 So, well, yes, Tommy say, my name is Mariana Janesa Teta, and I'm currently working in AstraZeneca as an associate principal scientist in the Advanced Drug Delivery Department in pharmaceutical sciences. And I did my PhD with Tommy, did a lot of neutrons, put him to wash a lot of troughs so I can recommend that you do that with your supervisors, but I did make sure that he got a nice dinner afterwards. Today I'm going to talk about lipid nanoparticles for mRNA delivery. So thank you very much Tommy for inviting me to this presentation, and I want to kind of summarize a bit what I would like you to learn from this presentation. So I want to have a, I want to start with a kind of high overview of what mRNA therapeutics are, especially kind of promising challenges and advances. Then I'm going to talk a bit how to make the lipid nanoparticles for RNA delivery, and then importantly how to characterize and the performance of these LMPs. And then, of course, this is a neutron core, so we're going to talk about how to use neutrons, especially a small-angle neutron scattering for the LMP development. And then I'm going to present two different cases looking at with use, with the use of sans, the effect of protein binding on the LMP structure and also how to use sans to functionalize the lipid nanoparticles or characterize functionalized lipid nanoparticles for subcutaneous administration. And I mean, if you want to interrupt me, I don't know how you do with the other presentations, just please go ahead and just unmute yourself and ask questions is fine for me. So I'm going to start with the mRNA therapeutics, promises, challenges and advances. So I guess that all of you are aware that mRNA therapeutics are a thing. They're very popular at the moment. So the promises is that we can produce proteins in vivo by administrating mRNA. And this is typically the approach that we want to achieve when administrating the protein itself is not viable because the proteins degrade, because the proteins don't reach the target, because the proteins will not have the right function. So then we would prefer to administer the mRNA to produce the proteins in vivo. However, I'm a fiscum. And when I look at mRNA, mRNA is a very long charge negatively charged polyelectrolyte. So that means that it's not very easy to deliver mRNA because it needs to cross an equally negatively charged cell membrane to enter the cytoplasm and actually produce this protein. And you can imagine that that's not an easy task for a negatively charged polyelectrolyte. Additionally, our body is made to destroy foreign mRNA. So then we need to protect from enzymatic degradation before it reach the target cell. And another very big challenge for mRNA therapeutics is actually finding a biocompatible vehicle. And typically when the mRNA enters the cells, so it crosses the cell membrane through different mechanisms dependent on the delivery, enter the cytoplasm, it's read by the ribosomes and this is producing the kind of polypeptide that will become part of the protein. So when people talk about the mRNA vaccines, this is not something that was developing in gear. This is something that has 50, 60 years of development. So first of all, the discovery of mRNA was done in the early 60s. And from that we have come a long way. So from the discovery of mRNA, we have come a long way to understand how can we translate both in vitro? How can we develop delivery systems? The first were liposomes. Maybe some of you are familiar with lipofectamine. It's typically used in a lot of cell acids to transfer or to deliver the mRNAs commercially for in vitro acids, but it's not very friendly for humans. It has been tested both in vitro and typically in animal models. Already in the early 90s, there were developments of an influenza vaccine using liposomes that contain mRNA. It's also commonly used for cancer immunotherapy. There's also other applications such as cardiovascular diseases. Already companies like Moderna were reporting in 2017 clinical trials using mRNA vaccines against the Zika virus. And all of those advances allow us to have the first two mRNA vaccines that received emergency authorization against COVID last year. So I think it's pretty exciting to see this actually become a reality. But then I'm going to talk about the lipid nanoparticles for RNA delivery because we have come a long way with mRNA, but we need to understand how to deliver the mRNA. And when we talk about lipid nanoparticles for mRNA, we typically talk about this specific technology. We talk about a lipid formulation that has mainly a cationic ionizable lipid. In this case, the lipid that I'm going to talk is called D-Lin MC3DMA. It's quite used in literature, but it's also a product for S-I-R-N-A. And these kind of lipids have a head group that is immune-based typically and has what we call an apparent P-K-A. That means that the P-K-A is measured inside the LMP. And that apparent P-K-A is somewhere between 6 and 7. That means that a physiological pH around 7.4, the LMPs or the cationic lipids are typically on charge. But when the pH decreases, because this is typically the mechanism when it enters the cells in the endosomes, the cationic lipid becomes charged. This positive charge will allow it to interact with the endosomal membrane and disrupt the endosomal membrane and deliver the mRNA inside the cell. So this is an important feature because when you have a constantly charged lipid, this typically alerts the body that there is a foreign system. So it's good to have an on-charge system before it enters the cell. But then you need to have this charge to actually be able to deliver the mRNA into the cell. Additionally, we have typically helper lipids. Those helper lipids are typically acetyl or cholesterol, a phospholipid like DSPC. And then we have a polyethylene glycol lipid that helps to stabilize, sterically stabilize the LMP structure. So as I mentioned, the cationic anisolipid helps to encapsulate as well the mRNA because it forms some kind of complex cationic lipid with the negatively charged nucleotides of the mRNA. It protects it until it reaches the target cell and it also helps to release it inside the cell by disrupting the endosomal membrane. LMPs were originally developed for SIRNA. And this already in 2010 there were reports in the literature about LMPs. And there has been a lot of attempts to understand how the LMP structure looks like, but also to be able to understand if we can improve its performance. And this was a representation from molecular dynamics simulations on how we look with an SIRNA. How do we prepare LMPs? There's a different approach to prepare LMPs, but the most commonly reported literature is the use of microfluidics. So what we have is typically the lipids, which are not water soluble, we have them in an ethanol phase. And the mRNA, which is not ethanol soluble, we have in a aqua phase, which is typically a low pH. And we mix this in a microfluidic chamber. And what happens is when we have the mixture, in this case, this is a specific system called a nano sampler, which is from a company called precision nano systems. They have what they call inside a mixed herringbone structure. That helps to create this kind of flow or caotic flow to help preparing the samples and also to help to make them a bit smaller and consistently the same size. So that's why we like to prepare the LMPs in the microfluidic mixer because we typically get very reproducible results with a very high encapsulation, more than 90%. After we get the LMPs, since I said originally we have them in ethanol lipids and in acidic buffer, the mRNA, we typically dialyze them against a physiological pH buffer. And if we need them, we concentrate them. And then we do some particle characterization. That's the most standard size and encapsulation. And this is a little like spreadsheet or how we look when you're calculating your amounts for your preparation of the LMPs. So we actually make, typically mix them in a one to three ratio. That means that for each part of the ethanol volume, we have three parts of the mRNA volume. We, in this case, we have a lipid concentration of maximum typical 12.5 million molar. And the reason is because when you try to do more and more concentrated, you have a more risk that this will get stuck inside the microfluidic chip. And you don't want to make it too concentrated either. And another important thing is the N to P ratio, the N to P ratio N starts from stands for the N or cut or amine groups in the cationic lipid and P stars stands from the phosphorus of the nucleotide. So it's basically a positive to negative charge ratio. But in this case, the N is not the negative, it's the positive. So when you say three means that you have three cationic lipids per nucleotide. This is very important for the particle formation. So if you start going to lowers and to P ratio to one to one, then this can typically precipitate and not form stable particles. You typically want to have them slightly overcharged when you made them. So more cationic lipid than nucleotides in a solution. Another important is to have the composition of your LMPs. This is the most standard composition that we have is 50% of the cationic lipid around 10% of the phospholipid 38.5 of mole percent of the cholesterol and 1.5% of the peak. And this is basically, I mean, I actually have the extension that I can share with you later, but this is what we use to calculate our preparation. So we put input our volume. Another important is to have that if you have the charge density of your mRNA or DNA or, or whatever you're using to input it here. So you can have the right calculations on the weight and the solution. So as I mentioned, we use a system in our lab, which is called a nano sampler. And the nano sampler is typically what we call a bench top. So it's not necessarily what it will be called what we would use in a large scale to produce the vaccine, but it's a representative of what we can do in our lab to make an excellent piece around one millilitre to 50 millilitre. And then we typically take them out with allies in PDS into some dialysis cassettes and we can concentrate them using amicone ultra centrifugation filters. Sorry, Maria, if I may ask a question here. Yes. Yeah, so I was wondering about like what are usually the sizes of this lipid nanoparticles. So I will come into that but for this preparation that I show you is somewhere around, I will say, if you measure by DLS the intensity average distribution will be somewhere between 70 to 80 nanometers. Okay. And I was also wondering about the structure. So do you have this bilia structure that encapsulates your mRNA or it's just a sort of a micellar structure which because in the chip that you showed, you had this ethanol where lipids are dissolved and you have mRNA and then they mix together and form the nanoparticles I was wondering like looking at the cartoon. What kind of structure it is. I can say that it's not a micellar in my definition of micellar and it's not a liposome either. So it's somewhere in between so it's not it's not just a simple bilayer that is filled with water where the mRNA is it's a more complex system. Okay, so but I will show you karate and because it's always nice when you can actually see them. And of course neutrons will help us to know much better how they look. So I will go. That was a very good question because now we'll go to the characterization and performance of the LMPs. So, when we talk about physical chemical characterization of the LMPs what do we specifically we talk. So I kind of we divide them here in waves and the ways depends on the complexity because we were not going to do the same characterization for all of the preparations that we do I mean we make LMPs fairly often so we cannot do the most complex techniques for all the preparations. So typically when we have our preparation, the first thing that we measure is the size and for that we use dynamic line scattering. A simple kind of instrument in the lab a modern system. And then we measure the encapsulation with a fluorescence acid called ribograin. Ribograin is a dye that binds to mRNA. So we typically measure what happens if we add the dye and then we kind of solubilize the LMP with surfactant such as Triton and measure again what happens with the concentration of mRNA to see how much is free, how much is total and then we measure the encapsulation efficiency. These are what I call the wave one characterization. Wave two is to look at more complex characteristics of the LMPs. This is an acid called TNS. TNS is also a dye that allows us to measure what we call the apparent PK of the LMPs. So typically when people reports in the literature the PK of the cathodontic lipids they always report them as I mean cathodontic lipids are not soluble in water they always report them in some kind of lipid nanoparticles so it will always be influenced by the composition of your LMP. So to measure the CETA potential that can be important but measuring CETA potential of the LMP is challenging. You have to, especially sometimes when if we are used to working in systems like the Malvern CETA Sizer is very easy to get a result but we need to understand if that result is actually physically meaning. So to measure the CETA potential of the LMPs we have to try to have a low ionic strain and low concentration so it takes a bit of tweaking to actually get good measurements of the CETA potential of the LMPs. So we don't do them as a standard control. We could also look at NMR, NMR can give also in information about charge you can also give information about the structure is a bit complex to analyze but it's possible we also have a lot of NMR instruments here in AstraZeneca. Then what I call the way through is the instruments that we actually don't have in-house and we typically have to go to the university or we have to go to large scale facilities. One of them is the cryo TEM. So this is a small picture of the cryo TEM how the LMPs look and I will have more pictures afterwards. So as I mentioned that most of the particles are here and this in this kind of small circle and they are not like but some likes they are actually electron dense. So they are, you can see that they are all the way through there like dark gray. I will have a better picture afterwards. You can also do both small angle x-ray scattering and then a neutral scattering as well to look at both the structure and the LMP distribution. And I will come more to a specific sample of how they look and what does the structure means. So this is a better picture of how the LMPs look by cryo TEM and typically the standard approach when we started to work this. So I did my postdoc here in AstraZeneca with Lipid nanoparticles and when we started looking at the Lipid nanoparticles we took the example as I said from the SI RNA. What they reported in the literature is that they will have 50% of the cationic lipid, 10% of the DSPC and then they will add more peg if they wanted to reduce the LMP size and they compensate this with the cholesterol. So that means that you typically have as a base 40% cholesterol and then if you add 1% peg lipid and you will add only 39% cholesterol. And as you can see as the peg lipid percentage decreases the particle size increases. You can also see that sometimes when they're a bit bigger you can find some liposomes but in the majority of the population are basically this kind of dark spheres that are all dancing the core. And then when they're small you can also start seeing maybe a smaller that are a bit more regular. We can also compare the size from the DLS number distribution with the size from the cryo TEM distribution and find a very good correlation. These are three typical preparations so that a lot of the sizes are going to come back around the presentation so we have some around 40 this is number size number size distribution 60 and 90 so the number size is typically a slightly smaller than the intensity average distribution. So when we started to look at this this LMP so we started looking at four different sizes between 40 nanometers and 140 nanometers and then we started doing in the process and we picked two clinically relevant cells. One is human adipocytes. So adipocytes are fat cells that are just under our skin. So when we have administration subcontinuous administration those are the first cells that typically the LMPs are going to encounter. And then another one so we took our induced pluripotent stem cell derived hepatocytes, which are liver cells and are also very relevant because we have an IV administration of LMPs this LMPs typically will end in the liver. We label in this case the LMPs with DSPC that had 3-tune in it to be able to track how much was inside the cells. And what we found was that the uptake was more or less the same independently of the size. We didn't really see a trend on the pen if it was smaller or bigger maybe for the adipocytes the 16 nanometers was a bit more but definitely for the hepatocytes there was not a big difference. But the interesting was not the uptake. The interesting was actually what happens when they're inside the cell. And what we can see is actually we measure here a protein called adipoprotein and this protein so when we administer the mRNA this protein will be produced inside the cells and it's actually a secreted protein so we can measure it from the supernatant of the in vitro experiments. And what we can see is that the 16 nanometer LMPs had a significant higher protein expression in both cell types compared to either smaller or larger LMPs. And we couldn't understand why are these LMPs better than the other ones I mean what makes it 16 nanometer because the uptake they all look to be up to taking up more or less the same amount. And then I came that was when I was fresh back from my PhD and I said oh why don't we do a neutron experiment and I drafted a neutron proposal as I was very well coached by Richard. I think I don't know if you had had that exercise already or you will have to write a beam time proposal but I think it's a really good thing I learned from from my PhD supervisors. And I mean I guess that I mean I could probably skip this slide because you had a much better introductions to small angle scattering at the moment but what we were interested was if we could understand a bit more about the structure of the LMPs. And if these could guide us to why 16 nanometer LMPs were better than smaller or bigger. And especially I was interested in not only use a small angle x-ray scattering but to use a neutron scattering because as you have probably seen already in the course is good to be the last day. We could do selected iteration of either the leaping components or the solvent to produce different scattering profiles. And we have significant we have many components we have the cathodic lipid we have the phospholipid. We have the cholesterol and we have the peg lipid plus the mRNA. So we needed to figure out actually what can we do trade to be able to highlight so if we have for example the DSPC hydrogenous DSPC the tails are have very close matching point to the to just pure water. And the head groups are very close to do trade the cholesterol but if we have do trade the DSPC then the scattering and it is much higher. So we could really highlight the DSPC. RNA also has a scattering and density that will change depending on the content of the to because it has labelled hydrogen second being exchange. So typically the match point is around four. So so we kind of match the first time we did was okay these are all components we calculated their scattering and density we see which molecules are available to do trade and we can start playing on how to design this LMP to be able to highlight certain parts both from the deterioration of the lipid process deterioration of the soul. But you can imagine that the first thing that we did was not sense because we needed to have data before so we actually did some sex. And interesting what we can see is that when we have a mRNA inside the LMPs we can see one peak at approximately one inverse nanometer that corresponds to a correlation distance of around six nanometers. But this is only in the presence of a morning we couldn't see it in the absence of a morning. And we could see the same peak independently of the size in the same position it was a different intensity or different order but it was exactly the same peak. But I mean, I guess that you have no background in scattering so you will know that we won't pick difficult to fit a thing so it could be anything it could be in the literature has been reported this is potential and inverted micellar phase. So this could be maybe a correlation distance between these micelles. Some people says that maybe this looks like an onion so a multilambular vesicle. Could it be the mRNA some kind of work like micelles and these are kind of correlation distance between this one line micelles. So then we went and why do we care about the structure of the LMPs I mean why was it important I mean what is reported in the literature is the LMP transfection and efficacy is very low is typically single digit in less than 10%. That means that if you administer a vaccine that has I don't know 100 micrograms of mRNA perhaps less than 10 micrograms are the ones that are making their, the job. So it's very small amount and a lot of it is just being recycled. And, and we want to understand if we can modify I mean the structure of the LMP to facilitate this in the summer escape and this is not something that is new. I mean a lot of companies a company that is facing losing Camus has been doing this for many, many years, trying to understand how the kind of liquid crystalline structure can facilitate both encapsulation and the delivery of small molecules and larger molecules. And so, yeah, so I, as I said, I drafted my nice sense proposal I got some beam time in in gargling in Munich, and we went there and I didn't know what to expect I just designed a few kind of particles one was deteriorated DSPC and partially due to the cholesterol that it can be fine found in Avanti so commercially. And we look at different kind of D2 OH2 oration and we found actually was something very interesting. We found that one of the lipids that DSPC was mainly segregated at the surface so we were actually able to fit five different profiles to a core shell model, where basically we can summarize it as we had an LMP core and then monolayer which was mainly enriched by the DSPC. We had on top a peg layer with solvent in and the peg was in a mushroom conformation. And this was very exciting because what it was in the literature was not this, when we look at the first SRNA simulations we, it was believed that all of the lipids besides the peg that it was still believed that be in the surface where distributed homogeneously across the LMP. So these were really the first ones to show that the lipids were partially segregated and the LMP surface, you cannot see that from cryo TM, you cannot see that from SACS. SACS was the only technique that allows to actually see where the lipids were located. With the first experiments that we did we had very limited information about the distribution because as I said we only have a few components that we could we had deteriorated so the DSPC is commercially available, and the partially other components cholesterol is you can find a higher level but it has to be in a deterioration facility and the cationic lipid was definitely not commercially available. We could still get an estimation that we have around 20% water, 24% water inside the core. So it was mainly lipids. But finally we actually, we had continued this work and now we're collaborating with the colleagues in Malmo University with Marite Cardenas which is also a former boss of Tommy and Federica Sevastiani who is also a former boss of Richard. And then we could actually see what was the lipid distribution by not only having deteriorated DSPC but our colleagues here in AstraZeneca could synthesize deteriorated cationic lipid in this case MC3 and our colleagues in different deterioration facilities. I think this was provided by ANSTO, the Australian source, could provide what they call match out cholesterol so it has a much higher scat and the density, and we could actually pinpoint that all the SBCs at the surface, the cholesterol is distributed both the surface and the core but it's mainly at the surface of the LMP in the same layer as the DSPC. The MC3 is both at the surface and at the core but it's actually mainly at the core of the LMP so it's depleted from the surface. The peg lipid we already knew that was at the surface and the mRNA is at the core. So we can actually put a number of how much there is at the surface and at the core of each of the components. Yeah, in the last graph like you were talking about like, so I just missed like how did you do this analysis, like how do you differentiate between like how much, like quantify how much of this is in this part and in the core or in the shell. Yeah, so I don't have the data, I didn't put it in this presentation I should have. But we did a lot of mixtures so we started to prepare samples where we have only deuterated cholesterol, match out cholesterol, only deuterated cationic lipid, we started to mix, we have two of the cationic, you know, cationic lipid and the cholesterol. So we did prepare many different particles and start looking at the scattering at many different H2O, D2O buffer ratios and try to find what was the matching point. So we got equivalent to what we did in the first experiment but we got much more data that we could feed consistently to actually put numbers to the volume fraction of each component at the surface and at the core. So it was similar to this experiment actually I didn't put the data but it should be in a publication that came just early this year out of the preparation so it was quite a lot of bintimes to collect all that data. Okay, thank you very much. I will also, isn't this article you mentioned down in the corner. Yeah, this one here. So this one has all the preparations. I mean, I said about Federica and I will, Federica did experiments and plan a bit the mixtures, I prepared the LMP so we did around, I don't know, maybe six, eight preparations to get all of this composition. So it was very many different contrast that we tried. Okay, thank you very much. So, and so now that we knew actually how the lipids were distributed both in the core and at this at the surface of the LMPs we really wanted to understand a bit more about the core so as I said we saw from sax one peak but that one peak tells you nothing. So what we did we was, we tried to do what we call a bulk phase and what is a bulk phase. So we take, now we know that it's mainly cationic lipid and cholesterol in the core not the SPC and opaque. And we mixed this with MNA in water but this will not form particles. This will fall more of a continuous phase and it's not water soluble. But we tried to understand if we can take this phase and analyze it and have a further information on how it is just the core, not the surface. First we put the cationic lipid, cholesterol and ethanol mixed with mRNA water in a dialysis cassette. We put it in a similar conditions as LMPs will typically form which is a acidic buffer in this case citrate and ethanol in a 3 to 1 ratio similar to what we do in this nano assembly instrument. Then after we have dialyzed this for like a day we take this out and we put it against PBS because we also want to know how it looks in a physiological pH because this is how the LMPs are typically stored. And this was also really interesting so I'm going to start from the pattern look of the sex data of the core phase and the acidic condition so the citrate, ethanol, 3 to 1 phase. So this is the data. First I want to point out two peaks which are pretty large here and those peaks are typically cholesterol monohydrates and we know that these phases actually have a bit of excess of cholesterol. This was done before we did the work with Federica. So we added a similar kind of cationic lipid cholesterol ratio it will be in the LMPs but the cholesterol has a limited solubility in the cationic lipid. That might be one of the reasons why it's segregated towards the surface. But if we kind of don't look at the cholesterol crystal we know that they're there and we know that they're in excess of cholesterol what we can see is that interesting. What we can see is a main peak around the one inverse nanometer so similar to the LMPs, but we can see additional peaks. And this additional peak so if we call this the q zero we have a q one and q two they come at a square root of three and a square root of four multiples of the original peak. So this is typically an indication of a reverse hexagonal phase. And then what we believe is that at low pH the mRNA is organized in some kind of water cylinder so mRNA is a very long molecule. So these water cylinders are have an hexagonal packing and this inverse nano one inverse nanometer six nanometers is the center to center distance between this water channels in hexagonal packing. And we also did some freeze fracture micro grabs of the face and we could also see this what we can believe are the water channels and we could measure the distance between the water channels to be approximately six nanometers as well. It was interesting what happened when we actually look at the bulk phase in the pbs storage buff. It's similar to what we have measured with the LMP itself. So we can see again is that when we have no money. We stop seeing that peak that we don't have any more that that that we have a bump but we don't have any more that very highly order peak, neither the further peaks we can only see the cholesterol kind of crystal. But when we have in this case we don't use a money and it's just because I'm an extremely expensive so we use a model molecules of poly A. And we can see from the poly A is that we can still see that the main peak that we saw before at one inverse nanometer although we cannot see anymore. With the same intensity that the Q one and Q two is there got so so we believe that is this is a less order phase but somehow related to the first hexagonal phase that we saw. When we look at the first fracture of the kind of upper face so the LMP similar with the poly A in pbs. We don't see any more this nice water channels we see more of a kind of mix structure. So what we believe is we originally I think call them in the in the publication at this sort of external phase, but that sounds very on. Oh, doesn't sound very straightforward, I mean it's an external phase but it's disorder what does that really mean. So that we started calling afterwards a worm like my son so it still is this water channels where the poly A or M RNA is located. There's not any more structure in the typical hexagonal packing but there's still some kind of correlation distance because it's a highly packed system of approximately six nanometers. And interesting, if we look at the empty LMPs as we saw before and the LMPs with the M RNA, we can see quite a lot of similarities between the bulk phase and the LMP or disperse phase. So I think we're still trying to understand and model this, this, this peak. So my postdoc advisor here in AstraZeneca Lena Linford he has been working quite hard into trying to have a proper model to quantify this structure to get more information perhaps about solving is not straightforward is something that is still ongoing and if you have any ideas I'm pretty sure that Lena will be very happy to hear them. But then I said at the beginning that I was going to say I was going to talk about how the LMP structure could help us to improve actually the development because we're actually care about the performance is very nice to understand the structure but if we want this to be a product if we want this to actually help patients we need to be able to develop better LMPs. So what we found from our kind of neutron scattering experiments was that the original formulation approach that I mentioned where we will add more pectin reduce the amount of cholesterol is this one in in blue, actually was making not only the size change but also the surface of the LMP change. So when we were having larger and larger and larger LMPs were having more and more DSPC and this at the surface compared to the other components. That means that the surface area per DSPC was decreasing. That means that the almost 140 nanometer LMPs were had a similar surface area of DSPC in a gel phase bilayer. So it feels that it was mainly just DSPC and not none of the other components. And then we started to tweak. So we wanted to have different size of LMPs, but that will always have the same surface as of our best performing LMPs, which was this around 60 nanometer LMPs. So then we said, okay, we changed the, we keep the cationic lipid cholesterol ratio constant and we change the other components we change the size and we keep the same surface as as the best LMP. And if you remember the, the kind of protein expression experiments that we did with the adipocytes the fat cells. So the original formulation had the best performance around the 62 nanometer LMPs. But when we changed the size and the surface of the LMPs then we started to see that actually protein production was increasing when the LMP size was increasing. We also had bigger LMPs had more copies of mRNA in them, but originally when the surface was just DSPC. There was not enough cationally perhaps to disrupt the endosomal membrane and help this to be released into the cell cytoplasm, but it had to be actually improve the surface to be able to deliver the package, let's say. So there we see it's not only size that matters for LMPs but also the surface of the LMPs that matters. Now I'm going to talk about two cases. So if you have any questions I can take now about just the LMP structure otherwise I just talk about two examples, and I'll leave some time as well for questions at the end. I have a question about the LMP structure. Yeah. Sorry my camera is not working. That's why I'm not switching on the camera. Yeah, so I was wondering about like have you looked into the structure of the CIL phase means lipid that you are like, you know, looking into your samples like you're using in your preparation. If I remember correctly you're using DSPC, and then you are using cholesterol, peg, and this cationic lipid, right? Yeah. Yeah, so in the cationic lipid have you looked into the structure of the cationic lipid as a function of like, you know, water content, how does it behave? So this is, I mean, this is a lipid so it's fairly water insoluble but we can look at the phase behavior in water. So it's typically cationic lipids for LMPs are designed more what we call a conical shape. So they have a very small head group compared to the tails. And the reason is because it's believed that this will help to disrupt the endosomal membrane in a more efficient way. So when we look at the cationic lipid at different pH, just like a bulk phase similar to what we did with the cholesterol, you can mainly see depending on the cationic but it's typically also an hexagonal phase. Because this kind of, let's call them conical shape lipids typically form hexagonal disperse liquid crystalline phases. So it would also be of course dependent on if it's charged or not so it will depend on the pH. Great also. But in your model that you have like, you know, obtained from the sax and sands experiment, sands basically, where the cationic lipids is inside the core that is inside the DSPC. So here I was wondering like how does it see the pH in this case like the change in pH because it's shielded by the peculiar and the DSPC. This is not, this is a bulk phase. So there is no, in this case, there is no DSPC or a PEC. So you take just the lipids that are in the core and you make, it's almost like a precipitate, the wide precipitate that you take. It's not an lipid and a particle anymore. So we're just trying to understand how the liquid crystalline, what's the kind of phase behavior of this mixture without the particle. So without the DSPC, without the PEC. Okay, I see that. Yeah. But it does, but it does, I mean, if you change the pH, like with the LMPs, it will still, I mean, the lipids are distributed across and there will be equilibrium of, you know, proteins in and out of the LMP. So if you do reduce the pH, it will induce a charge change inside the LMPs. So there is changes depending on the pH, on the structure. Okay. Thanks. And I mean, with that I will let maybe the future that you see that you follow Frederica and because we actually have some sense data of how the LMP looks a lot a low pH and it's completely different on how the LMP looks at high pH. But it's just still fitting the date. So I'm going to talk about the effect of the some, if you have any more questions, I can also take them now before I move to the next sample. I can also take them at the end. So the first is was the project actually that Frederica and I and Maritaz started to work together and my also my colleague Leonard effect of the protein binding on the LMP structure. And why do we care. The interesting is when once you administer and other medicines, especially through kind of intravenous administration and this is common for all kind of medicines that you administer intravenous or that is in the plasma, they will form what they call a protein corona. And I just believe that one of the main proteins in the corona of LMPs is a protein called apolipoprotein E, which is typically responsible for fat transport in our body, which makes sense because I'm leaving out a particle sleep it is typically fat. In the hepatocytes so this liver cells there are a lot of apo E receptors so that's why a lot of these particles and in the liver and hepatocytes. And since the LMPs are inside the they're taking in the in the hepatocytes via mechanism called endocytosis. Then the typically the endosome inside the pH decreases the lip cationally become charged and it's believed that this is what helps the disruption of the endosomal membrane and release of the cargo. So we wanted to understand what happens to the actually structure of the LMP when apo E is in contact with it, since we know that apo E is one of the most important structures. I don't have all the data, and maybe it's because I want you to go and click to the article, or maybe I can send you the article as well if you want. But we also did a lot of work to try to understand what happens when apo E binds to the LMP. And what we see is actually that the apo E, the apo E leads to a change in the structure of the LMP. It has been found in different studies that seems apo E selectively binds certain lipids. So it might be that the LMP can the apo E can selectively bind some of the apo E and potentially remove it. But what we can see definitely is following the binding of the apo E to the surface of the LMP, we can see that there's an enrichment of cholesterol in the surface. So this is the volume fraction in the core of the component of the volume fractions on the shell before and after apo E binding. And what we can see is that there is an increase of the cholesterol at the surface and there is a decrease of the cationic lipid at the surface and potentially an increase at the core. So there is there is a redistribution and why is this important. So as I said, we are trying to get as much of the cationic lipid to be at the surface because this will help disrupt the endosomal membrane. So if we are designing LMPs that have a certain composition then we add the product or we administer the proteins will bind to it and they will completely change the structure of the LMPs. This will affect how the LMPs perform. So we cannot, we don't need to, we need to understand not only the structure of the LMPs in our formulation before we administer, but we need to understand what happens when they're inside the body. Because we might have the perfect LMP in theory, but this might be a very different how it is inside the body. Then I'm going to end with the last example which is about the functionalization of lipid nanoparticles for subcontainers administration. So why subcontainers administration important for LMPs is because it's the most easy for patients to do in house. LMPV is more complex, I mean intramuscular is fairly, but patients for example that have diabetes or chronic diseases are very used to using auto-injectors. So subcontainers administration is a fairly convenient way for patients to self-administer. However to do therapies with mRNA for chronic administration, we know and I mean you might hear that LMPs can cause some kind of inflammation or side effects. And it's believed that this inflammatory response will be actually detrimental to be able to administer LMPs more frequently. So the idea was that if you could include an anti-inflammatory compound, typically an asteroid prodrug, this could reduce the inflammatory response. But to be able to actually reduce the inflammatory response, this prodrug had to be at the surface of the LMP to be able to allow to enzymatic cleavage before it can enter the cell. So a lot of the molecules were designed and this was also done with some of my colleagues here, Alexander who was also a boss of Tommy before. So we decided an anti-inflammatory compound that we could, that it was deuterated and we also look at how it looks using a small angle neutron scattering. So what we could find is actually find that the anti-inflammatory compound was luckily mainly on the shell. And that helped us actually to design an LMP that had anti-inflammatory properties that we could administer more than once and keep the same mRNA protein without eliciting an immune response in the body. So this was really exciting. It was also published very recently. So if you want to learn a bit more about the study details there's also this presentation here. You can also send papers if you're interested afterwards. I'm going to summarize and I'm going to leave some time I guess for questions. So I hope I convinced you and I hope maybe you didn't need to be convinced because you have seen in the news that the LMPs are the leading delivery vehicles for RNA therapy. To understand how to improve LMPs we need to understand that their transfection efficacy is not only size but surface composition dependent and this will also translate to other nanomedicines. I hope this is the last day of the science course that I don't need to convince you that science is a powerful tool for characterization and development of mRNA containing lipid nanoparticles. Also that binding of proteins such as APOE can cause changes on the lipid distribution and that's what we also need to understand not only how the LMPs look in the formulation but also how they look in contact with Plasman. And that we can actually design more complex LMPs and for example in this case we were adding an anti-inflammatory compound to reduce immunogenicity but it could also be a targeting ligand. And that can also help us to improve the LMP performance if we want to go to a specific part of the cell or if we want to get a different response in the body. And we can also use science to help us characterize these LMPs. And with that I think I am in okay time Tommy so if there is questions. Very good timing Mariana so please ask questions. Mariana I have one more question. Maybe you have mentioned this I missed it in your presentation but one thing is intriguing to me like when we are using this lipid nanoparticles as drug delivery so when you are administrating it like under the skin or it's directly into the blood stream. And how does it reach the target sort of what do you think how does it reach the target? I guess it depends on what's the kind of indication what are you trying to treat I mean if it is a vaccine which is more of a create an immune response so there is different ways of administrating LMPs typically is IV or intramuscular now for the vaccines. So you have systemic administration and you have also have local administration. Okay, so if local administration is more prefer when you know that to reach a target cell is more complex let's say I think maybe I should go back to the first of the mRNA development. So I mean mRNA although there is the most promising application is vaccines at the moment for mRNA, mRNA therapies have been developed for many different diseases because a lot I mean it's not easy to deliver proteins and diseases that are related for example in the brain in the lung. So for example, I think there is an example here from the 90s where they were looking at mRNA injection into the brain and this is an animal model of course for protein replacement I mean this is something that you would have to do of course locally because to do to cross the brain blood is very very difficult if you have to do this type of therapy if you had to do it in the lung then it might be easier to do inhale LMPs but then that will be a different way of challenge for both the kind of the development of the LMPs to be sure that things are going to work in the respiratory tract and also the LMPs are stable in whatever formulation that you have because it might not be the same type of formulation if you're going to do inhale, if you're going to do subcutaneous, if you're going to do intravenous. Typically I don't think that an oral LMP will be a product because that will be completely destroyed by the gastric acid. Yeah exactly, yeah that's what I was thinking also. Can I ask one more question? So I was wondering like if you induce any foreign particles like for example then already your immune system starts marking it as a foreign particle right and then you have this. I remember it really in terms of like you know when you are having this colloidal particles that are being put like you know that are inserted that you have this protein corona that like you know surrounds it and it practically then it cages the particle inside and then marks it for degradation. So in this case like means how does that this LMPs avoid that step of the immune system. They don't I mean. So your body will start identifying LMP so if you have like chronic or continuous administration of LMPs your body will start I mean it depends of course of everybody's immune system but they will start identifying the different components of the LMPs and they will start clearing them pretty fast your body is pretty smart. So it doesn't avoid the kind of like immune response. It does decrease it because compared to catechonic permanently charged catechonic nanoparticles. These are much easier to identify by the body than the catechonic and I saw a little bit kind of hides a bit in the kind of plasma but there are antibodies that for the peak. I think that's one of the most thing that it was hypothesized on why some of the vaccines had a potential side effects for some people like this antifelactic shock that could be related to the peak. So, so I mean, they can cause an immune response so we could think for example the first sRNA therapy that was done for a product that is approved at the moment on Patron. And this is done to basically knock down a protein production of a protein that is really unfavorable. It's a protein that is misfolding and forming amyloids. But you need to administer that product with dexamethasone, which is kind of like anti inflammatory drug otherwise your body will recognize it and will recycle it and stop the effect. That's it. Thank you. So any more questions? I see Trevor you have unmuted yourself so you want to say something? I think Trevor knows this work because he was also working with Ferik and myself. Well, thank you for inviting me. If you have any questions you can also reach out to me.