Thank you so can you all hear me.

OK good idea I don't know if this
Mike is supposed to be functional for

the audience as well OK thank you so much.

Much better I can see you you can see me.

It's an honor for me to be back here
I think about eleven years ago I

was standing at this stage
presenting as well and so

it's been an enjoyable time.

On campus today and meeting old and new
people and by old I don't mean old by age.

I'm not trying to say folks are old so
it's a pleasure for me to be here and

I thank you all for taking time to hear
some of the things we've been up to

over the last.

Sort of eight years that I've been at the
University of Michigan So again we're in

chemical engineering at Michigan and we're
interested in understanding how we can

look at the design of delivery vehicles
from the lens of chemical engineering and

in terms of how we can improve the
functionality of those delivery system.

For eventual translation into the clinics
I think when we started off working

in this area it wasn't clear to us which
piece of the policy was important to

to follow on our Until
we started to again.

Piece out the one layer at a time so

I'd always like to start in case there
isn't somebody in the audience that might

not have a good sense of the concept of
vascular targeted deliberate targeting

which is essentially has been the focus
of my life for the last eight years it's

essentially the idea of target
in Turkey to specific location

within the body by utilizing information
that we have of how proteins

are expressed on the vascular wall in
association with those disease model and

a lot of what we know about vascular
targeting we learn from the with

the interaction of the site with the end
of the all cells that lined the blood

vessel walls in many of this tissue and
it often involves the use of

some sort of carrier vehicle that
either look at Baseball America based.

All sorts of innovation has
occurred in the last decade

in terms of what the carrier
needs to look like and

in terms of the target in has a lot to
do with the understanding of the biology

of the disease model that you're
trying to go after and there's again

lots of work that's been done over
several decades in that area as well and

the idea of the again localized in therapy
is important because many of the diseases

that because there's still a challenge
towards a disease is that we need and

Tara puta X. to go after and so
localizing is important because it allows

us to use a high concentration
without necessarily exposing healthy

cells to those toxic interaction and so
we've talked about dark vascular targeting

in many important diseases cancer is going
to be the one that most people would have

experience but
there are other serious diseases.

Well in particular cardiovascular
diseases in which has been.

A central focus of the lab so
when we started this are pro-choice to ask

the question what are going to be
the critical piece in terms of design of

a delivery vehicle that's going to
optimize or have a high efficiency for

localized in to the vascular wall in the
in the tissue again the idea is localized

targeted delivery which means you want
to carry a system to be able to find

the specific location in a high level and
only.

Concentrate there so that you avoid
systemic delivery of the system so

again that's sort of talked about this
is a physical component there has

to be a carrier there's a range that
you can look at depending on the type

of therapeutic that you want to deliver
whether it's harder for big hurdle Felix

and things of that nature and
then you have to figure out the size and

size is going to be important in terms of
the drug payload that you want to deliver

is it important to have
a bogus delivery or

is it a control release
that's going to be used for

the particular disease again there's a lot
of work out here and we have a good sense

of how things would work once we have
a good understanding of the disease model

that we're going after size is
particularly interesting because when it's

vascular targeted that means the primary
route of entry into the body is going

to be through the vasculature of some sort
of injection into the bloodstream and so

then if you're going to circulate
the through the blood stream size becomes.

An important component because you have
to think about the size of the smaller

vessels within your body and
the idea there is that you don't want to

delivery vehicle that's going to interrupt
or disrupt the continuous flow of blood so

we tend to think about
nano sized particles for

that reason and anything smaller
than five and animators all the way

down to fifty nine meters is what you'll
typically hear about when we're talking

about to target a drug delivery system so
size constraint imposed by biology.

G. comes into play there targeting is

something we all think
about something we've.

There's a lot of work out there whether
the protein you want to go after I think

the literature now is more interested in
multiple targeting because it gives you

ability to improve your specificity going
after more to pull versus one protein

and then the third component for vascular
target in is the dynamic environment and

the complexity of the environment
that this particles have to play in

Once you inject them in and I think
this is this snapshot here of an S.T.M.

of a drop of blood was really what
nucleated or initiated the paths that we

took early on with our work understanding
that the environment that the particles

have to play in is a very dynamic and
high force harsh environment and

as the nanoparticle which I think
moved the block of us think about for

vascular targeting we have to ask
the question how are particle that is

very small and less concentrated that the
cells that are within the bloodstream How

is it going to compete in terms of finding
its way to the blood vessel wall for

interaction and this is also
complicated by the fact that the cells

that the particles have to encounter are.

Different in terms of
their physical shape or

size and membrane flexibility
the physics of the event so

it's very different from the physics
of the white cell and so

the particle has to navigate all of
this to find its way to the wall so

what we know in terms of blood
flow is that because of the.

Sheer number of the Red Cell the shape and
the different mobility the membrane

characteristic of the red cells when the
blood is flowing they tend to experience

a lift that pushes them to localize
in the poor of the flow and

you end up with layers here it's not shown
very well here I think I have pictures for

that down the present ation exaggerate
this what we call the self it's a sign.

A layer that could be between two and

seven Micron from the wall of the vessel
where you don't see red cells because

the red cells are again depend on the
force of flow tend to move in the core and

so this works in nature because the white
cells then are predisposition to be.

Localized at the war because
as the red cells come together

they deflect the collision and
deflect the white cells to the wall and

the platelets the wall as well so for drug
delivery it's going to be important that

our particles are also deflected to the
wall because you do want them to be able

to bind in many cases you're targeting
the vascular Walsall itself and

in other cases you want those particles
to find their way through junction or

interstellar interaction to get into the
tissue space but the first step is that

particle finding its way to the wall and
so the question we began to have

early on was what are the probability
that a nanoparticle can compete well with

this other cells to find its way to
the wall under this sort of high shear and

complex environment of blood flow and so
we started on early asking the question.

Then we looked at the disease models that
we people were typically interested in

going after we were interested in
atherosclerosis because it was the premier

cardiovascular disease that has high
incidence of mortality a lot of.

Clinical dollars go into maintaining and
treating and preventing

mortality in this disease the one thing
that very interesting with actress Gross's

is one of the few diseases that actually
affects the blood vessel itself so

there's a high opportunity for
vascular target in in this case because in

many situation depend on what part of this
disease you're targeting your particles

don't have to move much from outside
the bloodstream so of course this

occurs in typically in medium and large
ordinary So you're going to have borked.

Blood flow high pressure
highly personnel flow and

we reckon that that characteristic
of this of of this is these will

should have an impact in how you think
about designing nano carriers for

targeting that if you contrast that to
cancer which is again another important

disease model that we're still trying
to evolve better targeted tapi for

their issue and

the relevant circulatory system words are
going to be in the micro circulation be.

The news.

And more importantly
the capital raise themselves so

their vessels are much smaller and the
flow is more steady so the question is.

Can you use a one size fits
all design to have effective

localization of therapy into
this two drastically different.

Environment within the bloodstream and

so that big form the foundation of the
initial questions we asked about design of

dirty resistant the first question
is how does size matter I have.

A sterile particle which is
typical of what we think about for

vascular targeted delivery system
simply because before the invention

of many of the new fabrication techniques
that we have here the bulk of what we can

make successfully for
drug delivery were spherical particles.

Or.

Matrix based polymer system and so
the question then is the size of matter

in terms of how a particle a spherical
particle can navigate the bloodstream to

find blood vessel wall where the chemistry
that you've put on the surface of

the carrier can connect with the chemistry
that is expressed at the wall

interaction and our central premise here
is that it doesn't matter how beautiful

the chemistry that you've decorated and
your carrier if it's not able to come

close enough to the wall for the reaction
to occur then it's going to be.

Useless exercise and so
our model system is essentially

using microfluidics of different geometry
height and things of that nature and

work with it working with human blood
early on we wanted to keep things simple

because red cells make up ninety nine
percent of the cells in blood we

figured that that was going to be a good
place to start in terms of how a particle

can find is way through the tiny
torturous part that would exist between

red cells to get to the wall so the block
of the data I'm going to show earlier in

my talk is going to focus on work where
we took thirty to forty percent and

mostly thirty percent human red cells
in human plasma and expose in particles

that are targeted a lot of the work we did
early on was using polystyrene as a model

delivery system because we were interested
in size effect alone and we put enough

targeting light again on the particles so
that they can exist in what we call.

Transport limited Rejean which means that
if a particle can find it the way to

the wall reaction will not be a problem it
will bind so in those transport limited

regime which we can then access which
particle has a higher capacity to

be transported to the wall is not and
we can look at flow models and

I like to show this video just to give
you a sense of that self in there that I

talked about as the video starts you see
the red cells are moving and they're close

relatively close to the wall and this
particles that are bound to the wall and

our wall will have in that if your cells
you can see that the video is Clara Now

what happened there is the speed of
flow got much faster which tends to push

the red cells more into the core flow so
that the lifting away from the surface So

this highlights the our ability to create
that cell free layer even in our model

system so the question now is how does
size matter in terms of a particle.

Binding from blood flow particles of
different sizes are fed in the same

concentrate.

And then we assessed
the adhesion to the wall so

in here we looked at channel that is about
two hundred fifty microns in height and

this would be a relevant size for large
arterials are going to eventually branch

into the capillaries it's still relatively
so this would be say a medium size.

Vessel and what we see here and it's not.

Impactful in this data that I'm showing
when I look at the number of particles

that are bound to the surface relative
to the size going five five on and

on a meter is all the way to ten
micron we see that here the two

micron particles sort of intermediated.

Right here are a thousand and
three seconds which.

It's trying to get me to
connect to the Internet.

OK.

So as you can see here for
most of this year it

you look at the two micron in the five
Mike and particle seems to have a higher

level of binding ninety five an animator
particle The other interesting component

is when you increase the shear rate which
of course increase the speed of flow of

blood flow through the channel you see
that the micro particles are responding

with an increase in binding which
may be counter intuitive for

this audience because when we think about
flow and speed of flow we think about

force So what we've done is increase
the force in the channel yet

we're seeing particles perform better and
this has to do with this red cell core

that I talked about where actually the red
cells in the core provide some sort

of protection and a downward force that's
pushing particles towards the wall so that

there's not this particles are actually
not feeling that increase in force and

so that they can respond still with
the same level of binding efficiency but

you don't see that for
the smaller particles which we again.

Yes that there has to be something in
terms of the air transport but I think

the difference between the Nano and sort
of micro particle becomes more obvious

when we go down to a smaller channel
this is a twenty five micron channel so

this guys would be the last set of or
true before you start going into

the capillaries and there you see
an even more exaggerate difference and

we went into a smaller size
here two hundred nanometers and

this would be about the size that you see
more of what you look at drug areas for

vascular targeting and so
we see here that this two guys don't

a bind at a high level even though they
were fed into the bloodstream in the same

concentration as the larger particles
added to my comments against what seems to

be the superior performance difference
here eight we looked at the five micron

is not performing as well because we think
that in a smaller channel now the scale

of the diameter of the particle is very
close to the skill of the channel so

that there is on our voidable
collision with red cells that seems to

be working in a negative
way against a heater so

we can fix this problem by simply putting
more targeting light again on the five

micro particles we can restore their
huge and because now they have enough

reaction force to counteract
the collision forces and

shear forces that they experience when
we put more target enlargen on this guy

we don't see any kind of response in
terms of increasing the teacher so

that again starts to highlight to us
that this is not a reaction problem but

a transport problem so
what is happening to this particles where

are they going when we have fed them
in the same right concentration

the other interesting
piece about this because

usually when I talk about this people go
Well this channels are not capillaries and

when you're thinking about cancer
targeting and you're interested in

capillaries and it's not clear that this
dynamic is going to be the same because

in the capillaries you tend to see
the red cells lined up in single file.

Through the channel and so what I
often point out is that when you look

at a map of vessels in this
is breast cancer tissue for

example you see that a the ball of
the branch in a vessel is happening

just at the sides of vessels are branching
from the side of a bigger vessel so

what that means is that the fluid
is going to be essentially grabbed

from the wall side of
the vessel rather than.

So it's possible that if something is not
coming down to the wall that it's not

going to split into the capillary in the
in the amount that you anticipate it to so

we think that this could actually
propagate down into the capillaries even

though we're not looking at a channel
that has a cavalry size we've looked at

different flow parents in this or
exaggerated possible flow.

Forward forward and forward backwards and
we see that the doesn't change

the dynamics of the smaller particles
relative to the micro particle if anything

we just get more of the particles
binding than the nano sized particles.

In the same way we were interested
in flow that sort of research

because the point where you tend
to see accumulation of plaque in

this is areas where you have sudden
expansion of flow that creates eddies and.

You have high I.Q. Malaysian or
white blood cells here which is

an important component of generating those
plaque and so we wanted to see whether

this kind of the third flow maybe even the
playing field for all the particles and

more importantly do we see more of
the natural size particles doing better

in this situation so we can recreate a
model of this type of research in flow and

when we looked at the again we
find that if anything the five

micron particle is seeing a resurgence
of life here which is actually due to

the fact that there's enough evidence that
in this study because of the different.

Red cells in their size they
get trapped in the eddies So so

you get a low work concentration of red So
moving forward and here where you have

reattachment you use this line and you
see high performance of the five micron

because now they don't have to deal with
the red cells getting in their way but

again this is where we're interested in
and this float does not seem to again

impact the ability of these nano
particles to bind which we again think

has to be limits related to
the transportation to the wall and

so again the question that we would
often get at this point is well is it

the number of particles that's down that
matters or is it the volume of drugs

that you can deliver more importantly
you often hear just inject more and

you get more to the surface and
so we wanted to see whether

if we boost the concentration of this nano
particles do we actually see an equal

response in adhesion to the wall and so
we did an experiment where we increase

the concentration of the polystyrene
particles by five times and then we assess

a huge amount to see if we get a five
times response in that he shot it turns

out that the response is
actually less than five times for

particles that are less than
one micron as you see here but

when you go to one hundred five Micro
you tend to see the responses at least

linear with concentration and so
again this is telling us something is

happening in terms of the ability of these
particles to get to the wall and we know

this has to do with the blood environment
because if we repeat the analysis

in a buffer system we see that
these guys have the ability to

find the wall in the absence of blood.

As equal as dear micro micron
size counterpart and so

that led us to basically
take our system into.

A ditch.
Environment is trying to see if we can

image this this interaction so
again we think.

Blood is flowing in red cells tend to
move in the core that is possible that

in that process of moving to the core
of the essentially and trap the smaller

particles within those gap because
remember red cells are dimpled and so when

to read come together there's still going
to be space in between them and is it that

the space is just big enough for smaller
sized particle to sit comfortably and

where as the larger particles are squeezed
out and sent to the wall and so

we get in we set up our system we're
trying to conform microscope and

what we think will be in the self really
region to see whether we see the same

number of micro particle relative to
nano particles have been fed in the same

concentration and a deed when we look we
find more to Micron close to the wall

indicating that the localize very well
whereas we barely see the five running out

of mirror particle in this case so
of confirming this idea that.

The particles when you feed them in
the you tend to feed them into the core of

the red cells and only a few of them are
able to make it down to the wall OK And

we've gone in vivo and
injected particles of the of those

to event sizes in here we looked at again
the five hundred ninety two my God And

we tend to see more of them are bound to
the wall of mouse that has other Gross's

in their arteries compared I'm sorry
they're more of the two microns found at

the wall after thirty minutes circulation
compared to the five in an animator So

we know that this can this phenomena
that we're seeing in vitro does exist

in the even the complex environment of the
in vivo and I'll come back to this some

of this in vivo data later on to tell you
about some of the things we're looking at

in how our human blood
differs from animal blood OK.

So the smallest particle and it seems to
be one micron is this court of where we

tend to see a diminished ability to
bind to the wall from blood flow and

so the question is how do we enhance this
interaction because I started this talk

by making a case for why we want to think
about nano sized particle instead of micro

sized particles and so so we started to
look at the literature after some of we

got some of this data to see what
are the things that we can do to enhance

the operation of a smaller particles
nano sized particles I think for

long people have always think about
putting peg chains on on particles because

it shields the particle surface from
absorption of protein and that then yields

longer circulation time but we thought
well we know that there are enough.

Land on the peg chain that it actually
distances leg and that you're using

to do and he turned from the surface and
we asked the question can we use that to

increase the distance at which you can
capture a particle while it's in flow and

would that have a downstream
effect of improving at least

capturing more of the part was that you
make it to the wall so we looked at that.

A lot of the work we did was also around
the same time that we had very interesting

exciting work coming out of MIT to go
to a lab and I think this was done with

Julie looking at a long gated particles
and how McAfee's and go off them and

so we asked the question could
non-spherical particle also be

an important component here more
importantly if I have wrong is that a l

a gated could I disrupt the red silk or
another for them to at least escape and

I know that once they're at
the wall based on a lot of models.

Competition are worth I was out there that
once the Along get a particle was out

think I'd better try and
turn off my wife I hear.

Noise will keep having this.

If I can find my wife I.

See.

Or.

OK OK you.

OK And we're going.

OK so.

This was around the time where all
this competition it was coming out so

we thought maybe shape might be
a way to go with this as well so

the first component was we'll peg
chains on the surface help we

grafted the small of the five an animator
particle in this case with a high density

of Peg twenty thousand sites for Micron
Square which should put them in this

extended form and
we looked at Peg sized high as ten K.

Da which should give us a considerable
land and what we found there

was that had been pegged on the particle
did not seem to have an impact in

the number of particles there were bound
which further sort of solidified this

idea that each you with those particles
were transportation to the wall and

not necessarily the reaction component of
the work and so if you were like in system

is working fine pair does not improve the
number of particles that you have out here

and on to the surface what if you change
your life again system so this was.

A sugar that binds to select and that
expressed on and of yourselves when they

are activated and that like again receptor
system is very efficient at capturing

in high shear environment where you switch
to an antibody that has a slow reaction

you're not able to capture asked fast
enough then you start to see a slight

benefit of having peg but that level of
binding in general is not as good as.

The efficient like in Target in system.

But you do find and negative impact
of Paris potentially if you are not

high enough in your pack in the city
that you end up in sort of this mushroom

configuration that you could actually have
a negative impact on I'd hegemon in terms

of hiding the lie again so here you
could see that when you have no peg or

you have extended is about the saying but
if you're not putting enough peg on and

you end up in this transition region you
could actually have a decrease in your

adhesion which we thought was important
because people were designing delivery

vehicles in them that there needs to be an
understanding that you can't just put peg

on you have to make sure you're in this
sort of extend a confirmation Otherwise

you might actually be hiding some of your
target in lie again which tends to be on

the other free end of the peg so that's
one thing we learned in this data is for

the two micron by the way again for
the nano particles you tend to not see

improvement with peg so
we moved on to this idea of.

Surgical particles and whether that
was going to be of benefit and

the rods shape we've looked at this as
well but the Russian made the most sense

in terms of the probability of
disrupting the Red Cell core So

we looked at again Nano and
micro size particles and we find that.

The shape improves when
you have a micron sized

volume So basically the volume of the rod
is equivalent to the volume of a two

microns fears the rot is longer enough
you see and improve in our future but

when you come down to the small of volume
whether you are highly along it does not

seem to have an impact and
indeed this high performance of this super

micro Rod has less to do with the rod
coming out of the rectal core but

more to do with the improved surface
area contact with the wall so

you have what he savant to
be informed when we looked.

Take a microscope to look at localization
you could see that the localization is not

different between this rod and this right
in terms of being at the wall and so

that this increase adhesion has everything
to do with that increase chemistry

better affinity for the wall so

to speak so but again we were
interested in the smaller particles and

been elevated does not seem to
be useful again we go in vivo

to see in the off chance that there might
be differences in the performance and

we see the same exact trend
there's no difference between and.

You see a big boost with micro
Rod relative microspheres and

all of this were targeted interaction
because if there are no target in any of

the particles you see no adhesion So
again the trend we're seeing in vitro

with human blood is at least being
replicated to some extent in vivo and

again we look here and be able to see
whether changing the aspect ratio for

the the micro Rod gives us more and
more and better.

And it turns out that.

You do get to a point in vivo if the rod
is too long you decrease localization

to the law of the order because you
have high entrapment in the lungs so

there is a limit to what you can do
in terms of micro Rod strategy but

we get in we don't see an empowered
with a smaller are when we are.

So going to skip that there we've got to
a point where Peg has not helped us and

shape does not seem to have the impact we
need for the smaller sized particle and

then it occurred to us that a lot of
the work we have been doing where with

polystyrene and polystyrene is density
neutral with blood OK So a lot of

the localization of a poly time particle
to the wall is due to the active duty.

Placement that across ask the Red Cell
move to the core of the flow so

it's an active push into the wall which
is why if we flip our channel upside down

we would still see the same high level of
binding of our particle against gravity

because again the displacement to the wall
where the top or bottom is one hundred

percent mediated by the Red Cell So we
wanted to ask the question what happens if

we increase the density of our
particles will that have an impact.

In terms of binding so
we gain we moved from.

Again taking the five in the middle part
is fair and looking at the polystyrene

which again will be neutral density in
blood and comparing it to silica That's

two times the density and titanium there
will be four times the density and

those particle range were chosen because
they were commercially available in and

they're relatively biocompatible So
again we did this and Alice is in red cell

in writing this actually might
be a red zone buffer experiment.

So in buffer flow you see that the at
each one is lower across the board for

the five and then your particle but you do
see a tendency for binding to increase as

you increase the density of the particle
again this particles were all targeted in

the same way so that the theory the only
difference in fact that you're seeing here

should be the impact of the density of the
particles when we go into blood we see for

the first time a response form
a nanoparticle in terms of its adhesion

to the wall where as you move
to silica you see a significant

increase on the binding of a five hundred
nanometer particle compared to what we

were seeing with the density neutral
polystyrene in fact of level of a huge and

that we see here now for
the first time is competing very

well with the level of a huge and
that we were seeing with to Mark and so

this is where fire and meter used to be
this is now the same level that we see for

this not a particle so going to times
the density seem to be a good thing.

When we increase the density to the
titanium which is four times the density

we don't see many taint or
higher adhesion for this particle and

we think maybe it has to do with
the collision because again a lot of

the filtering of the particles to the wall
which has to be the case where you are in

an inverted channel because by not going
against gravity a lot of those movement

has to do with the collision of this
thread which is packed into the core so

that it's easy or the density of
the titanium sorry the silica is perfect

enough that where you have the collision
with the Red Cell the Red Cell doesn't

move much because it's the form of all
whereas the silica is actively displaced

and it's displacement is bigger than
say this polystyrene where whereas this

guy here it may be that because
of his density the impact with

Vettel is not large enough to displace or
change its trajectory from flow but

we're still trying to sort that out to
confirm that this is has nothing to do

with my turkey or
characteristic of titanium arm to at least

make sure the particles are still there
and when we run the experiment so

again density seems to
be working very well and

we did and now this is where we compared
the level of increase in that heat and

that we get in the presence of red cells
versus the buffer experience that I show

and you can see here that having red
blood cells in an assay actually benefit

the most are silica you see in terms
of looking at localization to the wall

because again we wanted to make sure
it's not the density that's helping in.

These silica in terms of
finding we do see that you

see a decrease localization in polystyrene
which makes sense and fits with the theory

that we had that the smaller polystyrene
particles were staying in the core whereas

when you have the two times density you
see a higher localization to the wall but

again the localization for
titanium seems to be reduced there.

So at the end of the day we again
go in vivo to see if we can

see this impact of density within
the mouth again look in order and

how divine and across the board
depending on the size of the vessel

we're looking at we will see that silica
is performing better than polystyrene and

in some cases you see that titanium
is not performing as well and

if we average across the entire length of
the order we see the same trend that we

saw in vitro where you see the silica
by any other better level than

the Palestinian although the gap here
in vivo does not seem to be scored or

as big as the gap that we saw
in human blood in vitro and so

we've been doing some analysis to try and
understand what the differences

are between blood in a mouse and
blood in human and that might explain some

of this discrepancy in terms of the gap
between the impact of density and

the impact of science there so we're
working now on how we can capitalize on

this new found information about density
obviously Silica is not going to be

the best foreign material for
drug delivery but

how can we make drug delivery vehicles
that have biocompatible biodegradable have

improved density again the bulk of
the polymer that we think about for drugs.

And things of that nature and those will
all be density neutral with blood so

we're working now to see how
we can improve the density and

expand on this potential benefit of
density for the small particles so

again the bulk of the data showed
you is with red blood cells

in plasma and
we tend to be at thirty percent and

we don't have other red white cells or
platelet in our system where we go and

do some of the analysis
we've done in hope we see.

A subtle differences right between
the two so a whole blood and

human red cell in plasma you see that
the gap between the two micron and.

The meter particle seems to be bigger

when we're in whole blood
relative to what we were seeing.

In.

The sort of simplified blood for the for

the five micron they perform better here
than they do here in whole blood and

we think it has to do with now that
white cells are present at the wall and

they're going to be collisions now between
the five micron the white blood cells and

the red cells that is depressing there
are huge but this gap is still big and

we think it may be because in the buffer
experiments we're thirty percent him

out of Korea which means thirty percent
red cell concentration and in the whole

blood the general ranges to forty four
human is forty to forty five percent So

that means the Red Cell core is bigger
in whole blood than they would be in our

system which would lead to compress
in particles through the wall and

lead to the Red Cell being closer
to the wall that's positive for

two micron A You see the impact of the
level of education and that's negative for

five nights on where we compare this
analysis to what we were seeing in

whole blood in vivo in my case we
don't see the gap between these

small particles a large part of the being
as big in most of the major segments of

the vessel that we looked at so
we're still not.

Having a good correlation between
what we're seeing in human or

blood in vitro and in vivo an animal and

we want to ask the question is this
discrepancy have to do with the fact that

we're in an individual system which
is a highly simplified system or

is there something different inherently
about the blood that we're looking at in

the mouse that might have in fact in
this subtle difference is that we're

seen in performances OK And so
of course one of the thing the key things

that we looked at right away was the Red
Cell characteristic between animals and

humans because again we seen for all.

The work we've done over the years that
red cell was a big impact in terms

of which particles can get to the wall and
which ones get stuck in the core So

we looked at data for the Red Cell
dimension for different animal models and

it turns out that there is a just
the difference in terms of the shape

of the red cell in a human
compared to a mouse the volume

is drastically different so
this is a differently shaped particle and

this guy is probably less the former
wall than the red cell in human and

if we look at other common animal
models you see here the rabbit and

the pig also have differences in the.

Red Cell characteristics of the pig and
mouse have about the same diameter but

there's a slight difference
in the volume so

again the shape of this two guys
are going to be different and

the rabbit against it's somewhere in
between there so we asked the question.

Do we see differences in how
particles bind in flow of blood

as a function of the changes in
the red cells here and we can look at.

Impact of diameter of the Red Cell we can
look at the impact of the volume when we

first did the analysis just looking at and
he there doesn't seem to be.

Discernible.

Trend here you do see that two micro for
example have a different binding pattern

going from Mouse pig rabbit to human and
that you see that that changes where

you're at the five micron level and that
when I change the flow of time from Lyman

to possible to resort to Lady you do
see differences in the pattern and

so instead of trying to filter through
different dataset and different flow

patterns what we would be useful is to
find a way to plot this in a way where we

can maybe find some sort of correlation
between particle would he and the German.

Of the red cells and so
one of the things we looked at was the.

Ratio of the volume to diameter of the red
cells whether that has an impact on

that heater that doesn't seem to be
a trend that fits the different types

of particles that we looked at and so that
we also looked at quality in this based on

diameter alone blister volume alone there
doesn't seem to be any correlation that we

could find the one correlation that we do
find is that the adhesion of particles in

blood flow seems to correlate well
with the ratio of the particle

diameter and the diameter of the red blood
cell that you're looking at and that for

every scenario there seems to be
a quadratic relation that where

you have a low particle diameter to R.B.C.
ratio you have huge and it's low and

that there is some optimum ratio where
you get maximum adhesion for all

the different blood and all the different
particles and then beyond that you start

to have negative impact that has to do
with a collision with a red white cell and

so that this optimal diameter particle
ratio I'm sorry particle to read so

diameter ratio can then
fit if you know the blood

type that you're looking
at in fact if we looked and

put a cow blood into
the system that also has

a different geometry the data actually
fits well on the score as well so

this is the same set of data
now I've added the cow and

you still see the seeing fit.

Before for the same blood flow pattern so
we can now back out which particle will

be what will be the optimal spherical
diameter of a particle to vine depends on

the type of blood so at this point
I look at time here I'm going to

have to talk a little faster
we focus on red cells and

we want to see that flesh out some
of that impact of white cells and

platelets by going into hope and then
doing a reverse direction removing cell.

Out of blood and seeing whether
there was an impact or an effect so

one of the things sells to other than
red cell that tend to impact adhesion

is going to be the white cell at
least that's the point where we see

most of the reduction in that huge and
whenever we have.

White cells present in whole
blood the blue bar for

the larger particles which can change
depend on the magnitude of flow you see

a reduction significant reduction in the
adhesion whereas this is almost no impact

with the smaller sized particles
compared to blood look a certain remove

the green bar is we've
removed white cells and so

we want to we probe that a little bit to
see whether this was white cells binding

to particles in flow that's preventing the
particles from coming down to the wall or

is it a physical interaction it turns out
if I change the chemistry on the particles

to one that I know should not interact
with white cells we don't see any change

we still see a reduction in the adhesion
of the particle when ever white cells

are present compared to non white
cell system so at the end of the day

we figured it had to be the physical
interaction between the particles and

the cells and so first thing we did was
say well if you look at local site removed

blood you will be essentially
creating space at the wall maybe that

allows particles to bind better than the.

Than when white cells were present and so
when we removed white cells we put back.

Particles that have the same
distribution in size of white cells and

at the same concentration so we wanted
to get at is is it just more space that

small micro particles to come down by or
is it something else going on and

we didn't see a difference
in the adhesion between.

Local site removed blood and little
site removed flood that we've added this

nonfunctional peeled back in but when we
going to hold that we do see that we do.

Reduction so we think that whatever's
happening has to do with the fact

that the particles and the white cells
are at the wall both trying to fall and

he's of bonds with the system and so
in looking directly at experiment

a video it turns out that
the issue hard to do with the code

that occur between the particle when
a white cell comes in collides with it and

it stays long enough to rip both the white
cells and the particles of the surface and

so what's interesting about this
is that both the particle and

the white cells were no
longer able to bind and

so now we're probing this as
a potential opportunity for.

Particles interfering with
white cell interaction.

So white cell matters and when we go
back into this impact of different

red blood cells by simply comparing human
whole blood to Mousehole blood relative

to just read lot cells in buffer you do
see that the optimum diameter is reduced.

Particle diameter to R.B.C. ratio is
reduced because of the negative impact of

the white cells on the larger particles so
essentially pushed your optimism to

the left in this case so red soled matters
in science in the sense that it's and

trapped in smaller particle into its core
density seems to be a way that we can

get those smaller particles out of
the core it's not clear right now to what

extent where rescue in that he should and
we're still working on that why still

white still matters but only if
you're looking at it through Micron.

Particle range the one piece of
the puzzle that we have not probed

it was plasma and really we didn't think
we needed to probe it because we were

viewing plasma simply a fluid in which
all of these guys were encircled except

to the point where the student I was
working on this was about to graduate and

she said to me is there anything else
you want me to do before I leave and

I said OK all your work.

Been with polystyrene and it's fantastic
book Palestine is not a delivery system so

why don't you go repeat some of
this experiment with Peel GA

which is a biodegradable polymer
that people tend to look for.

In the construct drug delivery
system she did the experiment and

she came this is a student who's from
Thailand and when she's nervous or scared

she sweats a lot so when I see her at my
door squarely in a pool of water I knew

something was wrong so she showed me this
data and say my heater on is not working

and I think I'm screwing up or something
of that nature the particle again.

Degradable biocompatible polymer hard
to say chemistry target enlargen

density polystyrene and they're the same
two microns fair sized that we know works

well with polystyrene and somehow as she's
running it in blood she's not seen and

he so I said go back and do this in buffer
to at least make sure your chemistries

working right and when she did
that indeed the adhesion of field

is higher and not only that it's higher
than polystyrene which makes sense because

in buffer appealed you will be
slightly denser approximately.

Polystyrene so that whatever is
happening in blood do with blood but

of course we don't think this would be due
to red so white so because those are from

all the work we've done those were
physical interaction not necessarily so

we couldn't see any reason why your GA
sphere of the same size and targeting

chemistry will behave differently
between in blood because of red or

white cells or so right away we pretty
much figure out that it's the last piece

of major component of blood that we've not
looked at which is the blood plasma and so

we constructed ass's where we can look at
particles in boffo particle employers and

particle in some sort of
whole blood scenario and

every time plasma was
present we see a reduction.

And yet he's one of the particle relative
to what you will see in buffer or

the Scots vote for which is essentially
a buffer that we put Dexter and

into have similar viscosity as
the viscosity of plasma and

this is where you see the drastic
difference where I have two micron

particles are either run
in pure plasma flow or

Part two micron in hope that
he is lower compared to

blog that we took out and
washed out the plasma very well so

that you just have cells are B.C.'s in
buffer and you see that the a heater and

it's fine so again it's not the collision
between the cells that's causing this is

some sort of interaction with the plasma
proteins that we know will be grabbed

onto the surface of the particle what was
surprising to us is the time scale that

this was happening was very quick and so
as soon as the particles are in blood for

thirty seconds we start to see a negative.

Reduction in a huge A and it seems to be
a robust by five minutes of the system and

we went back and checked to see if we
just missed in this polystyrene thirst or

by comparing people is styrene and
select and

titanium particles in red muscle and
buffer versus red blood cell in plasma and

you don't see any difference in the level
of heat and so this material types

are not impacted by whatever
we were seeing for Peel GA So

what we did was take polystyrene stuck
them in the same donor human plasma and

then strip off the proteins that are
absorbed onto carry a surface to see if

there will be any differences that
we can identify by a gel system and

we do see that when
the particle is polystyrene

there's a band that shows up here and
probably here that you don't see when

the particle is field GA So it is the
differential absorption of flowers won't.

The surface of the carrier
that's covering this effect and

if we zoom in on this one fifty K D
a partin size we see that the level of

that band intensifies as you
the longer the particle is in blood so

we think it might be this absorption of
this band that that getting in the way of

life against being able to bind the
interest in become part of this is that

this interaction also depends on the type
of donor that you look at in donor a donor

see donor since graduated made
a lot of money work with her.

Working with his blood or her blood you
see that there's a drastic reduction where

the part of who is in the way compared to
donor D. where there seems to be no impact

compared to buffer so this effect varies
with different humans and we don't see

those kind of variation again in
polystyrene So this has to be a material.

So face interaction I was still trying to
figure out what what about the poly star

GA makes it illegal to grab some of these
proteins that get in the way we have

says try to figure also how to get rid of
this protein negative fourteen interaction

again is obvious choice because that's
what people have put on particles for

getting rid of proteins and we saw we
looked at a decent amount of density

sixteen thousand paper Micron Square which
again should be in this brush conformation

and the distance between two
protection is about nine.

We don't see the impact on P.R.G.

adhesion from buffer to blood request
queue that we have since tried to put

more parrot we've got up to about fifty
thousand site that's it for parrot and

that's where we begin to see some impact
so it's all it seems like this effect can

only be made gated if we wipe out the and
we cover

the entire surface of the material so
that blood does not recognise fields you.

And it can see and so we're working
to try and this saw the timing how to

consistently do this for
particles of different argument again so

at the end of the day all of the blood
component play a role in terms of how

particles can interact with the wall we've
talked about the impact of red cells and

now we're focusing more on this
impact of crowds more proteins and

how we can actually be used to and
the design of a delivery system for

example I can say peel J. side I know it's
not going to bind to blood vessel wall

can I have a hybrid system of P O D N A
half polystyrene like for example where

now I can put the drug where I know it's
going to face the blood vessel wall and

see something to target other things in
blood that's going to face a wait for

the blood vessel wall so thank you for
your time I want to acknowledge to

students that are highlighted in post
yellow are the ones that did the work.

That I've talked about today and the bulk
of the work was funded by the N.S.A.

of career and the are all one is
funding us to look at this protein

material interaction
story there thank you for

your time it's been a pleasure telling
you about what we've been doing Thank

you.

Yes.

You.

Are.

Right.

There isn't any better or some sort of.

You know why.

We do you know.

That's actually that's not thought about
that but I can see how especially since

now there's the density
component to the work so that.

It's possible that a D..

I mean we've shown that a dense or small
particle comes down to the wall better

the question that I think you're
asking is if I have a big light

particle does that work as
well I think the issue that

you're going to run with on the largest
side is this collision with red cells

which if you light you just probably not
going to come to the wall number one and

number two that you're going to go pluck
clogged the smaller capillaries So

it's not clear whether that type
of correlation would be useful in

the practical sense of drug
vast majority of drug delivery.

Or.

Not in the time scale that
we look at here over time so

back in my graduate
study I was looking at.

And how much the diameter
changes over time and

over a two three week period maybe you go
from seven micron to nine might be OK So

then if you're smaller You're not going
to get that big in that sense so within

the timescale of that many of these guys
circulate in the bloodstream the P.R.G.

diameter should not change is much.

More.

Sorry about that.

One

more.

Word.

So what's getting bigger in your question

on the red zone the core of the particles.
Right.

Yes yes.

The size of the particle gets bigger or

the ratio between the two.
Who are.

OK.

So that is still a debatable.

Piece of the part of the puzzle is it that
blood begins to flow red cells actively

lift from the core and
grabs with it things along the way and

leaves behind thing is
that it can deflect or

is it that the red core red so
core forms and particles go in and

out of it the literature and
the folks the mathematicians and

the physicists that look at Red Cell
motion things is the former OK

that there's an active lift that occurs
with particles of different sizes and

different deform ability that tends to
work well for red blood cells whereas

the more rigid largest feral particles
don't experience enough lift so they

get left behind close to the wall and then
couple that with collisions along the way

with the red cell then the in their hands
they're delivering to the wall in which

case then it's the question is not that
some particles are able to go in and

come out and stays in.

I think has a question.

That's a great question that's one going
to be one major difference between us.

And polystyrene for example in the Piaggio
will have more roughness and it's possible

that that improve in the affinity of some
of this large protein on its surface so

we're trying to figure out how to
make poly start particles that's not

going to be in this sort of chain winding
process to create more smooth surfaces.

We're working on.

That question.

That's a great question
we are working on that.

Right now what I can say to you based on
our preliminary data is that that too is

linked somehow to the size of the particle
so that it's not clear to us and

so that's why it can't presented because
we don't have a clear trend yet in some

cases depend on size we see that the form
ability does not have any impact and

in some cases it's actually negative
impact so we're trying to sort that out

before but that's something
we're heavily working on now.

OK so I guess let me try and
tell you something else which may or

may not answer your question I think you
would say so what we figured out was

perhaps this idea that smaller particle
tend to circulate in blood longer OK

that one in the past we've thought that
it has to do with and track meant in

all these other organs filtration in the
liver and kidney it's possible also that

the difficult location of the different
particle based on size also help them so

white cells are going to be on the wall
large particles are going to be at the one

based on our data which means if somebody
is going to be cleared faster is going

to be the large particle whereas
the smaller particles if they are truly in

the red cell core then they are away
from white cells which may explain

their longer circulation or at least it's
an added component of to why you see

them longer in the bloodstream because
they are just not called localized and

with white cells for them to have
an impact in terms of their clearance.

I think that maybe answer the question.

Thank you.