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Particle Synthesis: Solvent Evaporation

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Videos:

Hello everyone! welcome to another lecture for Drug Delivery Engineering and
Principles. We are now talking about Micro and Nano Particles.
(Refer Slide Time: 00:35)

So, just a quick recap of what we did in the last class. In the last class we introduced
what are micro nano particles. So, these are essentially the miniaturized versions of your
matrix devices. So, fairly small, anything less than 100 micron, its micro particle and if
you are talking about nano particle we are saying even less than 1 micron.
We talked about what are the advantages over macro devices. So, several of them we
talked about first is of course, that you do not have to do any surgery now, you can just
directly inject it, you can get more targeting, you can get more leverage with them. So, if
you want to inject it in a sensitive organs, you can find the space, and won’t have to do
that surgery. Patient compliance is very high and then several other things we talked
about.

And then we discussed how they interact with kidneys. So, kidneys again when on the
major organ that decides what is the residence time of these particles and so, first we
looked at what size range is these particles flow through kidney and which don’t. So,
first thing we talk about size and what we found that anything which is greater than 6
nanometer, will not be filtered just because the GBM, the glomerular membrane, the
basement membrane is not going to let it pass through.
Then anything between 1 to 6 nanometer will pass through quite rapidly because the
basement membrane is fairly permeable and 1nm is going to pass faster through the 6nm
and then there is really no interaction. But once you go below one nanometer what
happens is the glycocalyx of the cells in the surrounding will start interacting with this
and eventually this will prevent the faster clearance, they will start interacting and we
will have a much more tortuous route through this glycocalyx. So, these were some of
the things we discuss in respect to kidney.
Then we talked about charge, so this again this GBM is negatively charged and so that
means that if there is a positively charge particle it is strongly attracted towards it and so,
the positive charge particles get cleared faster and the more dynamic the particle the less
is that clearance through the kidney.
(Refer Slide Time: 03:09)

So, let us continue and let us define some terms for particle mediated delivery. And so,
the first thing is particle, size and shape - these are really nothing but defining what is

the size in the shape of them; so how big they are, what sort of morphology they are,
whether they are spherical, whether they are rod shaped, whether the disc shapes and
whatever shape they might be. And of course, there are several techniques that you can
use to sort of get an estimate of this. So, you can either get a some sort of a qualitative
estimate - you can use microscopy we have now very powerful microscopes that can
image even 10 nanometers and lower.
So, and you can get down to even electron microscopes that can now even go down to
single digit size ranges. And then you can do this quantitatively, so there are several
techniques - coulter counting or light scattering techniques and that will scatter light and
you can get an estimate of what is the size that is scattering it. You can get a distribution
of what sort of diameters are present in the whole population, maybe they are not all
particles are the similarly sized and all of these can be used to get an estimate of the
particle, size and shape.
Another is polydispersity, so it links to this average distribution. And what it is, is unlike
these macro devices where you have quite a bit of control as to what is the size of the
device you are making - if making 1 millimeter you typically get it within plus minus 0.1

millimeter. But with these micro devices and nano devices that is not the case because-
and we will discuss about why this polydispersity- but then there is quite a bit of

polydispersity that you may find in different samples.
And so to define that we have this term where polydispersity that basically calculates the
size distribution. So, whether how many particles are there with, let us say, 1 micron,
how many particles are there with 500 nanometer and all this will define what
polydispersity is. And essentially it is just a measure of how broad or narrow the size
distribution is.
And then the carrier composition. So, first of all what is the polymer that you were using,
what amount of polymer is that you were using. So, how much percentage is polymer,
how much percentage is drug, how much percentage is other components like solvents,
and surfactants (if they are there) if there is any other additives that you are adding. So,
all of that is present in that case.

(Refer Slide Time: 05:52)

So, some more definitions. Next thing is the encapsulation efficiency and that comes in
when we are sort of talking about drug itself. So, what is it is how much of the drug that I
started with I was able to encapsulate in my polymer. So, let us say if I want to make
particles and I want 1 milligram of drug to be encapsulated in these particles. So, I make
these particles and we will discuss about various synthesis methods.
So, let us say the particle synthesis is done and at the end of it I end up with 100
milligram particles. Of course, this 100 milligram is essentially and the weight of the
polymer plus the drug and then what I do is I then dissolve this whole 100 milligram of
particle and see how much drug was actually there and I found that instead of one
milligram I could only get about let us say 800 micrograms drug in the 100 milligram,
so; that means, I have lost about 200 milligram of drug during the synthesis process.
So, my encapsulation efficiency is going to be 0.8 milligram divided by the initial drug
which is 1 milligram multiplied by 100 to get a percentage and that is nothing but
essentially 80 percent. So, in this case my encapsulation efficiency is 80 percent, but this
is how it is going to be typically defined.
What about loading level encapsulation ratio? So, this is another way to define to get an
estimate of how much drug is present. And what it is just the weight percentage of the
drug in the particle formulation. If I just take the last example and I said I had 100
milligram polymer (particle) and that was containing about 800 microgram of drug. So,

in this case now loading level is nothing, but 0.8 milligram divided by 100 milligram and
so, my loading level is actually less than 1 percent in this case. So, that is how it is sort of
defined. If I multiply this by 100, I am going to get 80 by 100, so my loading level is
only 0.8 percent. So, that is how it is typically defined.
And then we have stability. So, whether how stable the particle is, whether I can store it
for longer durations or not I mean this may had nothing to do with the drug itself or it is
a combination of drug in the polymer stability, but maybe my polymer itself does not
really remain stable. So, the drug will obviously, come out.
So, essentially chemical stability refers to as disabled the drugs inside the particle over
time. So, what environment you are storing it in, what are the different conditions and
the physical stability is what if the particles are degrading, they are absorbing water or
moisture from the air and then causing them to degrade and erode over time even before
its put in the body. So, that becomes important in terms of determining the shelf life and
efficacy of these particles.
(Refer Slide Time: 09:22)

So, briefly I had mentioned in the previous class that these particles can also be used for
intracellular delivery. And so, what do we mean by that? So, let us say this is a cell that
we have a picture of. And so, cells itself have sort of evolved different pathways through
which they can take up a external material- this could be glucose or any sort of energy
that they need or could be food in some pathogenic sort of cells. So, the several of the

ways through which they take large molecules like particles and this could be
phagocytosis, mostly present in immune cells.
And then best of the other other pathways that are defined here these are shown by
nearly all types of cells. So, this could be macropinocytosis which is nothing but the
membrane ruffling, so the membrane will ruffle and just sort of eat up whatever is in the
surrounding, it could be is a mediated by some sort of proteins present on the cell
membrane. So, this could be a clathrin protein and so, they form small pits all this could
be caveolae proteins they also form small pits, but they’re different proteins. And then
there are some other pathways that are not really well known and they are being sort of
clubbed into clathrin and caveolae independent endocytosis.
So, all of this will result in some sort of a vesicle being formed, containing particles that
are membrane bound and depending on what sort of particles you are using, what cells it
is, what stage this is being administered, these particles can escape through these vessels
and cause intracellular delivery especially in the cytoplasm. Or these vesicles themselves
can then be targeted different organs, they can be targeted to mitochondria, they can be
targeted to nucleus, they can be targeted some other organ or they might just be
transcytosed. So, let us say if I want to cross a barrier with cells on it and if the particles
do transcytosis, it means, that this cell is going to take up this particle and essentially just
throw it out on the other side. So, all of this is fairly feasible.
So, now, if these particles are degradable and are carrying a drug that is extremely
hydrophilic and would not have been able to diffuse through the cell membrane, now this
drug can actually do that. Because, now this drug is in these particles which get taken up
through these specialized uptake pathways and now it is in the cell where it can get
released. So, this is what we meant by intracellular delivery.

(Refer Slide Time: 12:01)

And then, since I mentioned here that these particles can actually escape from these
vesicles. What is the mechanism through which they can escape from these vesicles? So,
that is called a proton sponge effect - at least one of the ways that we can enhance this is
using a proton sponge effect and which is when particles that are taken through
endocytosis or phagocytosis.
They typically get localized and are entrapped in these endosomes and lysosomes which
is a machinery for the cell to degrade any kind of external particles or nutrients that it has
taken up, and these environments are actually very harsh, they have very low pH and lots
and lots of degradative enzymes are there.
So, if your drug is getting released there, unless you want to target those locations, you
don’t want the drug to come out because these are not conducive for the drug action and
it may it may even destroy the drug. So, what is done is to help the particle escape effect
which is called proton sponge effect is used. And so, what is proton sponge effect?

(Refer Slide Time: 13:09)

So, these particles are designed such that they carry lots of secondary and tertiary
amines. So, let us say if I have a polymer that carries lots of primary, secondary and
tertiary amines. What is going to happen is now these amines have quite a lot of capacity
to take up H plus ions (protons) and as I just mentioned that there are endosomes and
phagosome. So, this is a cell and here is my endosome.
So, the pH outside and the inside the cell is close to around 7, but the pH of this is now
maintained at around 5 and it further decreases at it as it goes. So, these are early
endosomes and when these mature, they turn into lysosomes, where the pH can drop
down to 2 to 3. So, for the facilitation of this process to happen from here the pH is 7
goes to 5 goes to 2 to 3 the way the cell does this is it has lots of proton pumps.
So, what it does? It takes up H plus ions or protons and pumps it in to the system. And
obviously, there has to be some sort of osmotic balance otherwise is these vesicles would
not be able to last, more and more water will also go in. So, now what is happening is
now I have put this polymer. If I zoom into one of these endosomes, so I have lots and
lots of primary and tertiary amines that are present, they are taking up this H plus ion.
So, they are taking up this H plus ions and not letting the pH drop. So, the pH is still let
us say 6 at this place.
Now, the cell does not like that, so it is pumping more H plus ions into it and this
continues till either you hit the saturation of your tertiary and primary amines or tertiary

second amines and if we assume that there are so many of them that they will not hit that
that quickly. Then what will happen is this H plus will continue to pump in. Because of
this now there is too much ions in your vesicles then there are outside. So, ions in here
are greater than the ions outside and so, that causes an osmotic imbalance.
So, now there is an osmotic imbalance, as a result of which the water from the
surrounding starts to go in and starts to maintain this osmotic balance and as it goes in
there is a some sort of capacity to which it can absorb the water, but eventually the
pressure inside becomes so high that these vesicles just burst. So, once they burst in
whatever particles are residing here, they come out and they are now in the cytoplasm do
not have to go through this harsh environment of 2 to 3 pH and that is essentially it is a
proton sponge effect. So, I hope this is clear.
So, H plus is going in to maintain the charge, chloride ion also goes in. Now, you have
lots and lots of H plus and chloride ions that are present in your system and because of
that there are lot more ions in your vesicles containing these particles and which then
release which then causes the imbalance in the osmosis, and the water goes in to
maintain that balance, and these vesicles swell, and eventually they burst after a certain
pressure is achieved.
So, one of the polymer is very widely used is polyethylene amine. It is a highly
positively charged polymer just because it has lots of tertiary and primary amines and
again very widely used. And then there are other mechanisms you can conjugate some
peptides, this is mostly adopted from a viral strategy. So, some of the viruses what they
do, is they have peptides that go and poke holes into the membrane.
So, these peptides will go and sort of make a hole through which your things can escape
when these vesicles may burst up. So, these are different mechanisms you can use to sort
of utilize this proton sponge effect or these pore creating peptides to come out from your
endosomal membrane.

(Refer Slide Time: 18:22)

Let us talk about some particle synthesis methods. So, first we are going to talk about
chemical methods. These, for polymeric particles, involve some kind of a
polymerization. So, you can take polymer in confined states, and start this chemical
reaction at the as the chemical reaction will proceed more and more of them will cross
link and by the time that action finishes is the end of forming a particle.
But for metal particles reduction, oxidation, crystallization, from salts is used quite a lot,
so you can get you can let us say have gold salt. So, you can have a gold salt and then
you can reduce it or oxidize it to basically return it to unit of state where they will start to
sort of interact with the other gold ions and form a particle in depending on what
concentration of the salt you have used you can vary the size of these particles.

(Refer Slide Time: 19:25)

And then there are lots and lots of physical methods much more widely used these could
be controlled precipitation of the polymer. So, basically you can have an emulsion
process and we will describe this in a little more detail as we go along, but you can have
an emulsion process and you, let us say, form these emulsion droplets with polymer plus
solvent. But this solvent is volatile, so it is it tends to just dry off and once it does this
polymer concentration is going to start increasing as well as this thing is going to shrink
and eventually all this polymer will just precipitate and forms a physical cross linked
network, which will then leads to particle and whatever drug that might be dissolved in
here it just gets entrapped.
And again, as I said we will describe this process in more detail. So, solvent evaporation
is again very similar method here. And there could be other methods that people are
using, you can have a complex coacervation. So, we discussed briefly about this during
ionic hydrogels the same mechanism can also happen at nano scale depending on the
concentrations and ratios or different things that are using. You can actually actively
remove the solvent rather than just relying on it evaporating out their process for hot
melt, spray drying, you can phase separate things out and that may result in formation of
nano and micro particles or you can use salt to sort of induce this sort of separation or
precipitation of the polymers.

(Refer Slide Time: 21:06)

So, let us talk about the solvent evaporation method one of the very widely used method.
So, first we are going to talk about single emulsion process. And so, what is typically
done is this contains two phases you have equal phase and organic phase, and in equal
phase you can have a distilled water with surfactant, in the organic phase you can have
some chlorinated solvents or maybe something else which also contains a polymer,
depending on whether your drug is hydrophilic or hydrophobic you choose these
methods in this case it is mostly used for drug that is hydrophobic and I will describe
why.
So, you add your drug into your organic phase because if it is a hydrophobic drug it is
only going to get solubilized into the organic phase and then you emulsify it and
emulsify just means you mix them and give it some energy. So, when you give that this
organic phase does not want to interact with the aqueous phase at all. So, what did we do
it will first if you do not give any energy, you will have them phase separate like this.
Where this is your equal phase or organic phase or this could be vice versa depending on
which one is heavier, and they will just separate out. But when I constantly give it energy
and force it to mix, they will mix, but they will mix very reluctantly. So, what will
happen is even though they have mixed these two phases will not want to interact with
each other.

So, what will happen is depending on which amount is more that will act as a bulk layer
and rest of it just is going to make these micro and nano phase separation, which is to
basically prevent bulk of the organics solvent from interacting with the aqueous solvent.
And so, the more energy I give, the smaller these droplet us will become, and that is how
they will sort of phase separate out.
And now let us say this organic solvent is volatile or as evaporates at a very rapid pace.
So, once this evaporates, what will happen is first of all these droplet us will shrink and
then eventually whatever the polymer is there will exceed the solubility limit because the
solvent is constantly evaporating out and that will eventually cause this physical
precipitation of these polymer molecules to result in nano or the micro spheres.
So, let us see, so you one of the examples that is very widely used for this type of process
is PLGA or PLA micro particles they are again fairly hydrophobic. So, if you are using
PLGA it has to go into the organic phase, one of the organic phase that is very used
either chloroform or DCM (dichloromethane).
So, this process is again referred to as oil-in-water and the reason for the oil in water is
because if the oil is in lower amount than this water, so essentially this oil droplets are in
water. So, it is very famously known as oil-in-water or oil-in-water emulsion. This could
also be oil-in-oil depending on what external phase you are using. So, you may decide to
use instead of aqueous phase you may decide to use another oil, but this oil is immiscible
with the other oil. So, in that case should be oil-oil, but again it is typically not used for
the applications related to the body because we always want whatever particles that are
made be able to interact with the water and so, for that one of the phase is typically
aqueous.
So, as I said, if I zoom into these small droplet us that are evaporating solvent what you
are essentially having is these polymer chains, when the size is decreased, these polymer
chains are coming closer and closer and then eventually they are just, there is no solvent,
all you have is this polymer chains and so, these will essentially represent a solid matrix.
There is no sort of capsule or hollow particles here, these are all solid matrix that are
formed. So, this process will result in a matrix type particle, not a hollow capsule.

The drug must be soluble and dispersible in the organic solvent phase that is why I said it
is used for hydrophobic drugs. If the drug is not soluble here then it cannot really go in,
only the drug is soluble here, it will also be present in these droplet us that are here.
(Refer Slide Time: 26:08)

And then the polymer is typically dissolved in some volatile organic solvent. So, as I said
one of the most commonly uses a methylene chloride or DCM. Other solvents like
chloroform and the ethyl acetate are also used, but they have to be volatile. I mean if they
are not volatile, then this process will take forever for the to evaporate and they had to be
more volatile than water because you do not want the water to evaporate first.
The polymer drug solution dispersion is emulsified again in large volume. So, as I said
this is going to be in excess whereas, this is going to be limited and then typically some
sort of a surfactant is also added. So, what the surfactant does is it just localizes itself at
the edge of these particles because these are surfactants have domains that want to
interact with the aqueous phase they have domains which want to interact with the
organic phase.
So, they sort of stabilize once these particles are formed. So, you do not have to
continuously give the energy during the evaporation phase. You can just give the energy
once for a certain amount of time and when these droplet have stabilized due to the
presence of these surfactants like PVA, you can then leave it and do not have to continue
to give energy to this.

And then this can be stirred under reduced pressure and elevated temperature, if you
want to increase this evaporation rate or you can do it at the room temperature and
normal pressure as well to let these particles harden. So, we will stop here and we will
continue more in the next class.
Thank you.