James Tour, Rice University | Nicolas Guiseppone, CNRS

9:39AM Mar 19, 2021

Speakers:

Keywords:

nano machines

cell

molecular machines

motor

nanomachines

materials

molecular

antibiotics

work

kill

polymer chains

rotation

effect

nano

drilling

surface

questions

ligand

polymers

bacteria

Hello, everyone, welcome back to what is now what is it the third meeting of this of this group formally and these evening meetings once a month. So we're continuing today with our sort of initial topic of like the advent of artificial or synthetic Micro Machines. We got two speakers today. So that means a little shorter about an hour. So we've got Professor James tall from Rice University, and Nicholas just pone from ISIS in Strasburg. So that talks about 20 minutes, we'll have the chat open for questions. Considering how things went last time, I think now we'll have things if you have any questions about anything related to the talks at all, please drop them in the chat. As you may recall, from some of the other sessions that groups had, we're very interested in thinking about how we can advance work in this community. So those questions may get prioritized, but any question of any type is is completely valid. So please put them in there. And then we can take it from there. So I'll take up no more time. Alison, have we decided Who's going first, actually, is there an order?

I think if we go with a proposed order, it's James first. And unless someone else has a different preference, I would say that's a good way to go.

Is that okay? With the speaker's? Yes,

yes, I'm ready to go.

Absolutely. Then. Games, the floor is yours. Okay.

Well, I'm glad to see here. Some of my co workers that have worked with me Gim devez. Has was a postdoc in my group. And then of course, then our grill and grant Simpson, have done, driven all of our nano cars for us. So, so good to have you here to talk about nano machines in medicine and also mentioned nanorobotics LLC, we had developed a number of these nano cars and showed that nano cars could could drive across surfaces. This STM work was done by Kevin Kelly, and we could show and that was the first nano collision ever recorded. And then we we worked with Leonard groves group and grant Simpson to to build and then race these fast nano cars. And we run we won the 2017 nano car race where grant took that nano car and drove it around that pylon around that pylon. And then through these two goalposts, that was 150 nanometers in 90 minutes. And the Swiss group came in second came in five hours behind us so so they in our grills group in Austria grass, along with grant Simpson, and Philip, Peter Meyer, really did tremendous work in driving these across across the finish line. We we've built a series of of nano cars and Leonard's group has has shown some of these related structures moving on surfaces and, and these, these are based of course on the firing of motors, which this particular motors not that fast. But other ones when you when you close down, this ring will spin it about 3 million rotations per second. So what we did is we showed that these could actually move in solution and we could track them using using these floor fours. And we could get get these became the fastest moving molecules ever recorded in solution, where they went about 25% 26% faster than the normal diffusion rate. So then the idea was to have these drill through lipid by layers, and kill. And I had worked with tried to work with several groups to do this and nobody can pull it off. And then I met with with Robert Powell when I was giving giving a one week lectureship at Durham University in the UK. And I tried to work with Professor Parker, he said, Look, I've got a I've got a former postdoc who's a new lecture here, and he's the best experimentalists I've ever seen. So Robert, and I talked for about 10 or 15 minutes. When I got back to Houston, I sent him the molecules and within two months he had this thing working he just utterly amazing. The idea is to put these molecular machines on cell membranes, and then turn on a light and have them drill through, see if they drill through and leave a hole in their wake. And these holes do not close up. Many people have studied this by popping holes in a if within AFM tip. They don't close up rapidly, they take minutes to close.

So we had done a number of experiments. This is just one of them that we did. We did patch clamp to show that when you put an electrode in a cell, you put another electrode outside and you're looking for current between the two electrodes. If you only when you have the nano machines that move fast, and you apply a light only when you turn on the light in the UVs excitable that after about a minute and a half, you start seeing current, then the current really increases. So again, indication that these were opening up cells. And so what we did is we took these nano machines, and if you just this is when we were using a UV visible, UV light activated nanomachines, if you put these in the presence of cells, just with a blank with no nanomachines, just the light will cause these cells to start dying at about 300 seconds. I'm sorry, start dying at about 300 seconds. But then they, they are fully dead by about 600 seconds. But what we found is that when we have the nano machines in there, they they would die by necrosis much faster, we could really get them to start dying, nearly admitted immediately and finished dying, where the other ones would just be starting. And then when we add propidium iodide in there, so we had a number of controls, slow moving motors, motors that didn't have any rotors, we had lots of controls. But we get the laser printer. So when we put propidium iodide in there, this propidium, iodide propidium. iodide only lights up when it intercalates with DNA and RNA. So you put that in the medium, and you start drilling and it starts filling up in these cells. And so you can see that you've drilled these holes and propidium iodide is followed the motor in. And so then we put some of these motors with peptide addons. And so these would target specific cell surfaces. And we could show that we could target for example, a PC, three cells, human prostate cancer cells, and you can see this blebbing that occurs, this blebbing occurs because you open up holes, and these are dying by necrosis, it's not a pop ptosis these all necrose and die very rapidly now. So we could target specific cell types. So we could also do this with two photon near IR. So so we could use two photons at 710 and excite these these nano nano machines that are 360 nanometers. Two photon is inherently inherently confocal. So you could, you could shoot right over one cell and hit another cell and propidium iodide will just pour into that cell and the cell right next to it doesn't even get hurt at all. And then we showed that we could we could target MCF seven cells, which are a, a human cancer cell line, another prostate cancer cell line, another, this is another cell line here. This is a breast cancer cell line, we could use different ad ns peptide ad ends, we could target one and not the other two, we could target the second and not the first or the third, we could target the third, not the first and the second. So we had great selectivity. In Vitro in a dish when you mixed all these different types together, then we switched to visible light activated motors to see if these would more easily translate. And we showed that these visible light activated motors would react this with was with human pancreatic cancer cell line, they would fill the cell membrane and some would get into the cell because they had inherent fluorescence, we could see this and then over time, they would work your way out. So they didn't kill the cell by themselves. But But if you put them in with the cells, and you turn on a light now, so you turn on the light, that you could really start killing these things. So if you put them in with the cells and you wash them clean, you let them clear for 24 hours, they don't kill at all. So when they're there and their cell surface associated, you could start killing rapidly. And this is the fluorescence of the propidium iodide getting in there. And here's the fast motor that that we were using. This is is we're exciting now at 405. So we're now in the visible.

Some people have said, Oh, were you just killing by generating ROI? Yes, and that is certainly not the case. Now we've shown that because what happens is you can use a photosensitizer and really induce RS in a cell. And what you'll do is you'll see you'll see a lot of a lot of kill there by the generation of Rs because we're using cell rocks. And so it's a green dye, it exhibits bright green fluorescence upon oxidation by ROI so you can generate a lot of Rs. If you then add an RS inhibitor and a LC this Rs inhibitor, it will drop it down. So in inhibits the RS. If you use light alone, here's here's the generation of Rs. But if you add the nanomachines they're the nano machines actually greatly reduce the RS. And that makes sense because these have a twisted double bond which are just begging to be hit by reactive oxygen species. So actually these are RS inhibitors. They are killing by a mechanical action, not by a chemical action in inducing Rs. They're actually RS inhibitors. And we started working at MD Anderson Cancer Center, which is the biggest Cancer Center in the world, just across the street from the university here, working with Jeff Meyers lab on skin and oral cancers. He working as well with Robert rangos lab, and then Ashish Komatsu lab on bladder cancer. And then the person doing a lot of the work is Dr. Cicerone, Allah, who was my former postdoc, and now he's working with this company, robotic nanorobotics LLC. And if you look at skin cancers, others the squamous cell carcinoma, then it goes to basal cell carcinoma is going goes deeper, and then melanoma goes very deep can start interacting with the lymph system. And that's when skin cancer turns into something that's, that's extremely dangerous. A lot of old people, particularly with lighter white skin, they have to have these scraped off and it's quite painful, because you have to, they dig quite far down and they the dermatologists will peel this off. So could we treat these with these nano machines by drilling into these. So this is the killing of melanoma and oral cancer cells in collaboration with the folks at MD Anderson that I mentioned. So this is using a murine melanoma, this is all in vitro. And then you can see that if you just use the molecular nanomachines, they're not doing anything, just we use point 1% dmso in water that does nothing, use dmso. And light does nothing but the nanomachines plus slight boom kills everything. Same with this rock one, squamous cell carcinoma cell line, just 150 watt milliwatts per square centimeter, and boom, everything's dead, we can kill the rock three. And and then if you use rock one to 300, again, it would killing everything. So they're quite good. And this is looking at the short term survival, two hours after use. This is again, in vitro, this is 16,000 cells per well, or 1600 cells per Well, when you have the molecular nanomachines. And light just really wipes these things out. So quite effective. Then we move we're moving in vivo. Now, these are recent experiments moving in vivo. So you inject this, this, this tumor line on the flank of a mouse. And then what we do is we let this thing grow. And after seven days, we will inject the nanomachines right on into the tumor, several points around the tumor, and then we shine the light, you can see just the dmso does nothing dmso just a light can can certainly cause an effect in having these not grow. But the molecular nanomachines are just tearing these things up. This is the the the size of the tumor now after after 16 days. So you can see the effect of the nano machines with light. Why does the dmso work? What alone? What is dmso? No, why does MSO work alone, in this case with light rather, with noise. That's that's that's the light effect and dmso It's interesting, the dmso with light is more effective? Yeah, dmso with light, the light itself has an effect on these cells on the cell line. It's not so much in the dmso as it is the light, the light itself. Now we've gone into bladder cancer, if you've known anyone with bladder cancer, they have to go in for treatment over and over again. It's really archaic, they will inject the bladder with with these micro organisms which go and they eat up the epithelial layer of the the bladder and in doing that they'll dislodge this, this these cancerous cells. But when those get past that epithelial layer and get into the bloodstream, there's severe severe

toxicity to the patient. But that's the standard of treatment. So we're seeing if we can inject the nanomachines just through the urinary tract into the bladder, they already have just like inserting a Foley. It goes right on in they even have lights that are already pre approved for going on in there. This is in vitro, we're able to kill a number of cell lines some better than others with these nano machines and light and so you can see the big decklid declination that occurs with light with the nano machines. And you can see the IC 50 measurements that even at two micromolar they can be quite effective in killing off these cell lines. So now we're just moving this now in vivo for the bladder cancer. Let's move on to bacteria. superbugs could kill 10 million people each year by the year 2050. So COVID is like a walk in the park for for what's expected to happen with with antibiotic resistant bacteria. So I tell students when you're my age, expect this to be happening 10 million people a year dying. There's already right now there are nightmare bacteria that the last resort antibiotics do not treat. And so what happens is, you have you have a resistant bacterium just because genetically it is it has something on it either something on the surface of the cell or something on the on the interior that deals with a drug where it's not as easily killed. So you end up killing off everything else. And that one persister remains with a little bit of, of some of the other ones remaining. And then what happens is these start breeding, these start multiplying, and you get now the resistant bacteria multiply and become common. And eventually, this is all that has evolved there is these these resistant bacteria. So that's how you get these drug resistant bacteria. Meanwhile, the discovery of novel antibiotics is not keeping pace with the emergence of new bugs. So this is the new classes of antibiotics that have come in nothing. And one of the reasons is there's no money in this because after four or five years, you get these persister cells, and you haven't made back your money. But your 1.5 billion of bringing a new drug to market and they're in the the the the drug is becoming effective before you've ever gotten your money back. Secondly, it's it's hard because these things have become quite resistant. So if you look at say a gram negative bacterium, it has a cell membrane like a eukaryotic cell, but it also has a peptidoglycan, which is a peptide with sugars. That becomes that makes this into a cell wall. And then you'd have another lipid bilayer gram positive, this would be on the outer side. But still, these are called a cell wall now not even a cell membrane anymore. And so we worked with Jeff Cirilo, initially, this was with our our UV active nano machines. And we showed we could just tear into bacteria. And when we put nano machines with outdated antibiotics, they killed everything. I mean, they've just killed an enormous rate, we could take outdated antibiotics, which were inhibited from getting through the cell membrane, we open up holes and they go in, then they kill. But then Anna Santos has been in my lab for a little over a year now about a year and a half. And she's done this now with bacteria with visible light nanomachine. So again, something that is more likely to get into the clinic, you look at killing these bacteria with standard antibiotics, very little kill. And now we have this is five log reduction with different types of nano machines that we've developed for this five log reduction. This is huge rates of kill. She's an expert in bacteria, she's come here and she said, I've never seen anything that can kill bacteria like this. And so you have this amazing effect. And then what she does is he generates the the the, the the, the persister cells. And what bacteria do is they also build a biofilm around them to help to protect them. And so you multiply, she multiplies now the persister cells, and then you look at the persister cells. And sure enough, we can drill right through those. These are five log reduction, the normal antibiotics don't touch these things. And we're just drilling right into that. These This is a mechanical action drilling right into them. Here's the standard antibiotics that are doing nothing. And these are doing these five large reductions. We've looked at the disruption of biofilms. So we're also able to disrupt the biofilms that depends which type of bacteria we're working with some work better than others, some we can just wipe out their biofilms quite well.

What we what she does, if she helps the evolution along she gets the persister cells, isolate spose and helps them to come along to really generate the persister cells. And you see the standard antibiotics don't kill the persister cells are nanosheets are killing them all nanomachines killing them all. And the these ones that are showing here, these are the typical, these are the typical antibiotics here at the nano machines are just tearing into these. So we've investigated the mechanism, we know the mechanism of these and the action occurs both on the inner membrane and the outer membrane drills right through the peptidoglycan. And you can see the effects on these of these molecular nanomachines on these structures. Again, they're drilling right through the cell wall and then they start disrupting the whole thing in the interior you in these experiments. We're not even using an antibiotic along with it just looking at the nano machines and how they can kill these. And you see the effects. This is E coli. Some of the pictures she's taken. These are these lines are because the E coli is dividing this is what they should look like. This is what they look like after treatment with the nano machines, they get all of these these this shriveling occurs. So the whole morphology of the ecola changes when they've been exposed to the nano machines. So the visible light activated industries display broad spectrum antibiotic action, they both in gram positive and Gram negative, it makes no difference to these nano machines. They disrupt biofilms, they eradicate antibiotic tolerant persister cells, they reduce the minimum inhibitory concentration of antibiotics with different mechanism of action potential waiting antibiotic action. And the mechanism of action involves membrane permeabilization, followed by leakage of the intracellular components and dissipation of membrane potentials, resulting in cell death. And this is necrotic cell death, there's no time for a peptic cell death. Alright, so the take home message is this. This is a fourth modality for medical treatment. One way of treating medical disorders is surgery, you go in with a scalpel. Another is radiation, which generates reactive oxygen species. Another is chemotherapy, which I'm including in that immunotherapy and genetic therapy, because both of these are chemical base. Now if you want to call these different fine, no argument from me, we can call these different. But these are modalities of therapy that are occurring. This is now a fourth modalities, mechanical effects at the nanometre scale, molecular nano machines are like nano scalpel, if if a cell can stop a scalpel, they be able to stop this, if they can't stop a scalpel, it's hard to stop this because it's a it's a molecular machine, it's a machine type action. Now, it could be that they might develop some resistance to this going on the surface. So you just changed the surface atoms, lots of nano machines are coming out now. So you can even change the structure of those. So the projections, potential applications achievable in the near term, and challenges preventing them. So we already have so what we what we need is we're going to in this one to five years, we will have clinical proof and translation is achievable in one to five years. Here's the challenges hiring synthetic chemists, since the COVID, shut down, I can't I can't bring in good synthetic postdocs, because so many so many places where we would normally get our postdocs are shut down, fundraising for nanorobotics has been slowed, because we just were going to start fundraising in January of 2020. And then then, so we have some seed funding, but it's it's slowed down a lot. Suitable scale up operations for synthesis and final selection of specific targeted molecular motors. Those are the challenges because we have so many of them that work, we're trying to find that the best choice here, potential applications in the medium term, five to 15 years. In this timeframe, we should have many of these molecular nano machines approved for clinical use. Certainly, in the middle of this this timeframe, we should have many of these approved, especially because we're dealing with cancers that you could go to last resort use and get it get it installed quickly. potential applications achievable and long term and associated challenges. Well, I think this fourth modality of medical treatment should be a mainstay approach in the coming years. Okay, with that, I end and open it up for questions.

Yeah, what's going on here? There we go. Thank you very much, was very interesting. And there's plenty of questions that have have come in as well. So no, forced me to select them. So I'm quickly quickly scanning through, but I suppose we can just do them in, in order. So I use my new hat. Okay. So

it says do you need one nanomachine molecule per cell? We have no idea I we probably need a lot more than one. You know, these are so small. We don't know how many. But I'm, I'm pretty sure it must be more than one. Because when you open up a hole, that's going to be about a nanometre diameter, you're going to have leakage of ions through that. But that that will close in about a minute and a half. So my guess is you need more than one. Let's see what's the advantage of nano drugs compared to targeted nano targets? targeted therapies to date are chemicals. This is a mechanical effect. So I think that you've seen by targeted therapies, for example, with these bacteria, it's hard so this is a fourth modality what's the advantage of radiation over chemotherapy, it is another modality so so it gives you more weapons in your in your in your box? How fast actually, by the

non unreal, so

not specific, how do you just attach it to the membrane?

You can no No, no,

no, no. I showed you several examples. Where we attach a peptide, and it targets to a specific cell type. So we can do it both ways.

People can do the same with drugs that don't require UV radiation,

correct? Correct, they can do the same with a drug, and then the drug gets to the cell and the drain does, the drug does a chemical effect, you can do the same with a drug for the bacteria. And it won't work. Because the drug doesn't work anymore. Alright, so it has advantages in that sense. Also, this gives you a trigger that we're can you use light, you can use light all over the skin, oral Gi Gi from top to bottom. So you get all of colorectal, you got all of urinary tract, bladder. So there's many ear I so many places that you can use light, and actually direct only at that place is that activated doesn't affect anything else. Only when you have the double activation goes to that site, and you have the light. So it's like a to trigger mechanism. device with the action of it. Let me let me get to some of the other questions. Drilling implies that the action of the motor is leading to translational motion to the cell membrane, do we have an understanding of how the action motor action leads to translational motion, we have some we have some we've talked about translational motion in some of our former papers in solution, and there have been models on how that works, where it clears things out in front of it and Brownian motion pushes up up behind it. But how it's doing that in the lipid by layer is is another fact I am sure some are going out the other way. You turn them on, if they're not cell associated, they never go through, you have to have a membrane associated before you turn them on. If you turn them on right away, they just bounce off the membrane. Do we still have more time for questions? Allison? Should I stop?

I think we still have I have. Okay, let's do maybe two more you're you're in such a go.

Okay, yeah, I want to try to get to as many as I can. What, what exactly happens to the cell membrane? Well, we've seen these cells, these membranes, we've seen the blebbing that happens the result of that occurring is in a mechanical force from the motor pushing into the cell like a drilling machine, or is it a chemical interaction with continuous rotating motor? Well, we do know it's not RS activation, it's not our OVA s activation, we can see these go right into the nucleus. So they're going through the nuclear membrane, because because propidium iodide just fills the nucleus. So once they get into the cell, they're still going, there's the motor have to turn you to directionally for this to work. It's much better unit directly when we use a flapping motor, so it doesn't have that other metal center to give you the dicier topic transition states, then it's going to flap in two directions. And and the efficiency goes way down. I would say goes down by a factor of four or five, if it's not unidirectionally rotating. What's the half life of the motors in the biological system? I don't know what the half life is. I mean, undoubtedly we can you shine these on single molecules on the surface. And if you can see a single molecule going for a minute or two, that's pretty good before they'll die out. So I don't know what the half life is. But presumably, a lot of them are dying out. But we have a lot of them in there. What are the pharmacokinetic properties of the nanomotors in vivo? So we have and what percentage makes it to the tumor of tissue? Well, when we inject it at the tumor site, we know it's right there. So there's, there's, you know, we've injected it, where we can see it. If it's injected globally, then we'd have to study that I presumed these are going to easily oxidized like many aromatics like this. And we don't have to worry about them intercalating into DNA in that they're much larger than just a planar system there are thoughtfully arranged, but I presume they're just going to get oxidized and go right out the kidney, since they're only about a nanometer in diameter, even when you have the peptide. And there's still quite small to be able to get into that does the motor attack mammalian cells he had text any cells, any cells so if you put this on melanoma on the skin, you're losing, you're losing other cells as well. If you're not targeting, you're losing other cells as well. But when you go in with a scalpel, and you cut that whole piece off, you're losing other cells as well. So So yeah, there's there's going to be collateral damage if you don't target. Allison, I'll end there. Okay.

Fabulous, thanks. He just rocketed through them brilliantly. I didn't even need my assistance taught. That's fantastic. So thanks very much for that. That's a really good so we'll move on to the next speaker. And then if there are any more questions for professor or you can drop them the chat or you should come to our galva session afterwards, where more questions can be asked in person. So we'll move to our second speaker, which is Nicolas just burning from ISIS. So if you want to share Your screen please. Fine. Excellent. Yep. Fabulous. So the floor is yours.

Thank you very much. Thank you for your attention. And I'd like to speak today about the possibility to integrate molecular machines in larger system basically to connect them with one another, in order to extract work at a larger scale than the nanometer one. So, for that, I'd like to start with the famous example, coming from nature, which is the functioning of muscles. And you can see that if you go down to the nanometer scale, you find in this sarcoma unit, these kinds of molecular machine which is a protein myosin head, which is able to continuously cycle motion to attach amazing filament to pull on it. And when all these machines are progressing together, then, because of the precise ordering of the myosin filaments, together with the actin filaments, they're able to pull collectively on the sarcoma unit, and to contract it by a distance of about one micrometer from cyclic motion of only a repetitive cyclic motion of only one nanometer. And what you can see from there is that by connecting molecular machines together with polymers and together with let's say, organization orientation, then you can reach what I will call here, I article mechanics, which can allow you to cross landscapes from the micro meter to the 50 of micro meter in the Mio face size, let's say to the centimeters case in the myofibrillar. And finally, muscular fibers. And this was very interested for us when we entered this field to explore the possibility to connect artificial molecular machines within polymer structures for him to change polymer networks and to study the possibility of amplifying them oceans. So, we started with this kind of see to daisy chain neurotoxins, which were first developed by Suraj in 2000. And, we also made use of the chemistry of Professor Stoddart, in order to design these, what will become a monomer unit based on this inter threaded let's say double threaded chrome eaters, which can be either located on this ammonium site when it is protonated. But which can then slide toward this medial triazolam site. When the amine is deprotonated, then this net motion is of about 1.2 nanometer. You'll notice also that each extremities we have attached here tip reading units, which we play the role of stoppers and also which we play the role of figures. So we'll make use here of Metallo supramolecular Super molecular coordination polymer chemistry to obtain coordination polymers as you call molecular polymers in particular, by using iron to ions. And then we were curious to see if we can detect by looking at the body machine, an integration of the contraction motion and extension motion of each individual molecular machine. So, this is what we proved is this kind of neutron scattering and light scattering investigations, we can extract from this data all the information about the single chains of the polynomial in the detailed regime in particular, we can see a cross section, we can see a contour ends or total length of the polymer chain. And what we can clearly see here between the contracted form in red and the extended form of these columns is the change in the intensity of the scattered light, which corresponds to a change in the linear mass density of the polymer chain. And we can really extract because of the sliding motion and the sort of telescopic motion within a single chain polymetal. This chant which also corresponds to a change in the micro meter landscape of the full contour lengths of each volume or chain.

We can go a little bit further we can aggregate these chains together by making use of further supramolecular polymers involving aged bones here, you can see that you can find by FM structures that are similar to the association of February's in muscles, let's say, but this is not over long range orientation just on domains. And there's still a lot of work to do on that. You can however, observe at miso scale changes in the morphologies by contracting and extended the rotax. Once again, further, you can cross link this for tax and in chemical James of Paul email and access now, macroscopic motions macroscopic protraction extensions of pieces of gel centimeter scale. So you can do this integration with values types of a sequence of regular balls, or you can also use covalent bonds, you can probably also use different kinds of molecular machines, we have worked mainly with the one that I've shown you presently, but obviously, you have also limitation or you have a real, fully reversible back and forth motion of these type of switches which work by changing the thermodynamic minimum of the medium that set of the system. And the second thing that we can still improve is this organization in IE oriented fibrillar systems. And this is the object as we asked me to present some perspectives, there is a project or European project, which is funded at the moment, cooperative project, which has the objective to really organize these kinds of see to daisy chain myofibrillar. rotax runs into myofibrillar, fibers, and artificial muscles and to control them with a robotic approach. In particular, we work in collaboration with roboticist here. And I think this is one of the main objectives now, with these machines to go further and to gain in efficiency directionality work out of these systems. But we were also interested to develop systems involving real molecular motors, it's a Brownian ratchets And for that, we chose to integrate possibly integrate the rotary on a unidirectional rotary motion of fingaz Motors. And this is not no views question, but finally, we thought that it would be possible again, to link such kind of models with polymer chains, in a way that for instance, in a simple system, two motors would be associated to two polymer chains. And by turning these motors, if they are fixed to any element here, then the rotation of the two motors should lead to twisting of the super image and these twisting should lead also to a decrease of the distance between the two models. So the idea was to transform a rotation event into a linear contraction event. And for that, we first developed the simple principle entanglement that you can have with a motor and 240 motion, which is a figure of eight polymer entanglement, which is obtained here from the derivation over seven years, not always too long polymer chains which are clicked here, in this figure of eight in pseudo diluted condition, you can image this kind of figure of eight by five and when you like it, what you choose to observe is the twisting of this full immersion and a sort of a collapse of the chain on themselves, which can be seen again by FM but more interestingly, again, in the reciprocal space by Sachs, where you can see that these kind of burn which is characteristic of the figure of eight increases in intensity by lighting,

which means increasing the density of polymer chains, and also with the maximum of the bump, indicating a decrease in the radius of gyration of this Metropolis conjugate. So we were also interested to use this kind of simple systems to possibly achieve Meccano transduction in living cells. And this is the work which has been done recently in collaboration with unshadowed combo in Germany, and Andreas gas here at Georgia Tech. So you probably know that mechanotransduction is a very important event in cells which can drive many cellular function but which can also be controlled in a number of battery geniuses. And the idea was here very simple and was to attach a motto by two arms two legs let's say at the surface and with the two arms bind cellular receptors such as integrants in fibroblasts for instance, through RGD ligand. So, this is the design of the construct you see here and molecular motor which can be attached at this your face and linked to these ligands and then incubated with cells and also a control experiment, which will involve your molecular motor, where the rotation is locked by the presence of this AP certified. So, first we can see here that by having a different concentration of motor the surface we can change the number of fibroblasts that bind to the surface and then, by irrigating zones of different zones of the surface with UV light, you can measure statistically that there is a clear effect of deamination of the metal with an increase of the addition of these fibroblasts at the surface, which is attributed to the pooling of the chains provided by the rotation of the model, you can see also here that the elimination itself is not responsible for this change of the cellular response as the control motor does not have any effect and also if you any bit dynamics of the magazine financing to the cells with this inhibitor, you also avoid the MC mechanotransduction in the cell. So, this was extended afterwards in T cells and human cells with a different construct here to bind a different react and you can clearly see here that when the Meccano transduction effect takes place, you have a release of calcium and then discussion gets imaged by fluorescent probe here, which can be verified in the presence of the lotto, but not in the presence of the rock model. So, this is an example of this transformation of the rotation into linear motion at the molecular scale, but can we again cross landscapes by designing chemical jams and networks of motorized unit which will play the role here of acting active reticulating a note in these gems. So, just by clicking the same derivative here in a concentrated system, we can then access microscopic gems that can then in the UV light either contract are bent as you see here, depending on the device you you set up and this contraction is the result of the rotation of each motors, which can braid then pairs of polymer chains and lead to a contraction of the material with the exclusion of the solvent. So, this is a very original let's say, process of contraction compared to other kinds of activation using body mass. So, we were able also to extract some quantitative data from this rotation in particular, we determined that the energy which is stored in the gel in entanglements is about equal to one kilojoules per mole.

Turn you see the frequency of the motto depends on the length of the polymer chains in the gel. We also determined that to have a maximum of efficiency in this kind of gel, which means a maximum contraction of the material, we have to crossing the gel at a particular concentration which is important for for finishing, which is the stock concentration which is the overlap concentration of the polymer chain before crosslinking. And we also evident that the variation of volume is equal to the variation equal varies linearly with the variation of the shear modulus in this matters and in particular, the free energy can be written as a function of the elastic energy chain here times the Chain number density which means the number of crossings that you create in the jail as you twist the polymer chains out to make this reversible because obviously, the motto is uni directional. So, when you have contracted your gel, you cannot come back. And this was the purpose of another study, which consists now in combining rota reverter with nada unit, which will be able to untwist the braids, let's say, a created by the lotto by also turning that at a wavelength which is orthogonal to the wavelength of the lotto. And in particular, we worked with directly Dean here that you see that can be locked in this planner conjugated form, by using UV so it means that when the most of times, this planner system can sustain the twists made in the polymer chains into the chemical network, but when you use white light, you can break these bonds and three leaves here to freely rotating sigma ground that can twist the brace that have been created by the motor under UV light. So now in principle, you have a reversible system. But I like to insist that the reversibility is not real reversibility to reset of the system push me to reset the system to this extended form. But the pathway that you use to winder for image change is not the same that the password that you use to unwind them. So you have a flow here of the different rotations. For the contraction and extension of the sister in particular, it means that they can work at the same time. So here are some results for different quantities of modulator. So the process is still very slow, but still it is reversible. And what it is interesting is that then you can work with two wavelengths at the same time, you can use a constant UV radiation, for instance, to constantly actuate the rotation of the Moto and the twisting of the polymer chain. And then at the same time, a very, for instance, intensity of the visible light to open more or less the population of the religion in order to release more or less the twists that are created by the motor. So if you look at the total system at the macroscopic, you can reach a steady state here, for example, for a given contraction of the matter, yeah, which is the result of a ratio of frequency at which the motors are turning, let's say clockwise, and which demodulator are turning anti clockwise. So you maintain your system here fully out of thermo dynamic equilibrium.

So what we have been interested in for a couple of years now for almost 10 years, is as I shown you the possibility to integrate either molecular switches into macroscopic switch is also molecular motors into either macroscopic sips, which is when the system is not reversible, but also a macroscopic motors when the systems can really free work out of thermodynamic equilibrium. So there is still a lot to do in this pathway. But I think at the moment, it's quite original if we compare to the set of work, because there are many, many works that are dedicated to the integration in particular, of molecular switches into macroscopic models, even not using molecular machines. And a lot has been done there in the past. But I think still a lot of new things have to be done by using more economical tools. And this brings me to the open questions possible applications that could be thought about during the next five years in particular. So we know now that IRT can make an exercise defined it before it's possible by using artificial animation, but still if we want to compete with other type of actuators in particular, also, polymer based actuators, we really need now to control the orientation of the actuation because this will help us to have on your workloads. And and for that we really need to tune the anisotropy of the change the bundles and networks, not only by chemical approaches in the bottom up approach I would say but also by using chemical engineering and top down approach is also a physical engineering, I'm thinking about many techniques now that can help us along this way. We also need to control the speed of actuation. At the moment, the systems are very slow. And this is not something easy to solve. In particular, if you want to work in materials without solvents, because we need some mobility of the chains to actuate our systems, we need maybe at some point or so to control the production of waste either to work with simulators that do not produce waste, obviously, for instance, the case of light, but there are also other kinds of machines that requires other kind of activation. And in case we produce waste, we have to release them. And this is also I think, a question when we want to investigate these objects into useful materials. Have you see that fatigue is a classical question for the design of materials, as well as the cost of these materials. chemistry that is developed here is extremely complex, and extremely expensive. So it's not possible to go

people from an industrial company and say, Look, I have this actuator, it's impossible to sell these on the market. And so these questions will come sooner or later. And then I think, as in the last example, I presented from a more fundamental point of view, the cooperativity between ratcheting at nanoscale and other kinds of ratchets, macroscale, there combinations are possible cooperativity interesting pathways to investigate five to 15 years, I think we have to answer the question if molecular motors when going to macroscale are useful. I'm not sure about the answer. Maybe we can do more things with simpler switching systems, maybe ncst polymers are very efficient to do many things. So Amato's really useful for that. And then comes again, the the classical question with molecule automation Can we do something is mottos in materials that cannot be made with any other actuator? I think one possibility is also and this would also answer the first question is to integrate models based on the consumption of chemical fuels, in particular ATP, and maybe to design from the interesting by your mother, so the ratcheting mechanism may be different. But still, there is no evident reason for me that we will not be able to integrate motors that use an ATP to produce I landscape actuation. And then the question we saw in the previous talk that now motors can go even in vivo? I don't really care ask Yes, when these biomaterials will go in vivo at one point making use of ATP to do something as implants or whatever. Can we make use of metallic materials also for another possible application, which constantly storing and releasing energy, when I spoke about this twisting of polymers, the actuation, and the work produced by the Moto is stored in elastic energy into materials, how can we make use of this elastic energy? I think we can achieve spring effects, which are very interesting in that case, and which can be then released very fast. And then this would be very something. There's something very interesting as well. Can we integrate molecular machines in complex systems? I'm speaking networks, sub networks compartmentalization, etc. And can we program them for sequential tasks? 30 years. It's a bit far away from me. But I just thought about when was awarded the Nobel Prize for Super molecular chemistry, it was 1987. We see only the applications, I think I'm speaking about industry applications. Nowadays. I don't think we would have predicted at that time that these applications will go in materials, such as self healing materials, rich, recyclable materials, which are materials of ice associate an impact nowadays. So I hope that the future of material and molecular machines will have such a high social impact in 2050. And this kind of striking application. I don't know yet what it could be. But I know that I know, I believe that it will require multidisciplinary approaches. We have now as machinists, molecular machines to approach other fields. We saw examples with biologies before. We have examples here with people working in material science. And multidisciplinary approaches will be important interactions with industry. It's not probably too early to discuss with with people there because we have to know about their requirements. And we have to discuss with them because I'm sure we can get interesting ideas from

their side. And finally, we also need specific training for the next generation of students who are sitting in our teachings, the domain of molecular machines have to be taken as a wolterman. specialization maybe. And I'd like for that to make a transition with another project that we are dealing with at the moment, which is this article, my project, which started a few months ago, and which involves, you may recognize some great people in the field of molecular machines here, and which involves as well 15 young, talented PhD students with the goal to study different domains interaction actually, with industry and partner, you see that surveys involved. We have also done on companies that are not used and that we are not used, this is the first time for them to be involved with people working on molecular machines. But I think this will stimulate very interesting distribution. And it is the price for new, interesting investigations in the future. So with that, I'd like to thank the people of my group here in red, who have participated in the work as presented today. External collaborations, as I mentioned, which are very important to give an interdisciplinary character to this research and financial support. Thank you very much for your attention.

Excellent, that was a fantastic talk Nicholas Thank you very much. Um, do you want to address the comments in the chat yourself like my Jameson Oh, God want me to read them out for you whichever is easiest.

To when does it start?

What since 49 6:46pm

six one I don't have the time 614

So, you got the is is the Meccano transduction

746 Okay, yeah. Is the mechanical transmission mechanism output consider the work? No, no, not at all, I don't consider to have the two ligands are still attached to the ligand. So, maybe this question is interesting in the way that obviously, you have a k of kin of K and K of the ligand which binds to the cell receptors, you can have some detachment at some point, but if the frequency of the motor, let's say Rafi is higher than the detachment, a timescale of the ligand, then you have a cleaning effect which can be maintained. So, there isn't no work in the sense of kidneys in or whatever what would you need to make a full artificial muscle out of these not a system I was strong with expect, would you expect it to be but for the the switches, I presented the approach of having myofibrillar structuration. So, this orientation first is a requirement and then the third link is another requirement to gain stiffness and to increase the workload that you can extract from such febreeze then what you need is a control. And this comes the question if you can work there on wet materials in solution cannot but can you still have some solvent to keep some dynamics of the polymer ocean? Can we really dry on wet matters and our dis control should take place this is also dependent of the presence of the solvent or not. If you want to nest a species that diffuses to actuate the machine you need a solvent. If not, you need for instance electron. So at the moment, we are trying to do that by electrochemistry in the neat material without solvent, then the force is if the force is is really I don't have values in my mind, because I'm not used to work at the macroscopic scale, but this was evaluated by roboticists and people like that. And there is no reason that you can extract a really iforce if you sell enough machines of use. And do you estimate the efficiency of the motion at micron or larger scales work out? Yes, one motor. So there is different values that you can give. It's about 12 kt motto itself. So this is related to the difference of energy between the unstable ethics and the stable Unix, this is more or less the talk of the system. And this is corresponds to about 10. pecan, you don't pay em nanometre. That's the polymer chain, keep it compressed shape after UV light, they switched off the termite. So it keeps it keeps the it keeps the shape, it gives the shape. We have now systems that if we pull on them, which relaxes to some extent, but but it keeps the shape. Here again, the directionality is given by the difference of energy between the unstable index and the stable. And what you do when you twist the pulley machine is that you equalize these two energies, and then you stop, but there is no reason that you come back at that point. Until you disrupted this balance of energy between the unstable and stable is, so you really have to pull back on the system, you want to do that. That's the wedding, but have you see when you have the modulator then you release the system. And this modulator is here rotating unit, but you can think about other ways to release the twist, you can have binding and binding effects etc.

What is the molecule mechanism, unlocking reversible systems. So at one point, you have a you you you have a long which is locked, which avoids the rotation of the the twist of the polymer chain. And you you transform these bonds to freely rotating sigma bonds. And you are able by this rotation to untwist the system. I need to explain that better with a skin but I don't want to be too long. And then I have Thank you. So I have nothing to answer, except to say thank you to you as well. If you have a question, I can take it, obviously. But I see that I'm late already. So I don't want to take your gathering time.

Sir. I mean, we started about five minutes late after all the intro. So you basically I think we're just we'll say we're just about on time and give ourselves a bit of generosity there. So Alison, join me to quickly share the PowerPoint slides I've got for introducing the next couple of

things. We still have to add those before. So I think maybe we'll, we'll leave it at that. Yeah, I can share in the chat. Two upcoming meetings in this group that we have. One of the one is and one that I mentioned already to you at the beginning of this meeting, which is an optional, just for fun brains on the Saturday which you should have gotten a Google Calendar invitation for in your inbox. And we just share the molecular machines bounty brainstorm form for that's the Saturday for those who are interested. And next Wednesday at 11am. We have visa fieriest of discussing the National Nanotechnology Initiative. And I'm not sure if these are here. But if you are, then we're very excited for that. And well, yeah, I think Thank you very, very, very much. And James Nicholas for joining. This was fantastic. Thank you all for your really great questions to the entire group. I see a lot of clapping hands, either via reactions or were actually people clapping their hands. So thank you. And I think that for now, it would be lovely if we wanted to meet for those of you who are interested in it in our in our gather room. And so I will share the coordinates for Just here. This is again, definitely optional. I mean, the whole group is as optional, but this is going to be a fun way for you to socialize with each other to meet each other. Cut the zoom, once you click on the link that I just shared with you in the chat, you should make a portal into our molecular machines lounge, many of you know that lounge already, just sit at a table and start talking to each other. This is from now on a space for you all to get to know each other. And yeah, thank you very much for my angle. James, do you have anything to add?

No, I think today's talks were absolutely fantastic. giving us more to think about and more to discuss afterwards and give us the group adding to the sort of well rounded view of molecular machines. I think we're hoping to get out of this seminar series across the year. So yeah, even though I have nothing to add a stop playing to say apparently so yeah, upsampling gather,

perhaps a nice icebreaker for setting your conversations and gather is I'm starting it with what you thought of the last few slides of the presenters, which is the more forward looking and speculation on what could happen in the next five to 30 years. So that way, maybe a cool way to kick off your discussion. And with that, I'll see you on gather I'm leaving now. I'll be a little bit in the chat in case people have trouble logging on together, but I just shared with you that information. So I'm hoping that I'll see you there in a second. Bye, everyone.