Video 1: Non Covalent Interactions
Good morning everyone. So, today we will be talking about Self-assembly and its applicationfor Tissue Engineering scaffolds. So, before we start with what self-assembly is, in thisoverall lecture, we will be looking about what self-assembly is, what are the interactionsthat facilitate this self-assembly process and how do we use them for our tissue engineeringapplications.So, I will be giving you a definition of two things today, what self-assembly is and whatself-organization is. So, self-assembly is the spontaneous associations of moleculesunder equilibrium condition into stable, structurally well-defined aggregates by non-covalent interactions.So, just as a question; so, everyone knows what a covalent interaction is and what anon-covalent interaction is? So, today in self-assembly we will be talking only aboutnon-covalent interactions, but why there is a definition of what self-organization comesacross is, these two terms are interchangeably used in the biological process.So, people come across both self-assembly and self-organization, but there is a verynarrow difference between what self-assembly is and what self-organization is. So, whenwe look at the definition of what self-organization is; it is a process in which order is attainedfrom a disordered state similar to what self-assembly does, but here in external energy is inputinto the state to maintain the equilibrium, but whereas in self-assembly there is no energythat is given to the system, it is a spontaneous process. So, self-organization comes up intoother macromolecular systems, where today we will be dealing only with self-assembly.So, a self assembly comes into a field of supramolecular chemistry. So, I am not goingto bore you with the more of chemistry terms today, just a little bit of how chemistryand biology are interrelated, we will be looking into it. So, supramolecular chemistry is definedas chemistry beyond the molecule. So, how do we say that molecules come up. So, we havean atom and then these atoms are covalently bounded to form a molecule; say you have yourC double bond O and the NH molecule forming an NH2. So, this is forming of a one molecule.So, this is what the normal chemistry involves, but what goes into beyond the molecules? whenthese molecules come together what happens, what is the aggregates that are being formed?When you look at the diagram there, you have a molecule that is a pictorial representation.So, this we say it, the initial supramolecular chemistry was termed up with host guest interactions,you take one molecule to be the host and another molecule to be the guest and then when theyall combine together, there is an interaction that is happening in-between and then theseform the aggregates which we called as a supramolecular aggregate or a supramolecular chemistry isthe term overall. So, unlike the covalent bonds that are being formed within the atoms,here there are only non-covalent interactions that come into play.Brief history of how this came across. So, we all know how this LB film is, your monolayeradsorption film that we have all studied. So, this is how an amphiphile or an oil displacesinto the water. So, this was a monolayer assembly, initially that was in 1774 by Benjamin Franklinand then there was another breakthrough in 1881 which is the cyclodextrin molecules.So, the image that you see there the 1881 one image, it was found in, by Villiers andHebd. So, these cyclodextrins are the most conventionally used systems for host-guestchemistries. So, what is a cyclin or what is a cyclodextrinis, the common polymer that we see, the glucose polymer is all linear in nature, but thiswas the first cyclic polymer that was found. So, these cyclins were of three differentclasses the alpha cyclodextrin, the beta and the gamma. And this depends upon the differentsugar units, the number of sugar units is the one that is varying. And then, there wasanother thing that they found was the substrate enzyme specificity that we all see in thebiological system. So, we have an enzyme, we have a substratethere is a very high complimentary binding that happens between an enzyme and a substratebecause of which they bind and then the product is formed. So, this is one level of a host-guestsystem that happens, which gives you a product formation within an enzyme and a substrate.And then, we have the Watson and the Crick model, that is the DNA which is the basicmodel of our human body. So, what runs across is the double helix model.So, you have two different parallel chains running across and then you have the hydrogenbonding within them. So, this hydrogen bonding is a non-covalent interaction that keeps thisDNA molecule intact.And so this, these were the three people who were given an Nobel prize for their supramolecularchemistry. So, these three molecules the crown ethers, spherands and the cryptands are allcaged like systems that was all formed. And these are organic molecules, this was oneof the first ligand metal process or a ligand and host guest systems that was initiallysynthesized. And this is when the field of supramolecular chemistry came into existenceand people started noticing what supramolecular chemistry is or how we can utilize them forvarious other applications.So, before we deal of how we are going to take these self-assembly or what supramolecularchemistry into tissue engineering, we need to know what are the interactions that comeinto play and how they are different from the covalent interactions. So, the five majorinteractions that we will be talking about are the electrostatic interaction, the hydrogenbonding, the pi-pi interaction, Van-der-Waals interaction and the hydrophobic effects.So, the first one will be electrostatic interactions. So, there; so all these interactions are nothigh energy involved unlike your covalent bonds. So, the covalent bonds will have around500 kilojoules per mole of energy and to break a covalent bond you need so much of energythat is also involved. But unlike interactions, electrostatic interactions or non-covalentinteractions, we do not term them to be bonds, we always say them to be interactions. Thereis very less of energy involved in all these chemical processes and they are mostly reversiblein nature. Just with a stimuli or any responsiveness they are reversible bringing them back totheir original form. So, the electrostatic interaction is the ion-ioninteractions. So, we know what an ion is; a cation or anion with a positive and a negativecharge. So, when there is an interaction between two differently charged molecules, we callthem to be as an ion-ion interaction or an ionic action. And then what is a dipole-ioninteraction? So, what do we say as a dipole molecule? You have a polar molecule and inchemistry we must have all learned that electrons move across and then there is a charge separatedspecies that come across. So, what happens is, there is a partial positivecharge and a partial negative charge within the molecule that happens. So, when thereis a partial positive and a partial negative charge, these ions are immobilized in differentregions and then this combine or interact with an neighbouring ion or a dipole, thenthere is a difference in the interaction. So, we have a dipole-ion interaction or adipole-dipole interaction depending upon where the dipole interacts.And so, the major thing that we will be looking about is hydrogen bonding. So, this is oneof the most important driving forces that takes these interactions or the non-covalentsupramolecular self-assembly taking forward. Because for tissue engineering applications,we use a very, very important biological molecules which all have an amide backbone or an NHgroup which is involved in that of the hydrogen bonding.So, let us first understand how when hydrogen bonding works and how you identify where thebonding happens. So, this results from the attractive force between an hydrogen atomcovalently bonded to a very electronegative atom such as nitrogen, oxygen or fluorinewith another very electronegative atom. So, let us take the conventional unit system thatwe take for defining a hydrogen atom; the water molecule. So, in the; in slides yousee two water molecules one to your left and the other to your right.So, there is a partial bond that is being formed in dotted lines between an O and anH. So, there is the oxygen which has a lone pair of electrons. So, that is the highlyelectronegative atom and then you have a hydrogen which has a partial positive charge. So, anyhydrogen bonding that you take, you need to identify what is an hydrogen bond donor andwhat is an hydrogen bond acceptor? So, the hydrogen bond donors are always the hydrogenwhich is linked to the electronegative atom. So, in this case the right-hand hydrogen,which is linked is your hydrogen bond donor. Whereas, the most electronegative atom isyour hydrogen bond acceptor. So, any hydrogen bonding that you come across identify, tryto identify where your donor and then your acceptor systems are, to know whether thesemolecules can undergo an hydrogen bonding. Because any hydrogen cannot have an hydrogenbonding; only if it is linked to an electronegative atom it can facilitate an hydrogen bonding.A CH bond will not give you an hydrogen bonding only an OH bond can give you an hydrogen bonding.And then we have the pi-pi interactions. So, these pi-pi interactions are within the pimolecules or the aromatic molecules that we always say. So, the pi electrons are alwaysdelocalized within your electron cloud in the aromatic systems. So, they are not stablethey are more in the electron cloud. So, when these molecules come into contact with closeeach other, they form a stacking kind of a nature.So, the pi-pi interaction is most commonly found in only aromatic systems, such as yourbenzene units and then your phenylalanine all your aromatic compounds. And this canhave a difference in the arrangement where you have a face to face pi-pi stacking orthen the T-shaped packing is a face and an edge packing and then you have a paralleldisplacement packing of the pi-pi nature.And then we have the Van der Waals interactions, the weakest interactions among all of them,that is claimed to be the Van der Waals interaction. This is also called as a London dispersionforces where people debate on whether both are similar or they have a similar property.So, Van der Waals interactions like how we saw for a dipole or an ion-dipole are notbetween polar molecules, are between non-polar molecules. So, we know that when there isa polar molecule, then there is a charge separated species that happens.But even in non-polar molecules and even at inert molecule such as helium might have thesetemporary dipoles or oscillating dipoles. So, when they are put in a medium, they dueto some probabilities there might be an electron that is moving and you will have a temporarydipole. So, these temporary dipoles are always oscillating. So, you have a very weak partialpositive charge and a partial negative charge. So, what happens is, at that moment when thereare similar molecules coming across to each other so, these partial positive charges.So, this is how the blue molecules is. So, you have a very negligible partial positiveand a partial negative charge in the non-polar molecule and when they all come across, theystart oscillating in a similar tune or a packing pattern and then they form a self-assembledarray. So, this is how a Van der Waal interaction takes place, but when you orderize or categorywise all the interactions, the Van der Waals interactions are the least among all the non-covalentinteractions. (Refer Side Time: 11:59)And then we have the hydrophobic interactions which is found in all your protein systemsand then the hydrophobic cavity, how the protein starts folding. So, these interactions aremainly because when the non-polar molecules when put inside water or an aqueous mediumhow they react, ok. So, what happens when you are put into a very unfavourable condition?You start to become very closed; you do not open up. So, this is what happens even whenyou put a hydrophobic molecule, which does not like water inside the water system.So, what happens is they become very close trying to minimize their energy and this iswhat; so, that is what, the water surrounding the molecules form a cage around the hydrophobicmolecules. So, because there is a change in all this, there is a rise in the entropy inthe surrounding system and the hydrophobic properties are being, inside the interiorburied deep. So, these interactions are very majorly found in your protein folding processesor your micelles and bilayer assembly where they all interact with your water system.(Refer Side Time: 13:01)So, we have seen all the different interactions that, non-covalent interactions that takeup all this. So, let us see how they drive our life. So, there are three important systemswithin the biological system that is well characterized and studied for all these non-covalentinteractions. So, they are the proteins, the DNA and the lipid structures.(Refer Side Time: 13:20)So, the first one will be of an hydrogen bonding in DNA. So, when you look at the DNA structure,you all know there is a DNA double stranded helix, as I have already told before. youknow there is a phosphate group and a sugar and a nucleobase.So, the major thing that is holding the DNA double helix is the hydrogen bonding betweenthe two nucleobases. So, we have an adenine and a thymine, guanine and a cytosine andalways the bond is between A and T, G and C. So, anyone knows why it is always betweenA and T and G and C? Why not is the other way?Because the interactions would not be stable at others.Ok, what interactions? Hydrogen bonding.(Refer Side Time: 14:08)Ok let us take an example. So, this I am giving you as your molecule. So, the common thingis adenine and thymine and then guanine and cytosine. If you are going to link the otherway; say you will have adenine and cytosine, guanine and thymine, how many hydrogen bondseach one will have? Two.Two for what? Both.Does everyone agree with this answer? Even in that, where hydrogen bonds it wouldnot be, it might not be planar. But the hydrogen bonding is more of a distancerelated phenomenon. Yes.So, when we look at adenine and cytosine say for example, look at your structures, youhave one donor and one acceptor system and then whereas, you have a cytosine you havetwo donors, one, two donors, one acceptor. One acceptor.But they are all misplaced. So, when you have an adenine and cytosine it will have onlyone hydrogen bond and thymine and guanine it will have two. So, this is the reason thereis always have to be complementary that, A and T will bind, G and C will always have,and that is the reason how hydrogen bonding plays a major role in keeping this doublehelix strand in place. So, when we design molecules also there can either be an intramolecularor an intermolecular hydrogen bonding. Within the molecules also there can be an hydrogenbonding, but for all these non covalent interactions the distance between the molecules plays adriving role. So, there is a very important distance, becauseit can either have a repulsion or an attraction. So, the distance between the molecules willhelp in facilitating the self-assembly process. (Refer Side Time: 15:49)And then we come across the protein folding. So, here in protein folding it is mostly ofthe intramolecular self-assembly system. So, the first structure or the primary sequenceof your proteins will be amino acids, these are all covalently bonded. So, you have theamine, amino bounds and then forming your long peptide sequence.And what happens when you go for your secondary structures? This is, the secondary structuresthe major role playing is the hydrogen bonding. So, this forms your alpha helix and then yourbeta sheets. So, after this alpha helix and the beta sheet they form with the hydrogenbonding, then comes your tertiary structure into play. So, the tertiary structure of theprotein is more about the protein folding or the intramolecular hydrogen bonding thatcomes into play. So, what happens is when you put them intotheir aqueous environment, the hydrophobic environment, interiors go inside so, thatminimizing the exposure of the hydrophobic cavities to the protein exterior. So, outsidewill have your lysine residues your cationic or your anionic residues. So, this is howyour protein folds and there comes the hydrophobic interactions into play. In quaternary structures,are all intermolecular hydrogen bonding where there are two polypeptides that come intointeraction, where all proteins do not have a quaternary structure.(Refer Side Time: 17:05)And so, the host-guest chemistry is one of the important mechanisms. So, basics, basedon this enzyme catalysis reactions only we have formed these cyclodextrins, pillarenesall these host-guest chemistry molecules. So, there is a very high specificity whenyou come for an enzyme substrate specific molecule and then this also plays a role inyour antigen antibody binding, where they are very highly complementary in each otherto them. And then the binding substrate has very specific pockets or sites for which youhave to design molecules to go and link to them for having an effect.(Refer Side Time: 17:40)And then comes the lipid bilayer; so, this is how your monolayer assembly starts. So,we have a hydrophilic head and hydrophobic tail and these molecules when put in waterthey start to self-assemble. So, either they form a micelle or they form a bilayer. So,the bilayer is when two hydrophobic tails come together and then they start packing,forming a bilayer. And then they might also have a vesicle kind of a thing forming withthat of the bilayer. So, similarly our eukaryotic cell membrane is highly composed of thesephospholipids and in comparison, with that of the phospholipids, there is also a verylarge probability of having the cholesterol in the phospholipids.So, the cholesterol also has a similar structure and shape and thereby this is how we havethis inflow of small molecules and then the hydrophobic interactions with other moleculescoming into play. Because these cholesterols are within the phospholipid membrane managingand maintaining all the inflow of small molecules and other molecules. And this is also oneof the reason why you say that hydrophobic molecules bind to your cell membrane, smallmolecules go through smaller diffusion process all this is all governed by how the moleculesare arranged and what is the bonding that they are going.
Video 2: Self Assembly for Tissue Engineering Applications
And so, after all these interactions and how they are in our life, our day to day activities.So, the major role of how our self assembly is in comparison with that of a non-covalentinteraction is that is it stimuli sensitive property. So, to forming a covalent bond thereis a lot of energy involved and to break them also, that is there. But unlike, in a non-covalentinteraction there is a very little stimuli that is needed either temperature, heat orany pH sensitivity to have an alteration in that of the assembly that we have been formed.So, this is how the biological process work and this is what we are trying to mimic inour tissue engineering. And then we have a tailorable biological properties and the nanostructuresthat we can form. So, all these self-assemble or self-aggregate to form stable structuresand these structures are mostly in the nano regime and they also have, even if they goapart the nano regime, they have a nanostructure; say if you have a fiber they have a widthin the nanometre regime. So, these can be used and tailored for severaldifferent application based on how we want them to be used. And so, then we will be talkingabout the self-assemble systems for tissue engineering applications. The peptide self-assembledsystem, the host-guest chemistry molecules that are being used, I am just giving yousome of the examples. There are actually a lot of literature reports which currentlydiscusses about all these systems and then the pi conjugated systems and the hydrogenbonded systems. (Refer Side Time: 20:24)So, the first one we will talk about an aromatic-pi conjugated system. So, the figure here; sothis paper is from a chem comm paper and you have an phenylalanine molecule. So, it isa diphenylalanine molecule. So, diphenylalanine or f-moc conjugated phenylalanine are knownto form hydrogels. So, these are small molecules when put in water, they start self-assembling,they form an hydrogel. So, how do you visualize the gel is by an inverted method. So, youhave a solution and when you know the solution does not flow, you term them to be formedas a gel. So, these alanines are known to form gel and what in this study they havedone is, they have conjugated an indole moiety So, the left one is an indole moiety to thatof a diphenylalanine to say; to see if this aromaticity increases the self-assembly property,increases its stability and since we are increasing a new compound, they are also trying to seehow the cytotoxicity has an effect. So, the initial characterization they formed a geland they have seen, they formed the fibers and so the storage modulus or the loss modulusare all rheology properties when we see for the gel.So, what it actually says on a clear perspective is, it tells you how the storage of the gelis whether it has viscosity property or a elastic nature. So, if the G prime is higher,it means the gel is more elastic in nature than that of a fluid nature. And these gelswere very highly stable because of the indole moiety.(Refer Side Time: 21:53)And they have also done a cytotoxicity tests in studying how the viability is different.So, only at very higher concentrations there is a reduction in the toxicity of the cells;saying that these can be used as a self-assembled system.(Refer Side Time: 22:06)And then, this is a study where they have five different molecules. They have a peptidemolecule and, in this peptide, they wanted to have glucose into the structure into thebackbone, they wanted to see if adding a glucose as an effect in the thermal stability andbiostability, ok. So, the major disadvantage of using all peptide systems are, these peptidesystems are highly degradable. So, the amide bonds and all because of theenzymes that are present in our body. When you take a peptide self-assembled system insideour system, so what happens is, because of the environment they are highly degraded,but they are very highly compatible because they are taken as to be our own system. Butso, to improve the thermal stability and the biostability they have tried using glucosemolecules and glucose is known to have no cytotoxic effects.(Refer Side Time: 22:55)So, there are five different molecules that they have planned. So, you have the 1, 2,3, 4, 5. First nap is a naphthalene molecule towards your left and then you has two phenylalaninerings that are conjugated and then you have glucose that is in the aspartate region theyhave modified to be glucose. So, two aspartates, one with each glucose. So, there are fivedifferent molecules each that have been changed to their glucose position. So, on the 5thone will have no glucose residue, only aspartate. So, that is to be taken as your control molecule.So, there is four molecules with glucose variations and the 5th one with no glucose variation.(Refer Side Time: 23:31)And so, they have summarized all the results in a table of how the thermal stability andthe biostability of the system varies. So, the MGC here stands for the gelator weightpercentage at the concentration at which the gel is being formed for all these peptidegels. And then, the pH at which the gel is being formed and then the width of the nanofibersin the fiber diameter. And the strain percent is the strain at which this gel becomes thesolution. So, this also increases or it depends based upon the addition or without the glucose.And then we have the biostability and the T gel. So, the biostability when you see forthe 5th one is 0. So, here the biostability is after 24 hours of incubation in an enzymefluid, they have checked what is the remaining amount of gel that is being there. So, inother cases you have a 48.5 percent or 29.4 percent, but without glucose there is 0 percentremaining saying that the peptide hydrogel was completely degraded.So, glucose increases the biostability of this system and then we also have a T gelthat is that gel temperature, the stability. So, the T gel of the hydrogels were studiedwhere the glucose ones were higher then compared to that without the glucose one. So, thishas a 53 degree at which it will degrade whereas, this has a very high thermal stability.(Refer Side Time: 24:51)And so, they have used two different gels; the first and the second gel to study theircell cultural activities. So, here they have done for NIH 3T3 fibroblast cells and forthe HUVEC cells. So, the A and B; A is for the fibroblast cellline and B is for the endothelial cell line over a 5-day period they have monitored howthe cell proliferation works. So, the image that the green image that you see is whenyour cells are stained with fluorescein diacetate, it stains the cytoplasm and that is the reasonyou have a green colour image that is being seen. And then we have the C and D where youhave the actin staining that is happening. And then they also calculate the cell densityand the density percentage at which over a point of time and to see how the proliferationhas increased over a period of time. So, all this shows that these peptide gels can beused effectively for our tissue engineering applications for wells, nice cell adhesionand then proliferation. (Refer Side Time: 25:48)So, this is another peptide self-assembled molecule, where they have a peptide amphiphile,two different peptide amphiphiles have been taken. And then, they have studied the initialcharacterizations for both the branched amphiphiles. And then, so they have tailored it with thehistidine and these two different molecules have been studying separately and also indifferent ratios to see how whether mixing the peptide amphiphiles has an effect in thetissue culture. So here, the figure that we see here is for 24, 72 and 96 hours in thefibroblast cell lines where they have taken a specific ratio of both these poly amphiphilesand then seen how the proliferation increases. So, this also shows that over a period oftime they have a very good proliferations for these fibroblast cell lines.(Refer Side Time: 26:36)Here since we have seen different interactions; so that was how the hydrogen bonding interactionsare you also have ionic interactions-based polymer gels for tissue engineering. So, herewe have an ionic co-polyelectrolyte system where the polymers are modified to have aterminal ion. So, one is a poly anion and the other one is a poly cation. So, when youmix this gel together, they form a crosslinked system, but these are not covalent crosslinksystem, but they are intermolecular interactions. So, the purple colour bubbles that they sayis where there is an ionic interactions between your two polymers. So, these are like thecovalent bonds that are being formed holding the gels together. So, when then these putinto the system we see how they have an effect, whether the crosslinking is effective or not.(Refer Side Time: 27:22)And then we have the host-guest inclusion complexes. So, this is one of the most importantmolecule; the cyclodextrins. So, the cyclodextrins as I have told has a glycan nature, here theyhave utilized two different ways of bonding them. One is they have modified the PEG terminalto be adenine and then the another PEG terminal to be thymine. As we have already discussedadenine and thymine have a very nice complementary hydrogen bonding forming an A T pair. So,what happens is the PEG molecules when they bind; they bind together and form an hydrogenbonded AT pair. So, the PEG both are having an intermolecular interactions. So, thesemolecules then slide into the cyclodextrin core also to give a well known intermolecularinteractions. So, this is how the gel is being formed theimage that shows. So, you have a solution and then mix them with cyclodextrin and thenyou see how stability is better. And then this is the SEM image of how the gel looksand here they have added the doxorubicin a cancer drug and see how they release. So,but this can also they have also done an in vivo study on it, on rabbits to see how theycan have an injectable effect of this gel on the rabbit.(Refer Side Time: 28:36)So, these are another small molecule gelators, where we have alanine as the core moleculeso, they have a small terminal modifications. Here the interesting part of the paper isthat they have very small modifications to the terminal segments showing that how smallmodifications or chemical modifications your system alter the self-assembly pattern. So,you have the first molecule to be alanine hydrazide, NH NH2 whereas, in the second oneit is an alanine CAM which is an C double bond O NH2 that is an amino bond.And then the other one is a protonated form of an hydrazide HYD plus. So, what happenswhen these are very small modifications when we do in a chemistry basis, but what happenswhen they move towards the self-assembly process. So, the first one is the amino terminated1 the AlA-CAM molecule did not form a gel in any of the concentrations. So, that wasruled out and then we have the alanine hydrazide and the protonated form of alanine hydrazidewhich form gels at different weight percentage. But when they wanted to take them to be ofan injectable system, these molecule gels are not that stable as a polymer gel wherethey are very soft gels. So, all this has a major rolein designing molecules for your tissue engineering. Thank you.
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