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    Study Reminders


    Hello everyone, welcome to another lecture for Drug Delivery Principles and
    Engineering. So, we have been talking about quite a lot about particles in the past few
    classes; basically we are going to continuing this discussion and most likely will finish
    the discussion in today’s class. Particles are again one of the buzzword in the field quite
    a lot of them being used for various applications.
    And there is quite a lot of excitement about it and it is fairly obvious. Why? Because you
    do not have to do any surgery and over a free drug you get a lot more sustained release of
    the drug; so that the patients do not have to take tablets several times a day, you can just
    get one injection or one shot of particle either orally or by some other mechanism. And
    that may be enough to treat some minor disease or in case of chronic diseases also, you
    may only have to take particles maybe once or twice a month or something like that.
    So, just depends on the application in the particles you are using, but there is a lot of
    excitement and there is a lot of tunability that it gives to the researchers; as well as the
    clinicians. And so we are going to continue that discussion, we had talked several things
    about particles, we have talked about how to manufacture them and we have talked about
    what are the different size ranges that are used in the literature.
    And why they are used; we are talked about certain classes of particles that are very
    widely used polymeric being the first one we talked about. We talked about liposomes,
    we talked about micelles, then we talked about some of the physical properties of the
    particles that is desirable. So, size is of course, one; so, spherical particle there is certain
    size range that we want. So, if you want it to sustain the sort of flow in the blood vessel
    or remain in our body we wanted to be greater than 6 nanometer because 6 nanometer is
    essentially the kidney filtration.

    (Refer Slide Time: 02:09)

    If we want it to be injectable in let us say blood vessels, then we want it to be closer to or
    at least less than 5 micron because the smallest vessels that we have are close to about 5-
    6 microns; so, we do not want them to get clogged.
    And we know that our body immune system is fairly good in clearing certain things up
    and what we have found is if the particles are less than 200 nanometer will flow for quite
    a long time because typically spleen and liver or organs that will clear anything above
    the 200 nanometer.
    So, these were some of the things that we talked about spherical particles, but now we
    have introduced a new concept and we are saying their particles which could be of
    different shape. And now when we are saying different shape we are basically saying
    that at least one of the dimension should follow these criteria, the other dimension could
    be different. And we are going to continue the discussion today as well to see what are
    the other properties we can change around while sort of still keeping in mind these
    limitations, but then circumventing them somewhat.
    So, in the last class we talked about particle shape, we talked about what are the
    synthesis method; so they were two synthesis method that we talked about. One was
    bottom up approach where you make particles from single atom and sort of accumulate
    them to make a certain size. Or you can have a top down approach where you use some

    sort of imprint lithography or you have bigger particle and then you break it down into
    smaller particles or different shape maybe.
    So, these are some of the approaches we talked about. And then we talked about some of
    the uses of them we found that uptake for is dependent on the shape. We briefly
    discussed one research paper on this, but then several research paper out there which
    actually further corroborate this point. And then we further found out that even the
    diffusion can be dependent on the shape itself (in the biological context). So, this is what
    we talked about in the last class. So, now you are going to look further; we also talked
    about micelles. Now we are going to look further and talk about the charge of the
    (Refer Slide Time: 05:01)

    So, we have talked about size and we have talked about shape; the next property we are
    going to talk about is the charge. So what is essentially charge? Charge is nothing, but
    what is sort of the electronic structure or how much of the positive or the negative charge
    electrons are available on the surface of a particle.
    So, as you know in body the particle will encounter all kinds of charge. So, the cell
    membrane that we have this is made of lipids that are slightly negatively charged. So,
    you can assume that all cells have slightly negative charge; the serum proteins that are
    flowing in our blood, they are also predominately anionic. So, most proteins that are
    flowing also typically carry negative charge; although this is not true with all proteins

    there are proteins which are also positively charged, but predominantly we will find that
    most serum proteins are negatively charged.
    So, now that we know that this membrane is negatively charged if I want to deliver
    something to the cell, I would probably want to have something that has a positive
    charge right. So, there is an electrostatic interaction; it gets attracted towards the cell.
    What will happen if I have a negative charge? Even though the particle may want to
    come close to the cell, there will be an electrostatic repulsion that will cause the particle
    to move away because of this negative charge and negative charge repulsion.
    So, for positively charged particles the uptake of the mammalian cells is much higher
    than the negatively charged particle. But then what happens in vivo is let us say if I inject
    it into a human or let us say an animal. Because I said that the serum contains lots and
    lots of proteins which are negatively charged; these proteins tend to interact with the
    particle quite a lot and they will tend to adsorb onto these serum proteins.
    So, we are going to talk about adsorption in much more detail in next class, but this is a
    phenomena that will start to occur. And some of these serum proteins are used by
    immune system to sort of recognize; if there is for an object and that will cause the
    clearance of your particles much more rapidly than say a negatively charged or neutral
    charged particle.
    (Refer Slide Time: 07:20)

    So, positively charged particles tend to adsorb so much and that is why also cause
    toxicity. So, if I have a positively charged particle there will be lots and lots of protein
    that will get adsorbed on the surface. And the structure of the protein will change, the
    function of the protein will change, the way these guys can coagulate with another
    particle and so on and so forth. So, their actual size once it goes in the blood may change
    and it may cause toxicity; maybe it will become larger than 5 micron maybe this will
    become 10 micron and that will clog blood vessels.
    What will happen if it clog vessels? If the vessel is going to the brain and if you clog it;
    the brain will not get enough oxygen and it will result in a stroke or it will cause a heart
    attack. What if the vessel was going directly to the heart? The heart will not get enough
    oxygen it will start pumping and that will result in heart attack. So, these are some of the
    considerations that we have to keep in mind while you are talking about charge.
    (Refer Slide Time: 08:32)

    So, neutral and slightly negative charged particles are typically preferred when you are
    talking about in vivo delivery. Because the positively charged particles can cause toxicity
    plus it does not really stay for quite a bit of time in the blood. And so even though for let
    us say if you want to just give some drug to the cell which is outside the body
    environment; you probably want to prefer a possible charge particle, but for a new
    applications you may want to look at slightly negative or neutral charged particles.

    But then again the understanding is still evolving every day; there is new and new
    research coming out sort of challenging all these concepts and proposing new concepts.
    So, it is still fairly dynamic field, but the general consensus is for a longer circulation
    you want neutral to slightly negatively charged particle to flow in the body.
    (Refer Slide Time: 09:27)

    So, the fourth property we are going to talk about is the elasticity of the particle and this
    is again a fairly nascent field; not much has been done in this area, but more and more
    people are now starting to look at the mechanical properties of the particles that they are
    So, again elasticity has been reported to have profound effect on how much the particle
    circulates in blood; actually the best known example of this is a natural particle which is
    RBC right. So, RBC we know is about 5 micron and they are highly elastic and very soft.
    And they have been known to circulate in the blood for about 2 to 3 months.
    So, this is by far as much circulation time as you will ever get with any synthetic particle.
    And what they have is they have a very low modulus and so what people have now done
    is started making particles which like RBCs have a very low elastic modulus and I have a
    large size and then studied how their effect is when they compared it with the RBC.

    So, compared to a synthetic particle versus a natural RBC cell that is circulating. And so
    here is an example, here these guys have used again top down approaches; so in this
    paper they have reported these top-down approaches, so in this case this is the template.
    And what they have done is they have a pre polymer mixture and they roll this pre
    polymer mixture to fill these templates and then cause the polymerization to happen.
    And then they can dissolve this template itself to sort of get your individual particles and
    as you can see the particles are fairly disc shape. And here they have reported sort of the
    bulk properties of this. So, depending on the amount of cross linker that they have added;
    so they can vary the cross linker from 10 to 1 percent their bulk material modulus will
    also change.
    So, here they have been able to change it by almost an order of magnitudes; so 10 times.
    And with that they also see that the half life has changed; so if you look at the half life,
    you are talking about a very elastic particle having a half life of only about 3 hours
    whereas, something that the very low modulus has a half life of close to about 95 hours
    or 93 hours. So, you can see what a jump just the modulus had on the circulation time.
    (Refer Slide Time: 12:24)

    And so this has been reported and one of the reasons for this will come in a few slides,
    but then there are other methods as well.
    So, here is another method to make these low modulus particle; in this case again they
    have used a hollow polystyrene spheres; what they have done is they have created

    proteins to adsorb onto these. So, because the proteins will adsorb on any exposed
    surface; what they have is they have this proteins which are essentially coated.
    So, let us see if I make this particle and then this particle gets coated with several
    proteins and then what do you do? You cross link this protein. So, now whatever these
    proteins were present on the surface well get cross linked and will form bonds between
    the neighboring proteins and hence becomes very stable; then you come with a solvent
    that is going to dissolve this polystyrene.
    So, then what you end up with is nothing, but a very soft hollow protein structure which
    is cross linked on the surface and is hollow. So, essentially extremely low elastic
    modulus and that you can then use to sort of get these RBC shape depending on the size.
    So, because it is hollow it just collapses and you get these RBC doughnut shape particles
    which are very low modulus.
    So, here is just an example where they are showing their actual particle and here are
    cross linked mouse RBCs they look very similar. So, what the authors are reporting here
    is they have been able to sort of mimic the RBCs using this particular method. So, this is
    another alternative to what we discussed in the previous slide.
    (Refer Slide Time: 14:33)

    And then what this show is the elastic modulus of the original PLGA particle is fairly
    high; so this is on the log scale. So, you can see you are talking about in the order of 10
    to the power 6.
    But once they have done their method and cross linked protein and dissolve this PLGA
    particle they get down to about 10 to the power 2. This is just similar to about what is
    reported here as to 2 into 10; number 20. So, still they have not been able to get down to
    the mouse RBCs, but they still are able to reduce the elastic modulus quite a lot.
    And here what they are showing is its fairly elastic, it can deform, it can regain the
    shape. So, now they have flown in through microfluidic channels which are actually
    smaller than the particle size. And what you can see is this particle and can actually
    squeeze through these microfluidic channels; just like the blood vessels will cause the
    RBC to squeeze through them.
    (Refer Slide Time: 15:38)

    So, we can mimic these properties; another example of making elastic particles is filo
    micelles. And these are nothing, but these are polymeric particles which are 20 to 30
    nanometer in diameter and they are about 5 to 8 micron in length.
    So, here is just a fluorescent image of this. So, it is nothing, but this is just like a thread
    or a one like particle and what they have shown is; if you can cross link the different
    regions and make it rigid or you can leave it like this. And if you see their circulation

    time, so if you use a lambda phage which is very similar in structure, but is a fairly rigid
    So, this is a rigid that gets cleared out by 2 days; so by one and a half day this get
    completely cleared. If you use stealth vesicles which are of the same volume, but they
    are spherical and are rigid; you see even then you can maximum get it to 3 days. But
    when they use this filo micelles; they have been able to circulate for longer than 7 days,
    this is greater than 7 days.
    Again just an example of showing how elasticity can cause this effect; so if they now if
    they use the same filomicelles and cross link it internally and this drastically drops down
    to something like a lambda phage where it gets cleared within a day or 2. So, what is the
    reason for all this? I mean, so we have talked about by elasticity is being or we talked
    about how elasticity is able to change the circulation time and able to flow quite a lot in
    our body, but what is the major reason that this is happening?
    And the major reason is the spleen. So, spleen is essentially nothing, but a filter for out
    body and what happens in a spleen is typically the blood which is flowing through the
    spleen will come out into the tissue from the blood vessels and then will go back into the
    circulation. So, if this was let us say a spleen vessel in the blood will empty itself into the
    interstitium of the spleen where there are lots of immune cells residing around.
    And then the blood vessel then there are other blood vessels which are fairly leaky and
    from these leaks all the blood will go in and squeeze through. So, if you have a large
    rigid particle it will not be able to squeeze through these gaps. And will just get
    entrapped in this region, where all these immune cells will clear it away. Whereas, if you
    have a soft and a large particle even though it might be larger than these gaps, it will still
    be able to squeeze through them and hence will have a longer circulation. So, this is just
    one of the mechanism through which we find that elasticity the particle plays a very
    important role in that circulation type.

    (Refer Slide Time: 19:03)

    So, far we have only talked about polymer and lipid particles. Another class of particles
    we are going to talk about is metal particles; typically as you might have seen through
    this course we have not really talked much about hard implants or hard particles like
    The reason for that is of course, even though they have been fairly successful and we are
    going to talk some more about them in the future classes. The problem is again you have
    to do a resurgery and typically; unlike polymers they do not really allow you to degrade
    the matrix and release the drug over constant time typically the drug is just coated on the
    surface or they are for structural support.
    So, they are not as widely used in the literature at least in the research scale. But then on
    the metal particles there is still quite a lot of applications and because these are small
    particles, they have been used for contrast agents, they have been just the surface coating
    of a drug will also cause enough volume of the drug or enough concentration the drug to
    be developed and so we will talk about metal particles.
    So, in this case you see some of the images and again metal particles; since they form by
    crystallization, it is very easy to get different shapes of particles in very large quantities
    using bottom up approaches and so essentially the same concepts apply for metal
    particles. So, you can have some of the ones that have widely used a silver and gold;

    gold again is one of the most widely used - you also have iron oxide, both of these are
    being used as contrast agents.
    You have quantum dots which is used quite a lot with imaging, mostly fluorescence
    imaging. The quantum dot also has some limitations because some of the metals that are
    used could be toxic, but then the field is evolved enough that they have been able to sort
    of make sure that; these are non-toxic at least for the time being that they are required
    So, one of the advantages with the all these particles is their optical response is fairly
    tunable. So, you can get different types of optical response based on the size in the shape.
    So, for gold particles for example, if you have a rod shape particle versus a spherical
    particle; you will find that the rod shaped particle has absorbance in close to about 600 to
    700 nanometer whereas the spherical shape is about 530.
    So, you can essentially tune that and depending on the length of the rod, you can further
    start to tune this. And then as I said, since the surface area now is quite large compared
    to the volume; for at least for the particle scenarios when you get to nano regimes, you
    can still load enough drug for drug delivery as well; so, those are some of the advantages
    with the metal particles.
    One of the disadvantage of course, is non degradable; so this is a disadvantage in most
    cases. If I continuously get an injection of these metal particles, they cannot be cleared
    out from our body because they are big they can degrade. So, what happens? They will
    just accumulate in my body in over time they might reach toxic levels.

    (Refer Slide Time: 22:33)

    And in terms of the synthesis first of all the synthesis results in extremely mono
    dispersed particles. I mean we are talking about maybe variation of let us say 1
    nanometer in each dimension at max.
    So, in that ways they are extremely monodisperse and good for research application. And
    again like the polymeric particles we talked about when you are talking about
    synthesizing them in different shapes and different sizes; they are well established
    methods. You can start from salts, you can reduce them down either by a chemical
    reduction or some other method to an individual atom which will then start clustering.
    And you can grow these clusters up to the size range that you want and people have
    shown this for all kinds of sizes, all the way down from 1 nanometer to micron levels.
    So, that is not a problem; you can take a bulk metal, you can do some physical methods
    or top down approaches, you can use laser ablation, you can grind it, you can mill it, you
    can reduce it further down to whatever levels you want. And so both these methods are
    well accepted and well used in the field. So, top down approaches, as I said, will include
    grinding and milling; in bottom up approaches typically requires chemical reduction.

    (Refer Slide Time: 23:52)

    And then their uses, especially gold is very widely used for contrast agents and for
    fluorescence. So, the spherical nano particles depending on the size range they will have
    different colors and different absorbance. And then similarly nano-rods will also have
    different aspect ratios, which is the ratio of length to width. So, if this is 1 nanometer and
    let us say this is 4 nanometer and then aspect ratio is nothing, but 4 by 1 which is 4. So,
    depending on the aspect ratio you will get different fluorescence and absorbance for the
    nanorods also. So, they have been used for various applications, they have been used for
    photo thermal therapy; so these things will absorb light and will actually heat up.
    So, this will heat up and the local temperature around these particles will increase to very
    high levels - up to let us say 60 degree Celsius, 70 degree Celsius and these can be then
    used to kill whatever cells are in the surrounding. So, you can imagine a scenario where
    these metal particles are accumulating; let us say in a tumor tissue. And then you
    externally give them some light; let us say it is a skin cancer and it is just topically
    applied particles, then you can just give them some light which will cause the disruption
    of the cancer cells; the death of the cancer cells because of the local heating of these
    particles and so that is one way.
    And they have been used for X-ray imaging because of course, they are non-transparent
    to X-rays. So, wherever they are accumulating they will give a lot more contrast at that
    region and they have been used in sensors quite a lot; photodynamic therapy as well as

    drug delivery. So, you can conjugate drug on the surfaces and release that out over time
    and to get enough concentration of the drug.
    (Refer Slide Time: 25:53)

    Another concept if we are going to talk about this particle hitchhiking. So, this is nothing
    to do with the particle property, but this is something that people are using to sort of
    make sure the particles are circulating for longer.
    So, what is done here? That you can actually if the particles are just flowing alone in the
    blood vessel. So, let us say if this is a blood vessel and this is your particle just sort of
    flowing alone; what can happen is any immune cell can come and sort of engulf this. But
    what happens if I conjugate the particle to let us say something which the body considers
    to be “self” - Let us say the body’s own cell, let us say the RBCs. So, what will happen
    is now the immune cells are not going to attack the RBCs because they think that this is
    one of their own and your particles can then circulate and eventually it is going to
    degrade to release whatever drug they are carrying.
    So, RBCs and immune cells themselves a very attractive target, various methods are used
    such as adsorption or chemical conjugation to these. Although we need to be careful not
    to affect the function of the host cell itself. So, here is an example; here you see that they
    had these green particles which they have then adsorbed onto the RBC. And then these
    have been shown because of this adsorption; they can circulate in the body for lot longer
    than let us say the individual particles them self.

    So, what you can do is you can isolate some blood, incubate your particles let them
    adsorb onto the RBCs. And then you can just infuse it back into the patient and these
    particles will then continue to circulate as long as that RBC circulating or they degrade.
    So, we will stop here; in the next class we will talk about protein adsorption.
    Thank you.