Dr. C. Kulkarni
University of Central Lancashire
Talk title: Beyond Liposomes: Hierarchically Organized Formulations
Liposomes, also known as vesicles, are one of the simplest but widely used forms of lipid-based carriers. However, recent advances in this arena have contributed some innovative carrier systems that do not only retain the properties of liposomes but also provide added advantages. These lipid-based systems constitute multilevel structural hierarchy, which enables them to exhibit special features such as high hydrophobic volume, high surface area, nanostructural fine-tuning and systematic control their physical forms. These emerging formulations will be focussed in this presentation.
Kulkarni, C.V.* (2012) Lipid Crystallization: From Self Assembly to Hierarchical and Biological Ordering. Nanoscale, 4, (19): 5779-5791.
Dr. Chandrashekhar V. Kulkarni
Group Leader: Lipid Nanostructures Laboratory
Centre for Smart Materials
School of Natural Sciences
University of Central Lancashire
Preston PR1 2HE
Aug 4, 2021 Presentation at the Global Conference for Lipid Nanoparticles & Other Non-viral Nanocarriers
Beyond Liposomes: Hierarchically Organized Formulations
T_T Scientific_ Dr. C. Kulkarni_ Beyond Liposomes_ Hierarchically Organized Formulations (UCLan)
Nima Tamaddoni, Ph.D.
Hello, Dr. Kulkarni, it is a pleasure to have you here. Thank you so much for your time. It is a pleasure to have you. Dr. Kulkarni is Head of the lipid. Sorry, I had to, Lipid nanostructure laboratory at University of Central Lancashire and he is joining us from the UK and yeah, they are very attractive topic beyond liposome, hierarchy organized formulations. So, I will pass the mic, I apologies for the slight delay. This is a first major move on our events. So, we apologies for any delay, I pass it to the doctor.
Dr. C. Kulkarni
Thank you very much. Can you hear me now? And can you see as well?
Yes, we do hear you. We do see you and we do see the slides.
Dr. C. Kulkarni
Okay, perfect. Yeah, thank you very much. Yeah, thanks a lot for your kind introduction, as well as allowing me to talk in this very nice forum. I quite enjoyed the previous talks, as well and it looks like there is a very good lineup of excellent speakers all over the world, from industry and academia who bought, so it looks like exciting forum. So, let me tell you, take you really to beyond liposomes as my title suggests, you know, because, yeah, there were two talks, and you had Tamari best LLPS lipid nanoparticles, and of course, they were based on liposomes, so you had lipid loaded, so mRNA loaded liposomes. And, so LNP is when we are already load mRNA into these liposomes, we are already moving beyond liposomes but what I am going to talk to you today is some more hierarchically organized formulations. So, you can see. So, let me start with liposomes exactly, of course, you know what liposomes are, but let us refresh ourselves. So, Lipid amphiphilic and when we mix them with water, they form some self-assembly. So very simple self-assembly is by layer and when that by layer is enclosed, like, is rolled into a sphere, it forms like liposomes and you can have various liposomes to see small, large and giant but these are uni lamellar. But nowadays in last like about a decade or two, so these MLB and oligo vesicular vesicles are coming up as well. Why because these GUBs are good as model cells, but nowadays, what people think that you need compartments because you cannot have just one sort of water reservoir, you need several compartments and these MLBs and OVBs are coming up, but these ones which I am showing you are structural types of liposomes or vesicles, they call them. So, but there are some more like, you know, mRNA loaded LLPS, these are again liposomes, then you have a peg, peg highlighted liposomes or tender liposomes and so on, you can have a cholesterol elite and so on.
So, various lysosomes but all of that starts from the very basic lipid structure because you know, head group is blue in color here likes water and then hydrophobic part dislikes water. So, you can see that and that on the right-hand side when you put a lipid sort of sample on water, you will start seeing that the tails go away and that from the basis of liposomes but much more than that they form the basis of self-assembly. So, what is the difference between these two, you know, liposomes, yes, they are self-assemble, they are not directly self-assemble, because by layer is the structure which is self-assemble and that is thermodynamically stable structure. But liposomes are kinetically stabilized structure. So it is already hierarchically organized, okay. So, lipid bi layer forms liposomes and that you are moving already towards hierarchy structure liking, but more than that, if you have a different shape of lipids, and those shapes come from chemistry, so if we have a large head group, if you are let us say more polar groups, you will have a conical shape and if you have a less conical less polar molecules, but you have long working chains or maybe alkyl chains with unsaturation or maybe more than one alkyl chain, then you will have different shapes.
So, there are conical sort of shape liquids, like for example lycopene, then there are cylindrical type liquids like the DOBC, you have inverse conical, which is DOP hospitality canola mine. So, these liquids are of various shapes, you know, and they compose, sell and cellular membranes. So, how do they form self-assembly, you can see that the shape matters in this case because if you have larger shape groups, you will have normal micelles, type one and that is what we see everywhere when you have surfactant in water for example, you know, SDS is a surfactant for example, and sometimes some lipid form, but major developers will form planar micelles or they will form inverse micelles and there is a huge variety here, you can see that these simple ones will form spherical micelles or hexagonal cylindrical mindset, but these are H1. So, these are normal ones, but if you go on the right-hand side you do have those H2 and L2, what in this case is you have water inside these phases and the main structural components, structural integrated is given by the lipid phase itself, lipid itself or lipid arrangement itself. So, as lipid gives the structural identity to the phase you can have a variety of phases you can have cubic phase which are on the top, IM3MPMRI3D, these are, excuse me, by continuous cubic phases, what is bicontinuous? They are continuous in water as well as their continuous in lipid, which means you can put something in lipid phase well as in water. So, and that is why they are buying continuous there is a very good diffusion. So, you can see that this phase of three dimensional and they are really ordered, you can really define them using mathematical equation, you know, this cubic phase is. Sponge phase is also bicontinuous but it is disordered face okay.
So, these are all faces, but there are some intermediate phases LC, L-Beta, these are the names of polymorphs of lamella face. So, LC is something like when you have a frozen structure, there is no real moment in the bio lab; L-beta is a gel phase or L-Beta prime, L-Beta touch star, L-Beta prime these are another polymer subject with. P-delta, sometimes you can have interdigitated or tilted while S and L-alpha is a true lipid crystalline phase which is very similar to the lipid bilayer. Okay. So, right, these are the level of phases but maybe biologic people or maybe people who see some phases using confocal microscopy, they will not find any of these phases in their vesicles for example, or their structures. Why? Because they are named differently but if we have to compare those phases, we do have there as I said there is similarity or there. So, L-alpha is your LD phase, which is LD, D means disorder phase, okay. So, that is L-alpha, where you have like a fluid bilayer, where there is always like a dynamic in the chain region. So, L-beta is a gel phase and that gel phase is your LO phase which is ordered phase, okay and this sometimes seems like you have a cholesterol domains or sphingomyelin domains and these come from these L-Beta ordered phase, okay. So, these are various non-laminar phases and these all I am talking about are thermodynamically equilibrium phases, okay.
So, they do have, you say that we can put them on a scale like zero dimensional one dimensional two and 3D. So, 3D is like you can see really like a folded membrane, you know, so, if we compare bi membranes, you see ABCD or the large bio membrane structures, ER, Golgi and mitochondria, here okay. So, this is a I3DLPL3 do mimic structurally, mimic these dynamic membranes you know, interesting little members how, so, this G and D is a gyroid and a Double Diamond, these are mathematical minimal surfaces, you can define by equation as I mentioned, so, at every point there is a zero mean curvature okay and on these minimal surfaces, these lipid cubic phases will be formed. So, the bi layer will drape on these minimal surfaces to form these three dimensional phases, okay and if you take projection, two dimensional projections at several points, some projections look like this and if we compare TM, high resolution image of organized smooth endoplasmic reticulum, you can see the S part here, S is a sinusoidal and the sinusoidal structure looks similar to the projection on the left hand side, it means these three dimensional cubic phases have very similar structure to that of highly complex intracellular membranes. So, we were until now, we were just mimicking cell membranes or compartments. But with these phases, we have a great potential to make highly complex membranes which are internally organized, you know. Moreover, these are folded, yes, the cubicles are folded as well, there are three dimensional, they are highly dynamic, and they are accurate channels and lots of them. So why I am telling you this because this is one point of lipid based systems which are hierarchically organized, but there are some more points and these ones, you see we encounter in daily life, these all these self-assembling phases, hexagonal cubic, yes, they do form in our stomach as well in GI tract, because when bile salts or lipids attack on the limits of the fact we age, then they do form such phases. So, we have enforced in applications like in medicine in drug carriers, or of course, as I mentioned in biomedical applications in biology and some hierarchically order structures. So, we do have these, so how do they look like, if you for example, if you start making formulations of them, they look very different than liposomes. How do they look?
They look highly viscous and you need to do something with them. So, but these are very different and that is why they look very different and they are very difficult to handle and that is why they are not really in the market yet. I mean, there are few products in the market, but liposomes are something people talk about. But there are certain advantages of these formulations because they are assembled bimetal structures, these are food grade or food itself, I will tell you an example of the food itself. They are biogenic they are biological origin. So, these monoglycerides which we use to form these phases are found in our stomach when triglycerides are digested okay. So, these are amphiphilic so, you can put hydrophilic, hydrophobic and hydrophilic molecules in them, they are good for civilization, pure poorly soluble components and very interestingly at the very last, one we have presence of lipid in GI tract triggers the secretion of emulsifying and digesting molecules, they will trigger the secretion of bile salts, lipase and they are needed, you know, if you want to release the drug or release the component then they are needed you know, you need to digest the lipid molecule there okay.
So, as I mentioned, they look very viscous. So, that is why people do not use them. So, these are the cubic phases okay. But then people, I mean, these are gels, so, you can use them for topical application straightaway, but if you want to use them for some other applications, you can convert them into different formulations, you can prepare particle dispersion using high pressure homogenizer but to use high pressure homogenizer is usually difficult with these viscous faces you need different like a probe ultrasonic okay. So, what do we have, we have oil in water emulsion and oil is a lipid phase here in this case, okay, you can lower the drug hydrophobic drug in the lipid itself and that we form an emulsion? This is a milky fluid emulsion, what do we have there? We have particles and those particles are my cells themselves, stabilized at the interface using block glycol polymer okay. So, you can return hexagonal phase or cubic phase in the interior. So, now, we have an advantage of lipid phase plus you have an advantage of having an emulsion. So, you can also put do multiple loading, you can put hydrophilic as well as hydrophobic plugin, okay and you can find you can find you using all those faze, whom we saw earlier, like the self-assemblies, okay. So, these are oil in water emulsions, but these are, if you have a cubic phase as the interior, you can call them as cuebosomes, you may have heard these terms and hexagonal phase then you can have hexosomes, okay. So, there are certain advantages, what are the advantages, because how large hydrophobic area as compared to liposomes, is a main advantage. Okay. So, we started with comparing these two systems. So, let us see if we have a gel phase as is or cubic phase, how do they compare? So, we prepared a setup for drug release, we just loaded aspirin as a model drug and check how I mean, I am just showing the representative data here.
So, cubic phase, and then you have a cubosomes, which will have a fast release, you can see the rate of release, you have fast release in cubosomes home and there is obvious reason because cubosomes are about 200 to 400 nanometers, the drug has only has to pass that 100, 400 nanometer area, not much. But cubic phase is like a whiskers gel, it has to come out of it and that is why it is a bit slow. Okay, and because of open channels, there is already initial release, rapid release. Okay, so sometimes lipids are very expensive. So, we thought okay, let us use some liquids which are like, like available, cheap, and abundant. So, we thought, okay, let us use butter and butter oil, and we prepared emulsion out of them. So, you can see on the right-hand side here, the stable emulsion was formed using a glycol polymer. Okay, so we have advantages there because it is a food itself. So, you can use them straightaway there is no issue about food grade or not food grade or if there is toxicity or not. Okay, we then use different stabilizers because these F1 to seven which is a peg is usually an issue these days, you know, people are trying to find some alternatives to back and then we thought, okay, let us use something which is already in the body bile salts, okay.
So, we prepared the lipid bile mixtures and we were able to prepare very nice emulsions out of them. You can see the milky fluid at the bottom here, okay from the cubic phase, but while doing that, what we got is we of course, bile salts are good stabilizers, but while doing that, we understood something else. From our body, you know, physiological phenomena. What we understood is not only emulsification is a role of lipids, these bile salts but there is another role what do they do? They interact with liquids in such a way that they convert them, they are highly complex structure into very, very simple structure. So, we had a cubic phase at initially when we started or when we eat the lipid fat, we have a very complex structure, but when it goes inside, bile salt interacts with them, and then they form vesicles. So, when they form vesicles, vesicles are simple. I mean, you have only one bilayer; there are maybe many multiple bilayers and bilayers are easy to attack. Lipase easily attacks it and digests it. So, to aid in digestion, bile salts help not only in the emulsification, but also in the conversion into simple structure that is another role. Okay, so, these structures, I am showing you these different for different systems. So, these have very good potential for drug loading and of course, loading of some other molecules like injury genes, okay. So, we prepared some hybrid systems. So, we took Kappa carrageenan, which is a vegetarian source of hydro gel, you can see that upside down bottle it forms a very nice transparent gel and loaded our pure fluid emulsions into them and then we had a mod, prospects mod we prepare the team pill, which is number two here, and we just loaded drug in it. So, we and then you can see the kinetics here. So, when we had gels only loaded with drug, we had this red curve, the drug release was not efficient as well as drug does not want to come out and we had a lipid particle loaded with into hydrogen then we had a sustained really slow release over there as well okay and there was efficiently. So, more drugs were released in that case.
We also prepared some formulations, these lipid particles using carbon nanotubes, because carbonate is because there are there is a community who say that okay, carbonate are toxic and there is another community who says that they are organic molecule they are not toxic, but yes, there are toxic, I mean, these carbonates are shown to be toxic, but if we load them with coating with lipids, we have a possibility that we have a reduced toxicity because then the hydrophobic part is coated with liquids, which are biogenic and then they may easily get digested into the body or maybe they at least will not be hated by the body, okay. So, we prepared those we also prepared some formulations using fullerene. Fullerene is good because they are monodispersing and then we also prepare those hybrid particles on this one is published in case you want to visit and see the details. So, what are these, where are these useful is for multi-drug loading and this is a potential application of these hybrid particles. So, these are all particles, which we saw were oil in water emulsions, but there is another type called is water in oil. So, now you see the colors are reverse, what does it mean? Actually, the water in oil emulsion looks like this is what is on the right-hand side.
So, the lipid itself which is yellow part forms a thin film and inside there are water pockets, these are water reservoirs. So, and these ones look like I will show you how do they look like but how can you prepare, we have some customer machine in this case on the lab scale. So hydrophilic face and lipophilic face, we shown by the color and then there are pistons which push them into pre mixing chamber, high mix, high speed of the mixing chamber and then then they go into rotating horizontal cylinder with the gap of about 100 to 200 nanometer and because of that shearing at high temperature those get mixed very well and we can prepare creams like this and very interesting thing to note here is they can hold up to 90% water and with some fine tuning we can go up to 95 to 97% water and the beauty of them is they do not need a surfactant stabilizer because these creams are used for on the face for example on the scale, you may not like a soap you know surfactant or the stabilizer So, these can be made without surfactant and soaps as a stabilizers okay. So, these are very good for several applications. So, and you can see that there is a structural hierarchy in both of these emulsions, okay, you start with the molecule at about, you say that is two or less or two nanometer lipid, it forms a hexagonal phase or any other queries, then you form an emulsion and other hierarchy then you go to the next level and then you can see, visibly you can see those faces, there is a confocal microscopy and there is a TM image of that as. So, you can see that these are hierarchically organized structures here in this case. So we can use some two representative examples. In next two minutes, about two minutes. We prepared some high value formulations and these are right now in preparation for patent. I mean that is almost in the final stages, now. Cur cumin has an inherent problem of solubility in water or dispensability in water and that is why they are not much, cur cumin is not much bioavailable, but its applications are known and many new applications are coming as well and you see in COVID era, I mean it is sold as immune booster, turmeric and cur cumin.
Cur cumin is the active ingredient of turmeric in case if you do not know about it. But there is a very good potential because the market is coming up and there will be a really like a good growth and this is old data but there is new data which came up about two weeks ago and then it has even more even better potential for this. Okay, so there is a great demand. So, we prepared this formulation, as you can see on the top and this is just as I said is a representative example, these are gels, but we can prepare their crease. We can prepare pairs, we can prepare dry fields, powders, and rest everything but what is good to know about from these formulations is these are lipid waste, and these are biogenic food grade lipids and that is why whatever is dissolved will be bi-level. Okay, and that those studies are underway. Other formulation we recently prepared is a copper as per lead, these are copper complexes, and these are good for topical applications. There are several papers published on antimicrobial applications of them, but, again, there is a problem of solubility; they are not soluble in almost organic solvents as well.
It is difficult and that is why we are prepared these and very good potential okay. So, if I take you, now knifeless tech, because I told you so many different formulations and different lipid systems just on the last slide, I would like to show you here. How do they compare with liposomes? So, liposomes here we have aqueous interior and just one bilayer of hydrophobic lipids, okay. But in this case, if you make particles you can have like whole interior made up of hydrophobic lipid, but that also has accuracy interior in them. So, you can also learn hydrophilic as well as hydrophobic particles in there as well as unbefitting and they are good for membrane protein crystallization for instance and there are certain advantages, I mean lipid-based formulation. So, I will not go into all of those, but you can see that these particles do show a play of properties of liposomes, but they show some special properties and I mean, there are several properties that are listed, I will not read all of them, but I read some important ones. Because similar structures are seen in nature, they have high lipid volume per particle, and they have very good potential to how to get good potency, high potency and then you can solubilize as hydrophobic molecules because that of course, as you may know, are good for major diseases. So, I would like to thank my collaborators and funders and thank you for listening and I am happy to take questions.
Nima Tamaddoni, Ph.D.
Thank you so much, Dr. Kulkarni, we will get to a question. We have a really short time again guys. If you have a question for Dr. Kulkarni, you can put it in a Q&A and you can answer with video or with text. But we do have one question that can be probably answered. We interviewed two gentlemen.
Yes, thank you Dr. Kulkarni. One of our questions is can you briefly describe how the formulation of an emulsion may vary from an LNP? With like, with standard ethanol injection?
Dr. C. Kulkarni
Yeah, of course! I would say, I mean, these ones do not contain any other smaller one than water. I mean, you do not need ethanol for this, you know, you can have completely Aqua system and that is probably what is accepted by the body. That is first difference. Second difference is you can have a highly concentrated system because vesicles you need, I mean, if you take a solution of vesicles, you have our LNPs, you how usually dilute system. In this case, you can make really like 15, up to 15 to 20% lipid volume in your emulsion. So yes. So, these are two major differences.
Nima Tamaddoni, Ph.D.
Thank you so much. That was that was very helpful and so yeah, we have about 70 people for us and Dr. Kulkarni has two books that you can, he has authored. You can learn a lot from, it is very attractive. We have already order it to come and take a look. But, Alex, you have another question?
We have, yes, this one is come in, how stable are the cubism in general, in the Olympic context? There is a question about the lamellar or inverted hexagonal phases and the challenges of keeping it in the cubic phase. So how stable are they in general?
Dr. C. Kulkarni
Yeah, that is a very good question. I mean, cubesomes, if you prepare cubosomes, they are stable at room temperature up to six months, about six months and they will stick your songs if you do not really add anything into it. Because if you do not change temperature either if you do not go below 10 degrees Celsius, you do not know how to store them in the fridge. If you do not go below 10 degrees Celsius, they would not change the phase and if you really keep it in the fridge and bring it back to room temperature you need to go just about 20, 25 degrees Celsius, they will come back as a cubosome. So as a cubosome, they are stable, but they would not usually change phase on its own unless you add something to it. You know if you add some component, let us say if you are another lipid or if you add another drug, they may change a phase but then drug to lipid composition you know that mixture proportion you need to study separately before you prepare cubosomes and if that still retains the cubic phase as a bulk lipid crystalline then it would not change usually in the cubosomes as well.
So, phase only change just by keeping it at home temperature, it will only change if you change the additives into it and if you have a hexagonal face at the start, we will keep hexagons, and hexagons will stay as hexagons. Laminar phase also forms such phases because laminar phases usually form vesicles, yes, like liposomes, but they also form internally self-assembled particles when you have a particle similar to this cubosomes but instead of cubic phase you can have a laminar phase and in that case also it is possible to get high hydrophobic volume and if you make a laminar phase, it will stay as a laminar phase unless you change the composition or for example, PH, you know, at some point if you change PH, it may change. But if you study that beforehand, you have a possibility that it will stay stable as an original phase.
Nima Tamaddoni, Ph.D.
Thank you so much. It was a pleasure to have you. It was great.
Dr. C. Kulkarni
Excellent! Thank you very much for allowing me to be in touch.
Nima Tamaddoni, Ph.D.
We will be in touch. Thank you. Bye.
Dr. C. Kulkarni
We will talk soon.