is a huge field. So I'm just selecting three problems to share with you. Select a problem in mechanics of cell nanomaterial interaction. First problem, first class problem, I say morphology. This is an area where we question the mechanics of cell entry of a 1D nanomaterial. And we can just give you an example here. Second problem I'm sharing with you is called packaging. How a nanoscale material is packaged in an intracellular vesicle. So this has to do with cytotoxicity of a flexible nanofiber. Third problem I'm sharing with you is the membrane active antibiotic. There we do energy mapping, showing how physical science can help. This is really a biological problem, but I'm just giving you an example of how physical science is helping us understanding the mechanism by mapping the energy evolution as this nanomaterial interact with cell membrane. So the first problem, I'm showing a little bit of mathematics, so only two slides. So bear with me. So here, for example, I'm just giving you this example. Suppose we want to know how a nanofiber, such as carbon nanotube intercells. So we can use a physical science approach by writing down the free energy of interaction of the two objects. One is a cell membrane, as I draw here. One is a nanofiber, which is the intercell angle theta. Theta is orientation. So there are the free energy. We have two term. The free energy consists of, first term is the bending energy, deformation energy. Because in order to bring this nanomaterial inside a cell, the cell membrane has to bend, curved around its nano-objects. There's a deformation energy. So here, the curvature, H is the mean curvature of this membrane. Kappa is called bending-rigidative membrane. So this term represents the deformation energy of the membrane. The second term is the membrane tension energy. So the membrane subject to some tension is maintained by the cell. Because the nanomaterial, stick of nanomaterial, change the area, change the membrane surface area so that has a tension energy. Anyway, so you're looking for the minimum energy configuration of these two objects, assembly of these two objects. It turns out this problem is governed by a single parameter. This parameter is a non-dimensional parameter. It's called normalized tension. It's defined as the real membrane tension times the square of the diameter of the fiber divided by the bending rigidity, a single dimensionless parameter. That parameter is given. You know the behavior. You know what is the minimum energy configuration. It turns out the critical number is this 2 pi over 5. If this dimensionless number is bigger than 2 pi, which is 1.26, if the normalized membrane tension is bigger than this number, then the minimum energy configuration is shown here, having this fiber just like a tree, like a tree planted on the ground. This is the minimum energy. The nanomaterial like to stick perpendicular to the cell membrane. Another case is, as soon as the normalized membrane tension is bigger than this number, 1.26, then the favorable energy configuration, we call paralleled hearing, is having this nanomaterial just fall down. This tree just fall down on the ground with a very shallow angle. This is very simple. The conclusion is very simple. So I'm not going to go into the depths of mathematics, but it's just simple. So here's two examples of free energy evolving. So here shows one example is normalized tension for the 1, which is in a subcritical range. So here I'm plotting the red is the bending energy. The first term, the membrane bending energy, how the bending energy change with the entry angle, with the orientation of this nanomaterial, which clearly shows the bending energy is minimized at 90 degree when this is normal to the cell membrane. The tension energy is showing the blue curve. The tension energy prefers a shallow angle, not 90 degrees. Actually, the energy increase. But total energy, the net, if you add these two terms together, the bending energy dominates. So this is total energy, the black curve. So total energy favors this mode, perpendicular entry mode. For on the other hand, if this normalized tension equal to 3, which is a supercritical case, which is this case, you see the bending energy still prefers 90 degree entry. But tension energy become more stronger so it dominates total energy. Total energy now prefers the paralleled hearing mode. And there's a lot of math details I'm going to skip in here. But let me give you a simple explanation how to use in the mathematical geometry. To understand why there's a perpendicular mode, or interaction mode, when the normalized tension is smaller than this 1.26 critical number, consider the bending energy. Bending energy of a membrane deformation is given by the bending rigidity of the membrane times the mean curvature. Mean curvature. If you still remember the calculus, the analytical geometry in your college class, there's a special mathematical surface called catenoid. Catenoid is given by this surface here. It's drawn exactly the shape. As I draw here in this figure, this is the shape of the membrane called catenoid. Catenoid is a very famous curve, soap bubble related to, and things. This mathematical curve has two principal curves. Every point has two principal curvature, kappa 1 and 2. They're equal in magnitude, but opposite in sign, which means if I have a mathematical surface like this, then any point along the surface have zero mean curvature. Because a biological membrane, the deformation energy is proportional to the mean curvature of the surface. So in other words, this curved membrane have zero deformation energy. Even though it's curved, it's highly deformed. It looks like deformed, but it actually have zero deformation energy. This is a tractor existing nature. So provided a perfect mechanism for our cell to take in food and nutrition, it has this special shape. You can curve in so the food and everything can be transported in and out of the cell without causing too much deformation energy. This is an attractive state. But unfortunately, this profile dominates only when tension is not too big, but a little critical normalized tension. If tension is sufficiently big in a supercritical stage, then tension energy dominates. Tension energy is proportional to the increase in membrane area as a result of sticking fiber into the cell membrane, which prefers a shadow, a parallel adherent node. So that's why our food particle is always break down our body, our stomach, right? Our body process a breakdown food particle nanoscale so that we have this mode. So a cell absorb all the nanoparticle nanomaterials through this channel by deform itself into a catanoid. So it can receive this material at a low cost, a zero cost, in deformation energy. But if the material is big, if the nanofiber is big, then it actually can evade this pathway and become a parallel adherent. So here's some experimental evidence. Sorry, let me just say one word here. So this condition, when sigma smaller than sigma c, when they normalize tension smaller than this one, is equivalent to, say, a critical size effect. If the nanofiber, if the fiber is smaller than AC, where AC is given by this, square root of five, membrane tension divided by pi times bending rigidity, this is about a length scale of 40 to 150 nanometer, depends on the cell type, depends on the cell type, in various, so the critical dimension. So this critical state is also equivalent to a critical size for the fiber. If the fiber is thin enough, if the nanofiber, really nanofiber, if the radius of the fiber is smaller than this critical number, then it prefers the normal perpendicular entry. If it's big fiber, then you prefer to lie down. So this is what I'm gonna interpret. So experimentally, we did experiments on my collaborators. We feed the cell into various nanomaterials with different chemistry. So here showed, the micrograph shows a carbon nanotube. Okay, when A is, we consider small enough fibers, small enough radius. So here shows a carbon nanotube, right? And so you can see they're nearly perpendicular. This is a scanning electron microscopy. Okay, showing these nanotubes are standing perpendicular to the cell surface, as we expected from theory, right? And when our paper was submitted, the reviewer was saying you gotta check this is not a chemistry fact, right? So we changed the gold nanowire. Okay, here's actually a bundle of two gold nanowire, but still thin, okay? You can see it's a normal perpendicular to cell surface. Asbestos fiber of completely different chemistry from gold and carbon, also stand in perpendicular to cell membrane, okay? Now, for bigger bundles, okay? We find that when a fiber bundle together, you form a large enough bundles, when they exceed the critical size, then they fall down, they lie down on the cell surface, okay? So 1D nanomaterial, for example, CNT's nanowires, asbestos, fibers, intercellular tip-first perpendicular entry mode, as predicted by our theory, and fiber bundles adopt the horizontal motive interaction. This is kind of consistent with what theory predicts. But ideal experiment, ideally, if we can design experiment, we should have a growing fiber. The fiber growing diameter, our theory predicts initially fiber would stick perpendicular. Now it will lie down at a critical size. But unfortunately, we couldn't do the experiments. So we took a look at this again, this phylo-podia, phylo-podia. Phylo-podia is like a growing fiber. It's not, except this phylo-podia protruding out of the cell membrane instead of going from outside to in, right? Instead of entering cell, actually it's poking out of the cell. But the free energy function is the same, okay? We write down the free energy function for two problems, even though they're physically different problem, but they're mathematically, they're analogous to each other. Okay, the phylo-podia are considerably protruding microtubular proteins, right? You can see this paper, it was 15 years ago, showing how phylo-podia, the forming process phylo-podia, have this microtubular protruding out, then they merge together, right? So it's like a fiber protruding out of the cell membrane, and that fiber is growing, right? But before, nobody was able to explain why phylo-podia, the size is always limited. They don't grow too big, okay? Phylo-podia grow to be like 100 nanometers or so, then they stop, you know, it's pretty stable, right? So here's a movie I showed already. Okay, if you look at, they do transition, right? They do, they do actually, right? You can see they actually fall down. They have the transition between standing up and lying down. Okay, so again, our theory indicates the 1D protruding nanostruction cells, they should become unstable at a critical condition, which is given by this critical diameter. So this movie combined with this explains why these fibers are unstable, right? These fiber structures, phylo-podia, initially they want to protrude out, right? But then when they grow thick enough, they lose their mechanical stability, okay? Okay, so, of course we can also, we have a theory, we can always check by simulation because these days, computer simulation becomes so powerful, we can conduct simulation to check this prediction. So in one simulation, here's a molecular dynamic simulation, we have a cell membrane with receptor molecules interact with carbon nanotube. In one case, we pick the membrane tension, normalized tension to be subcritical, okay? In this case, we see, we give it the initial condition as the fiber is oriented some angle with respect to the membrane. We see that the interaction, the physics, okay, just the physical force alone. There's no biological motor in this model, right? The fiber is spontaneous oriented to be normal, perpendicular to the membrane. If you take a supercritical case, normalized tension is bigger than critical than they actually lie down, right? So here's a movie I show in a subcritical case. We try to explain, okay? So we try to explain this experimental observation for various type of nanomaterial that tend to stand perpendicular, right? So in this simulation, we randomly orient a fiber with respect to cell membrane. We show, so biophysical energy, just purely physical forces, the membrane's spontaneous orient them. Again, this is because of the catanoid, right? The biological membrane prefer in this catanoid configuration, which is a minimum energy, minimum deformation energy. Okay, so let me show you a little bit a connection to disease, okay? For many years, it's known asbestos, nanofibers, asbestos fiber, they cause lung cancer, right? Because asbestos was used a lot last century. For construction material, they're cheap, they're fireproof, they can use insulation, they have very good insulation. But today we don't use them anymore. Most of the manufacturers asbestos have become bankrupt because of lawsuits, right? So this connection is known, they're connected to disease. But why? The mechanism, the mechanistic origin is not understood. So our model shed some light, right? So we said, there's a critical dimension, okay? If you make the fibers thin enough in a nanoscale, nanofibers lower than this critical size range, then the cell cannot distinguish a fiber from a particle. The cell only identified a tip of the fiber, the thing is a nanoparticle. So the cell tried to take it in, okay? Without worrying how long it is. Now imagine the fiber is very long, right? The fiber is actually very long, right? So you get it in this state, right? This is a famous paper probably in 10 years ago, Nature Nanotechnology. This is actually not asbestos fiber. This is a carbon nanotube. Here's an immune cell, okay? Immune cell, right? So you can think what is happening, right? Because this important size scale exists in nature, cell interacts with a 1D nanomaterial only with its tip if the fiber is thin enough because it's catenoid, right? Try to take it in. Actually immune cell is trying to kill it. It's trying to remove it out of our body, right? But this is a huge fiber, right? This is like you're eating a lollipop which is longer than you. So you're getting into trouble, right? So cells overeating, so cell getting in trouble. When the immune cell get into the stuck like this is called frustrated endocytosis or frustrated phagocytosis. The immune cell what it does is the immune cell will call for help, right? You activate your inflammatory mechanism cause a permanent inflammation in the target body many years later evolving to many health issues, okay? So this is like a priest try to arrest a criminal but the criminal turn out to be too strong, right? So the priest constantly call for help. For enemy that shouldn't, couldn't be killed so you get lots of problems. Your body turn into an agitated state for years. Okay, so no surprisingly, so this paper studied carbon nanotubes to the different chemistry from asbestos fiber the pathologic, the phenomenological, biologic concept is very similar, okay? So their conclusion exposing method, method the lining of mice to long multiple carbon nanotubes resulting asbestos like lens dependent pathogenic behavior. That's our main conclusion. So it doesn't matter, it's not a chemistry driven, it's actually a mechanical driven phenomena. If it's long and stiff and you have this toxicity, right? Because it's a, the material, the geometry of the material such that your immune system have not learned how to deal with that, okay? So the second problem I'm gonna share with you is the packaging issue, okay? Imagine nanomaterial such as carbon nanotube intercell, okay, so where do they go? Okay, they go to this important compartment for lysosome. Lysosome is an intracellular vesicle which is responsive for digestion, okay? Our food particle taking to cell and end up lysosome being digested. Lysosome is a highly acidic environment, contain many, many enzymes, okay? It is digestion, degradation, autophagy, metabolism is very critical. The size of lysosome range from 0.1 to 1.2 micrometer. So highly regulated by cell, right? So we are trying to address when a fiber is a problem because a sphere is no problem, right? It's known that if you just feed the cell the sphere, spherical gold nanoparticle, no toxicity. But if you feed them with a lung fiber, then it's a problem arise. So where's the critical transition? How big is big, right? How big is the toxicity starts? So that's a question we address. Here's the key engineering issue we identify as a so-called bio-soft and bio-stiff. So what is bio-soft? Bio-soft we define as the following picture shows, the following movie shows. If the nanomaterial is lung slender and compliant, flexible, then this vesicle, right? It's trying to wrap it around. So it cause, because there's a pinch force, okay? The biological vesicle can apply a pinch force on this confined nanomaterial. If nanomaterial buckles and just nicely package inside the cell, we call it a bio-soft, okay? That means it's in a biological system. It can be nicely packaged, okay? And so this pinch force is about 20 people Newton, right? So we identify. Now, what's a bio-stiff? Bio-stiff if you feed the cell with a lung and stiff nanofibers, okay? You can see this membrane cannot fight against this. This is too rigid, too stiff. It doesn't buckle anymore, okay? It doesn't yield. So instead you have the membrane form a tether around. Deform around this fiber, form a tether, okay? Form a membrane tether. So you have a persistent contact here and here, right? It's like you have a pinch force persistent contact. That persistent contact cause problem, okay? As I will show you here, it's carbon nanotube. Because the fibers material have a high density, high atomic density, okay? There are these material have a high density of atoms here. They have a strong interact with lipid membranes. If you have a persistent contact of a nanomaterial, nanofiber with the membrane, it can extract lipids cause lipid molecule to be extracted along the wall of these fibers. That cause make this lysosome permeable. Make them pores, create pores in the membrane, right? So anyway, so the conclusion here is the persistent contact needs to local membrane damage due to lipid extraction. So we published paper a couple of years ago on this one too. Now we wanted to do experiment. We did my collaboration again. We did experiments to verify this, okay? Again here, we choose the material with the same chemistry. We choose carbon, okay? Same chemistry, all the materials, various geometry, okay? For example, here shows one micrometer long, stiff multi-wall carbon nanotube, okay? In this case, we see that here's a lysosome. We see this fiber is long, right? Cannot be deformed by lysosome actually sticking out just as we, our model shows, okay? This fiber is protruding out, membrane deformed around it, okay? So here shows carbon black, same chemistry. Carbon black is like very small particles, okay? And they become nicely packaged. 0.5, half micrometer stiff, same stiffness, but it's short. It's also become nicely packaged within the lysosome. A long, a soft carbon, like these are, these are, these are with only a few walls. So the nanotubes are flexible, right? You can see this flexible tube are being bent, okay? Being nicely packaged and bent and packaged within lysosome. It doesn't cause this kind of issue. And tango or soft multi-wall carbon nanotube, even more flexible ones, there's no problem. And carbon nanohorns, these materials like a horn, right? They have sharp corners, but they're pretty short. It's small in dimension, they have no problems. Okay, so the short story is we only find this case toxic, same chemistry, right? For this case, you, the lysosome is not capable of packaging them completely. This can cause problems. This is our definition of bio-stiff. All the other one we call them biosoft, okay? So our experiment, so we further verify this permeability. So we tack on an important protein that's usually contained within the lysosome. It's called Cotapsin B. Okay, Cotapsin B is an important protein that's normally within the lysosome. It doesn't permeable. If the Cotapsin B is released into cytoplasm and the cell die, okay, it's the trigger of apoptosis. It's a very famous signal, upstream signal that calls cell death, okay? So we feed different carbon nanotubes, carbon material here. We find only in this case, stiff multi-wall carbon nanotube involved cause substantial release of Cotapsin B. Okay, this is a clear evidence that lysosome become permeable. A lot of things being leaked out into cytoplasm. Okay, here's a number of cells with Cotapsin B release shows this long and stiff multi-wall carbon nanotube show those dependent release of Cotapsin B. The more such nanotube you have, the more Cotapsin B is being released into the cytoplasm. Okay, just our theory predicts, right? You have the persistent contact, damage cell membrane causing the leaking, causing the leaking, and then we can correlate with cell death. Okay, we can correlate the Cotapsin B release to cell death. Okay, so we monitor the Cotapsin B release. Let me also monitor how many cells are surviving, a surviving cell. Okay, we count down with 24 hours later how many cells are surviving. Okay, so here shows the dose dependent survivability. Okay, for example here, let me show you flexible, for the flexible carbon nanotube, so basically surviving, right? Most everybody is surviving. The only exception of a substantial cell death is this red curve and this green curve. So remember the green curve is the one micron carbon nanotube showing this picture, right? So this red curve is even longer. Okay, red curve is even longer. It's more than one micron long, so-called cell death. Okay, so this again correlates the geometric interaction, the packaging interactions to the cell death. Okay, so based on this insight, based on insight we draw a phase diagram, cytotoxicity phase diagram. Okay, phase diagram. Here we plot two parameters, vertical axis is the length of the fiber, horizontal axis is the diameter of the fiber. Here's the effective diameter because this nanotube is multi-waters, inner hole and outer surface, right? Effective means we're gonna condense with the solid fiber. What's the effective diameter? This curve is the buckling criteria, okay? So this, what is the critical condition for this fiber to buckle? Okay, under this 20-peak-one-second pinching force. Okay, so we did a lot of experiments. Each of these data represent experimental measurement, okay? Red means they're toxic. Cell are dying from the viability, assay. Cell are dying. Blue means the cells are surviving, the non-toxic. Okay, so in this range, this between this buckling and a critical length, this length is given by basically less or some size, okay? Less or some size. It's about one micrometer. And we bounded in this range when the length of nanotube is bigger than one micrometer, but they're stiff, it's in this range, okay? So we did a lot of experiments showing they're all toxic. In this upper corner, they're flexible, okay? They're, this range are slender fibers, they're flexible, they're packaged, they're non-toxic. Okay, there are two data here. Okay, and here, okay? In this range, the fibers are short, right? They're smaller than, the lengths of the fiber are smaller than one micrometer. They can be well packaged, okay? Because you don't have persistent contact. There are thermal fluctuation, the statistical fluctuation, right? The thermal forces would cause this fiber to basically do in brownie motion within the cell membrane. So you don't have persistent contact. You don't have persistent contact, it's no problem. You don't get a kind of a lipid extraction. So it's easy to understand in this range, they're non-toxic, right? Including a little part of the spherical particle, they're non-toxic. So this is a cytotoxicity phase diagram we proposed. Okay, so my last example is I'm gonna talk briefly about energy mapping, okay? So here, the challenge is to develop, try to help our medical school colleagues to develop membrane-active antibiotic, okay? The critical challenge here is these antibiotic molecules, whatever you select, has to be non-toxic to human and animal cell. Has to be specifically talking about bacterial membrane, okay? So there's a lot of choices, right? For example, take a nanoparticle, the dimensionality, whether you take a 1D or take a sheet or take a particle or take a tetrahedral, there's many choices. In terms of shape and size, right? How big should it be? Polarity, whether you put electron, you put polar groups, non-polar groups, and stiffness, right? How stiff these particles? There's a range of choices. So a lot of physical questions you can pose. Each of these physical dimension and parameter probably will influence how they interact, how the nanoparticle interacts with bacterial membrane, okay? I'm gonna show you some really simple experiments showing their specificity, okay? So here I'm showing three different molecules, okay? They are chemical from, they're all 2D sheets, sheet-like particles, okay? Have these benzene rings. We put them interact with the vesicle, right? In the first case, in this case, the membrane stay intact, right? Not much damage, okay? The cell membrane is completely okay. In this case, slide change, O to OH, we see the membrane's been damaged. There's segregation and poor formation on a cell membrane. In this case, let me play this again. In this case, actually, even more extreme, right? We start with the membrane, then we have a huge aggregation here, then the membrane burst, okay, the membrane burst. So that means our nature, bacteria and the membranes, actually very sensitive to all these physical parameters, okay? Yet, we do not understand, we do not understand fully how each physical parameter are influencing this, right? So that's why it's important to study, to map out the energy of interactions. So there, we use a methodology that's been widely used in the chemical engineering, about chemical engineering, look at how molecular interactions is called steered molecular dynamics, okay? Look at how a molecule in the process of going to cell membrane, how the free energy change, okay? Essentially, you map out the energy evolution as this particle go into the membrane, go into the, so I'm not going to go into detail here, given the limited time, but here's our calculated free energy evolution, okay? As a distance from a bilayer center, from a center bilayer, okay? So take an example of adipoline, adipoline is this molecule, the first one, adipoline is this case, okay? Adipoline is this case, we see that it doesn't have much effect on cell membrane, doesn't have much effect, okay? So here that corresponds to this green curve, this green, light green curve, right? Adipoline, you can see that as you bring this molecule toward the cell membrane, the energy is increasing, all right? So there's a strong resistance, strong barrier to penetration, this particle actually doesn't easily penetrate into cell membrane. Take a CD437, CD437 is a second molecule that causes substantial change in structure in the membrane structure, causes aggregation, the membrane aggregation, okay? Make them pour, make them leaking. And in this case, we see that there's a small, as you bring into the cell center, small energy barrier, but then there's a big energy well here, okay? There's a strong motivation for this molecule to be inside the membrane, all right? This red curve is the one I showed cell burst, okay? In this molecule, you can see initial causing aggregation, then it caused the burst, the whole cell, the whole membrane just ruptured, okay? In this case, it corresponds to the red curve. There's a huge energy well, right? That means there's a high density molecule would rush into the cell membrane, oh, it's the membrane interior, okay? So here's some simulation, okay? In this case, NTZ, let me show the movie here. Okay, so this is the red curve, simulation of a molecule interacting, molecular dynamic simulation of interact with cell membrane. This is the corresponding experiments. You can see eventually that the molecule will get into the interior, and okay, the middle one is the CD437, CD437, right? So have this blue curve, it goes to the cell membrane. Okay, both of them actually get into the cell membrane. In this case, you have this segregation, okay? Causing permeable, and that pulling is the green one, corresponding energy curve, okay? This one, there's a huge energy barrier to penetrate into the cell membrane. You can see this one actually doesn't go into the membrane, so the membrane stay intact, okay? Just slight change in chemical. Remember, these two molecules deviates only by a hydrogen route, okay, a proton. Very tiny difference in our chemical structure. Have a huge difference in our interaction with cell membrane. Okay, so we've been interacting with our biochemical colleagues from medical school, Brown Harbor Medical School. So we test these two molecules as new membrane active antibiotic, right? So this was reported in Nature paper a few months ago, we show three things, okay? We show these two molecules, indeed, they're fast killing on persisters. Remember, persisters, they're not synthesizing anything. They're sleeping bacteria, right? The only way to kill them, because they're not, there's no synthesis pathway, they're not making anything. So traditional drug doesn't work. But we say that these new molecules do kill them rapidly, right, giving enough dose. They have low human toxicity, right? They have low resistance probability, right? Remember, we're compared with a drug on the market, Cepro, right? So the traditional drug, develop resistance, substantially, this is 100 days of experiments, along experiments, right? The traditionally drug on the market, develop resistance, show resistance after two weeks, okay? After two weeks, you get bacteria, bacteria become resistant, right? Become resistant, this drug, you have to constantly raise your dosage to be effective. Now, because these drugs are targeted on the membrane directly, right? So within 100 days, there's no indication of a substantial resistance, okay? Anyway, so this is the beginning of this work in this area, so there's a lot to be done. So I think maybe there's an emergence of a simulation-based platform to speed up a drug discovery optimization. Because currently, most people in this field are doing combinatorial chemistry and high-soup drug screening. They're just combinatorial, try, try on air, right? Making this, making that, and cooking just like a cook, right? We're just trying to see whether they have any effect. But simulation does reveal the mechanism, right? Tell us why, tell us the rules, revealing the rules behind this, how they're by mapping on the energy, which we have the computational tool to do that, that will help us greatly reduce the time that's needed for the drug innovation, right? So there's a lot open question, of course, in this area, right? There's a multi-scale from all atom simulation to theoretical modeling, the core screen MD. Actually, this is one of my proposals I'm writing, right? I'm proposing this, a lot of open questions in the field. So modeling and simulation, facility screening, assessment, design optimization of synthetic antibiotic. Anyway, so this is a new thing. Let me, how am I doing in time? So, okay, let me just conclude my talk today by summarizing, okay? So rapid events, experimental computational tools, as well as unprecedented bio-imaging techniques that are facilitating quantitative mechanics-based model of cell nanomaterial interaction. Okay, as I showed this bio-imaging techniques even five years ago, lots of questions we were even possible to begin to explore, right? To study, because not biology so far has been qualitative, not quantitative. But with high resolution bio-imaging techniques, biology started to be more and more quantitative. This is where engineering can make a closer bridge with some of the biological problems. And I show three problems today as a representative examples of study contacting my group. And first example I gave it is the morphology of cell nanomaterial interactions, okay? Here I show that one-D nanomaterial below a critical diameter intercells while tip first entry pathway leading to a pathogenicity mechanism for CNT and asbestos nanofibers, right? This is because there's a critical size in fibers. Below that size cell only interrupts a tip which is very dangerous, okay? Cell only capture tip so this material cell gets stuck, stuck in the endocytosis. Get frustrated. So the second example I gave the study on vesicle packaging of flexible nanofibers. Here the key concept is bio-stew versus bio-soft. So when you say biology is soft but how soft is really soft, right? So we give example, we give a concrete criteria, right? Which is for fiber. For fiber, if the fiber undergoes 20 people Newton it buckles, then it's bio-soft, okay? We are very quantitative here, okay? So shows the persistent tip contact can damage the lipid membrane leading to cytotoxicity. We develop a mechanics-based cytotoxic phase diagram and we validate it by experiments to distinguish between pathogenic and biocompatible behavior for carbon 1D nanomaterial. But anyways, but this one is not limited to carbon 1D material. Can be extended to all the other different type of nanomaterial. The third study I showed is on energy mapping of nanomaterial penetrating the cell membrane, okay? Here the example is how this kind of work help us develop a new antibiotic, right? As our paper just published shows, indeed we were able to identify these two very promising, highly promising compounds that can kill bacteria. Even though we still do not fully understand the physics behind it, right? But so we're working with our colleagues on this. So thank you very much. Let me stop here. Thank you. Thank you. Thank you. And actually I want to make one statement. After the discussion, there's a reception and it's called a mayor. So please, you're welcome to join and have some discussions. Any questions? Okay, any questions? Yes, yes. We're going to make a few... I mentioned his grandfather, the only grandmaker in the back area. Right, right, right, right, right. So far we only have grand positive, oh, grand negative material. We're working on grand positive material. Grand positive material, yeah. So because they have a different membrane structure, they have a different structure, that's obviously very important. How much can the newer patients that you mentioned before... Yeah, so the criteria is to have a very low acceptable toxicity to the human cell, but enough to kill the sufficient to kill bacteria. So these two common eye shows seem to meet that criteria. But so far it's still, lots of things are not fully understood, a lot of physics, even though we're doing a combination of a combinatorial chemist to try different things and see how it works, right? So there's a lot of work to be done. It's not fully understood yet. Yeah. What I showed simulation today mostly are full-atom. Mostly full-atom simulation. Oh, coarse-grain, yeah, yeah, yeah. Depends on, right. Because a full-atom simulation is very expensive, right? Very expensive. But for example, one of the things is coarse-grain I showed today. Yeah, these things are all full-atom. But one thing that coarse-grain, this is coarse-grain, right? We're trying to simulate the interaction between a bilayer and a carbon nanotube. So the deformation is actually large-scale, large-scale. So it depends on the scale of interest because in order to model all the atoms, then it's too expensive to do this. So the coarse-grain depends on the problem, I would say. If you're interested in problems looking at the morphology, looking at a configuration from a physical point of view, some coarse-grain is necessary. But if you look at the molecular interactions, if you're focused on the energy mapping, how a single molecule goes into cell membrane, then the full-atom simulation is useful. Right, right, right, right. There's a various level of coarse-grain. Essentially, it's your condensed a cluster of atoms into one particle, right? Then you'll try to capture the energetic evolution. From energy point of view, you're trying to make them more or less equivalent, right? From free energy point of view. Just like our model, you can see it's ultimate coarse-grain model, right? We condense into one parameter. This is actually an awfully complex problem if you do an atomic simulation. So we just condense into energy functions, right? Giving a single parameter. This is like ultimate coarse-grain, right? You reduce the degree from into one. We're talking about the length. What about the geometry of the point at the end? Oh, yeah, yeah, yeah. Right, so you're giving a tip here, right? The tip here, so in this particular example that I calculated here, we're showing tip is always rect tip. So as the fiber changed in different orientation, that tip is always being fully wrapped, right? So they don't come in to this free energy function tip because they're always covered, right? But in the initial stage, it will play a very important role. Like let's say a particle just first touched the cell and it started becoming important role. I also published a paper in those, saw the initial stage of interaction. So the geometry, the key geometry are important at different stages. Once the fibers in or they're already, they don't influence energy. But the initial stages actually create dominant. I just didn't have time to touch this question. I gave a, it's not in my one example, right there. But they're very important, very important, but not important in this question. Yeah, you mentioned that there was gonna be a product research involving bacteria and let's see how three new interbiotics, but I was wondering if this could also be important to other areas, like other kind of sicknesses, like for example cancer or even maybe viruses. It's also part of that. Yes, yes, right now a lot of people actually use nanoparticles to do cancer diagnostic example, right? Because the cancer cell membrane also have different characteristics compared to normal membrane. There's a lot of receptors. They have different electromagnetic properties. So you have the nanoparticle, you can code in this cancer cell. It's very important for diagnostic. So nanoparticle actually, so I just didn't give an example here because there's a lot of people in the community who does that. Cancer treatment is one of the very important application areas for nanoparticles. I was just wondering, so are you sure there's no receptors involved? I wonder why. Sorry, sorry. I wonder why the end of biotics which are really non-toxic or less much less toxic to human membranes? Oh yeah, yeah, yeah, yeah, yeah, yeah. Very good question. Yeah, I didn't have time to discuss it here. Human bacteria, human membrane, the bacterial membrane, they have different compositions, right? So bacterial membranes have different lipids. Human membrane, for example, have cholesterol, have many other. So they're competing different. So we did the same calculation for human membrane. Okay. And this free energy mapping, right? If you consider human membrane composition, they're completely changed. Thank you guys for the experiment. We don't take an actual living, we just make the vesicles which have the composition, you know, the polyglycerin present in the leaves so we can see what's in the back. We have done a summer work but not complete. Because I want to do this, this is a separate version of the stuff, right? That's right, that's right, that's right. No, we have not completed this at this beginning. We have, there's so much open questions, so many open questions. So it's amazing, yeah. Right, right, right, right. Yes, yes, yes. A lot of things worth looking into for this. Yes. So you were talking about this one traditional antivirus, what do you find? Oh, Wendy. Right, right. So what we call one-dimensional material is that one dimension is dominant in your lung, fiber-disciplinary. If it's 2D, I mean, two of the dimensions are lung, like a sheet, I think, we call it 2D material. So like a plane, right, you look at plane. The thickness given, thickness prescribed. But the other two dimensions you can design. Like one that you can design with lens. The cross-section pretty much, it can still be contuned to some extent, but pretty much it's clearly, right? But the other dimension you can play with, right? So this is, this is like jargon use in the animal-material community. See the blood in that, they work out in material. Oh yeah, yeah. They're just a sub-family of 1D material, right? Well, but they're very important in terms of flexibility. Single or multi-platform, very different flexibility. Thank you, brother, for your talk. I have a question on this slide. I mean, when this cell, so the red curve is for the cell with bars, right? Yeah. Okay, so point goes to minimum and then goes back up. Like when it hits the minimum, is that the point when the cell bars? Right. This name on here corresponds to, this is from center, right? It corresponds to some place like here. That means when this part goes in, this is the minimum energy state, not right in the center. Right in the center, because this is bilate, it's bilate. Right in the center, it's a hydrophobic region, but that's the high energy point. The lowest energy is right in the middle here. Okay, why the burst is a very good question. I didn't quite address that, right? So amazing that the energy map in this interpathic already reflected the consequence, right? With the burst, I mean, that's totally rational kind of investigate. That requires some more bigger scale model. For example, we use free energy based modeling to model the aggregation and deformation of how the membrane developed force, right? So that's, this is a completely open field. Completely open field. Where we do not fully understood how this vessel burst, okay? Even without anti-matter, you know what I'm talking about. How the force formation, the rupture in the membrane, it's a lot of people are working on this. But here we say they're highly sensitive to nanomaterials. If you put different materials, you can cause the membrane to, you can destroy the membrane, you can keep the impact, you can cause the leakage. There's a lot of things, there's a lot of biophysics that needs to be explored. Do you think bacteria will actually form a chemical resistance to this kind of like the chemical resistance to other active gases? Yeah, so thing we see here, we put them together in 100 days, we do not see a clear increase in resistance. While we do see, you can see the slightly, of course on this scale, it's almost flat. But there's a slightly increase in resistance. What we find amazingly, the bacterial cell membrane become more negative charged over time, okay? So I think the bacteria is trying to, trying to develop some resistance, but not so successful yet. So we don't know if it's long enough, maybe there's, it completely changed your composition in cookie, right? Bacteria learn how to put some other molecule in and change this energy mapping. But so far, we don't know you, this is a good question. But they do become more electronegative, more negative charged, right? They change your energy interaction a little bit, but not sufficient enough to develop substantial resistance, yeah. So they're trying to do something. Yeah, yeah, yeah. Anyway, evolution is a strong force. It's a very strong force, yeah. We don't know what eventually it leads to, but it's certainly a lot of interesting open questions. Sometimes when cells absorb some of those nanomaterials that they ask for the help of other immune cells in order to take care of that nanomaterial, is it possible to actually trigger an immune response by using nanomaterials that get stuck in the membrane of the cell membrane? Right, so in this case, right, the frustrated endocytosis is a trigger to immune response, where it should not, right? Because there's no, because what cell, what an immune cell does is trying to clean all the dead cells, bacteria, all the foreign substance away from the body, clean them up, right? In our body, one beating cell die each day, right? So the immune cell always working very hard, right? So, but if it gets stuck like this, it triggers a great immune response for no reason, for no good purpose. Because our immune response cannot kill this, no matter what you do, right? So this immune response is unnecessary, and this leads to problems, right? You've got a, like a permanent inflammation somewhere in your body, right? So this is what the problem triggers, a lot of mutation, a lot of other things, problems, right? So the fibers are pretty dangerous to our body, in that sense. We've got to be very careful. Oh. No, the trigger-ready immune response is trying to get rid of it. Oh, yeah. You can't swallow the whole cancer cells. Oh, that's a great idea. That's a great idea. You know, the immunotherapy, just one Nobel Prize two days ago, right? This is a great idea. Yeah, yeah, yeah. This is a great idea. Maybe nanomedicine can further the messed up view, as well. That's a great idea. Yeah, I haven't thought about that so far, yeah. It's a great idea. Yes. In the industry, occupational exposure to nanofibers, especially those biologically long biologically set points, has there been an increase in the fragmentation of the disease? Sorry, I didn't capture your beginning of your speech. There has been an increase in disease and occupational exposures using nanofibers. Right, right, right. Oh, in occupation, asbestos is one prominent example. Asbestos is one, but there is a lot of new ones. Right, a lot of new ones. I don't think there's enough data. FDA regulation on nanomaterial currently focuses on chemistry. They haven't focused on the mechanical aspect because there's not enough data there, right? So far, they say, this is toxic. This is not toxic. Mostly, almost always based on chemistry, based on the chemical composition. But the mechanism effect, people start to recognize that. But haven't been to the FDA regulation yet. It's important work then. Yeah, yeah, right, right. Thank you, thank you, thank you very much.