Well

first thank you all very

very open

very very very

very very little over you.

You'll

thank you.
Next to make had a good opportunity

to get back at me because I had
called him during as this is defense.

So I'm glad he didn't
take that opportunity.

So it's nice to see a lot of faces here.

You know I was going to do it myself
I said that when I was growing

up the breakfast club was a movie about
people who get sentenced to early

morning detention and
it's a form of punishment and judging from

the amount of students here I can see that
it's still early morning punishment but

today I hope that this isn't too bad for
you guys.

I am still fighting the flu
a little bit last week so

bear with me but today I'm
talking about all the great stuff

that we've been doing in our
lab over the last couple years.

I chose a broad title novel strategies for
treating cardiac dysfunction or

treating my cardio infarction and
I intentionally broad because I wanted to

give people a flavor for all the different
things that we do in the lab.

So today instead of
getting a big long story.

Like Ulysses you're going
to get the Canterbury Tales

which is just a little eggs
are from different projects.

The big important thing here
though is that heart failure

is a leading cause of death.

So this is not anything new.

It's the leading cause
of death in the US and

it's about me it's the leading
cause of death row wide and

it's only growing so you can see here
some statistics is from the lips.

Two thousand and nine website and

you can see here five point seven million
Americans suffer from heart failure and

I'm going to say that it's probably
one of the biggest costs to

the health care burden so I think
the estimate last year it was over five

hundred billion dollars in health
care costs for this country a load.

Leading Cause of hospitalization for

people over sixty five years
of age you can see here.

About three hundred thousand people
die each year due to heart failure.

Which is a subset of the eight hundred
fifty thousand died due to cardiovascular

disease.

So you can see here about each year five
hundred thousand people will develop

new or recurrent my cardio infarctions.

I would show the heat map it would
only depress me because it's all in

Georgia Tennessee Kentucky the southeast
here due to die it will be C.

the genetics things like that but we're in
the largest concentration of heart disease

in the country probably in the world.

I'm so this is really a growing problem.

You can see here.

Currently the only cure for heart failure
is cardiac transplant Taishan about

forty thousand patients require
cardiac transplantation each year and

you can see here only twenty
five hundred donor hearts.

And ultimately about thirty
one third of all patients.

Listen for transplantation survive
until the heart is available.

So what they do now is this is the heart
failure this is the work cost so in missed

work in this labor it's about thirty seven
billion dollars but in health care costs.

It's much more.

So.

This is really really
a growing problem and

it's only getting worse as our population
ages and as our diet gets worse.

We see joke when I was in graduate school
that the first thing you see when you

walk out of Grady is a McDonald's So
a lot of people they go to Grady for

their heart disease of a they have their
acute my and as soon as they're stable and

they're released they get to go
right back out to more fast food.

So this problems only and get worse as the
population ages and our diets get worse.

I always like showing this because this
is one of the reasons why I started doing

cardiac regeneration at
the Brigham I went there.

This is a this is a study this is a review
actually from two thousand and two.

But this is a study from
the one nine hundred seventy S.

showing that new hearts can regenerate.

So you know that right now if you grab a
salamander the tail falls off it will grow

back and I'm sure you've cut up lots
of worms in your biology classes to see

the two headed worms and everything grows
back here but you can see this Newt.

When you make an incision in the heart.

What happens is there's a clot forms.

Then the cells at
the borders differentiate so

they go back into the stem cell fate.

They proliferate and they re differentiate
to give you a brand new heart here.

So this this happens in.

And a few other species as I say
in all my talks were not newts So

our hearts don't regenerate the heart
you're born with is generally the heart

you're you're going to die with it's kind
of depressing but all the cells in there

that are there when you're born about
eighty to ninety percent of them are there

when you're nice and old so our heart is
considered terminally differentiated.

Another important thing to understand
about cardiac healing is that.

Well we called Heart it's funny
because it doesn't really heal but

it happened through several stages and
I'll show you the importance of this when

we start to design biomaterials and look
at our other studies but and Jay made.

It made my job a lot easier but generally
what happens is you have this arterial

occlusion So you have a clot formation
either somewhere in your body or usually

in the corner arteries what you get here
is a blockage of the corner artery and you

get this scheme examined here this unhappy
with the product of nutrients and oxygen.

Usually it can happen over the course
of decades but what happens usually is

there's an acute event where the clot
moves and it lodges in a certain area and

you get this blockage scheme assume no
oxygen no nutrients what happens then is

the patient gets rushed to the hospital
they run upstairs to the cath lab and

they use a catheter to
go in there give them.

Angioplasty which clears the vessel.

That's great novel what happens
when you clear the vessel.

Despite restoring blood flow and nutrients
to the tissue is you get this in a kit

inflammatory response so oxygen now
is the final electronic scepter and

electron transport and
you get tons of reactive oxygen species.

What happens is your body basically
treats the area as a wound.

Because there's all this acute damage.

So you get this acute inflammatory
response which is macrophages neutrophils

monocytes all come to the area and
somebody knows the job

of the inflammatory system is it
necessary to repair it is to destroy.

Pathogens.

So what happens is these inflammatory
cells will go to the left in trickle

in such a destroyer tissue.

That leads to chronic inflammation so what
happens then is that you're now that your

macrophages have gotten there and that
all this damage is that now it's time to

repair all this damage and
the way your heart does that is by.

Signaling to the fiberglass to produce
extracellular matrix and divide.

So my say as I said a terminal
differentiated they don't divide.

But you have a lot of lost Mass in
your heart you have billions of my

sites that were lost.

So fireblast secrete this college and
scar which gives the heart stability.

Unfortunately it's a non
contract while scar.

And then eventually what happens here is
that over time because the college and

does not contract your heart can't pump
enough blood eventually the my sites

undergo a high perch.

Which is they grow bigger
to try to compensate for

the fact that there's fewer of them and
they need to do more work and

that eventually will
lead to heart failure.

Because you can see here.

There are specific stages and there's
a temporal signals and cues that happen at

each stage away from the clues in
all it's the onset of heart failure.

So it means that when we're
designing therapeutics and

we're looking at therapeutic approaches we
have to think about some of these things

going forward.

So we did this study at Harvard.

When we were trying to figure out
more about cardiac drug delivery and

why a cardiac drug delivery was so
hard and it turned out that the answer is

pretty much staring us in everyone else
in the face these are mice that were

injected with a dye that states and
that the illegal cells green.

The red borders of the cardio mine site so

these black areas are actually
the cells and blue is nuclei and

then we sacrifice the animal fix
the tissue and sectioned it out.

And as you can see here.

The heart is really vascular you didn't
really need a picture to show you that but

it helps when we're doing some
modeling is that the heart.

Each My estate is completely surrounded
by a network of capillaries you can see

about five or six capillaries each
which you can see that this is three D.

and there's even more
interaction as you move down.

So what does this mean.

Here it means that it makes retention
of things that are very small extremely

difficult and

there was a paper here that we did when
we were at Harvard and this was done.

Actually with the collaboration of
the law from Berger lab all of you

who are really modeling papers of E.G.F.

and all those wonderful things can see
all the work that is done an MIT but

in short what happens is they model what
happens when a cell in the heart produces

a local growth factor and this case
there was heparin binding E.G.F..

Because it is the.

Well from her lab and
as you can see here this green cell.

Is the cell that's producing
a factor which is a kind of mice and

these red areas here are the capillaries

what this picture shows and
this picture here also confirms

if the capillaries act is a huge
convective force is that anything.

The highest concentration of any drug
that you're going to find is going

to be in the capillaries even if
they sell itself is producing it.

So it means that anything you deliver to
the heart will likely be carried away

in a matter of seconds or minutes so
that makes it very difficult when you have

a long process like my own fortune which
can take days or weeks to manifest

itself in the injuries so you can see
here there's really a strong need for

retention of local therapeutics in the
heart and when I came to Emory this is one

of the things that I want to focus on and
when she know it that I happen to be a fan

of Mary Murphy from some of the work that
he done with that state and over there.

You don't read done some similar things
here and just so happened that he was

working on a polymer which I thought
might be able to do this for us.

So what we had been using in the past

is hydrogels which are hydrophilic
they're great for large proteins but for

small hydrophobic molecules
they don't do you much good.

So I teamed up with the rain who had
recently developed pollicie tell particles

which were so named here for
this ph sensitive key to a linkage.

So traditional molecules are not going to
say this but traditional molecules such as

peel J. They degrade into lactic acid and
other and sitting by products in fact it's

lactic acid things like that to
degrade into glycolic in lactic acid

which can cause tissue inflammation which
is probably not a good thing when you're

dealing with already a nice
level of tissue inflammation.

So these are neutral.

You can see here this Quito linkage
which is acid sensitive it doesn't

really degrade water.

He said when.

Exposed to sort of conditions you
get break down into a dial and

acetone the main one that we use is called
P.C.A. DK which degrades into cycle have

time ethanol which is the dial which
is an F.D.A. generally regarded

a safe product as a food additive and
acetone which is also an F.D.A.

approved list you make it naturally and
you breathe it out.

So this polymer here degrades under
acidic conditions into neutral byproduct

which is just great.

And you can see it summarized here and
also the hydrolysis is tunable I didn't.

I'm not going to show you this.

DAVID But but Stephen Yang and
her integrate paper showing that you can

change this our group you can change
is our group up here by changing

the hydrophobic say of the molecule you
can totally make it for a half life of

days weeks months and I think they had
one that would degrade even in a year.

So here's some of them here you can
see we make them micro particles and

explain why in a minute but you can see
here when you create this emotion you

get these really nice vertical
particles here in the meaning and

there's probably about
two to five microns.

So the initial study we did was reach
shows this P thirty eight inhibitor and

I'm not going to like I said this is
the Canterbury Tales and this will be

the Millers television with that but
we're going to keep it really simple.

I want to show you the eight thousand
pieces of data that went into this but

we use this small molecule
her hair to be one of these

S B compounds A P thirty eight hamburger.

Thirty eight is a kind that's
involved in inflammation.

It's implicated in cell death.

Inflammatory response and in fact
mice that have a dominant negative P.

thirty eight Alpha survive better
after a skinnier perfusion.

And there was a lot of papers showing that
if you gave mice this inhibitor orally

over the course of a couple months that

they actually improved function
following M.R.I. unfortunately in the.

Paper they pretty much washed

with inhibitor every day twice a day
every day for about three months which

would basically mean that you probably
walking around and drinking it constantly

all day which really isn't feasible so
we thought wouldn't be great if we could

figure out a way where you can actually
get the same effect just doing it once.

So we took the P.C. eighty K.
which is shown here.

And here's the breakdown products.

And you can see combined it with our
small molecule inhibitor in a single

emotion because the inhibitor itself is
hydrophobic in the polymers hydrophobic

we're all put them in a single
emotion over over some P.V.A.

you can see here we homogenize
at a very slow speed.

So you get these kind of big particles and
really is joke that this isn't

really nanomedicine because
it's my comp article so

we call micro medicine but
sometimes nanomedicine isn't really good.

So you can see here we
get a permanent occlusion

of the left descending coronary artery so
we took we verified all these wonderful

things in cell culture they want to
macrophages macrophages would release it.

So what happens here I didn't mention
that these particles will be internalized

by inflammatory cells and

once internalized into the fact as ohms
with low ph particles will hydrolyzed and

it will disrupt the faggot zone and
release into the cell.

So you get this intracellular delivery.

So these were delivered after
permanent inclusion of the L A D.

So basically what we do is we just open
up the chest of a rat tied off the corner

a to simulate a heart attack and
then injected these into the borders and

try to simulate what we think might
happen in a clinical situation so

moch can get into all
the nitty gritty details but

what happened was that we saw
after three in seven days that our

inhibitor significantly decreased
P thirty eight phosphorylation and

the infarct to compare with the free
inhibitor or the empty particles and

that got us thinking well we just want to
make sure that these particles are so.

Really there.

I'm so it is we look at these
particles of the bring which is that

a nice fluorescent dye is hydrophobic and
we injected these into the heart after

a senior profusion and then we harvested
the three seven in ten days and imaged it.

And you can see here this is a three D.
reconstruction of a section so

these were sections slides that were
stained green which is for potent

which will stay in the sarcomas of the
cardiac my sites and this yellow big giant

yellow thing here is a loaded particle and
this is several days after injection.

So member I say we make the micro
particles we make the micro

particles because we want them to stick
around for a long time so you can

see here this is three and seven days in
here you can see a little bit better.

There's a bunch of little particles
that are sitting around in the heart.

So we're pretty sure that when we
inject these particles in the heart.

They're actually staying
in the heart which is big.

If you want to create a drug
reservoir in the mike heard him.

I'm not going to show the data but

we looked at ten days and
we still saw them there after ten days so

we're pretty sure that not only
are we delivering it to the area but

it's sticking around and
it's releasing the drug over time so

to cut to the chase and
have a happy ending.

Here we looked at function.

So we looked at lots of other markers like
inflammation and decrease inflammation.

But ultimately all that really matters is
what happens to the function of the heart.

So this is a fractional shortening which
is a measurement of how well the heart is

contract ing these were done by M.R.I.
echo and see here this

is two hours the white bars here day seven
and the gray bars are day twenty one.

You can see here on the one axis is
the percent of fractional shortening so

you can see here these sham animals they
contract about fifty to fifty five percent

which is what they're supposed to
do by measuring peaks sisterly and

diastole areas.

You can see here when you have an M.R.I.
alone.

There's a significant
decrease both at seven and

twenty one days there was no
effective our P thirty eight P.

thirty which is the free inhibitor and
my plus P.K. which is our MT particles.

There is no effect.

Now interestingly when we looked at our P.

thirty eight load of particles
as P K P thirty eight.

You can see here at seven days there
is actually no functional improvement

which got us a little worried and
start to panic a little bit that

despite all these positive
results nothing was happening.

However we looked at twenty one
days you can see here there

was a significant improvement this was
also significant against the Am I alone.

So you can see here over time
the animals improved their function.

And for their viewers a number for
those that are in my class

if not all the wonderful things
that reviewers always make you add.

They wanted us to show that we could beat
the industry standard which was this.

So we created size matching
loading match particles and

all the plumber data from that but
basically they

themselves induced P thirty activation
in a lot of cells and you can see here.

There was no significant improvement
in function so the take home message

from the slide here is that although there
is no acute improvement at seven days and

twenty one days there was
a significant improvement in function.

So went back and looked at all of our data
and we realized that it had been T.N.F.

and super oxide and

prophesied all these things that perhaps
we are having an effect on remodeling.

I don't think this actually worked.

I tried to load some movies
in here because the movie

was worth a thousand words but
it didn't seem to work today.

But this is how we get our images
here is that when we look at M.R.I.

slices from Sicily and saline if you were.

If I had some magic now you'd
be seeing these contract and

we can measure the areas each one.

But we were looking for
a reason why these animals.

Got better and
when we sectioned the tissue.

We looked at college and staining So you
know I showed you the initial slide that

it had longer term things being
remodelling hypertrophy and

things like that and you can see here
remember when I told you guys that

the heart basically turned
into a college and scar.

You can see here here's
the left ventricle of a regular

mouse this is right this is three weeks
later the red is college in the yellowish

these my sites that are still alive and
you can see here about most

of the wall turns into this college and
scar which is not a contract.

We actually measured a lot more than this.

This is just one area to show you.

But we measured this in lots of sections
and it's about thirty to forty percent of

the heart of the left ventricle is a scar
as you can see here percent fibrosis.

If we have our free inhibitor.

There is nothing to forgive our load
in had our empty particle which is P.K.

there is no effect.

But you can see here a loaded
inhibitor shown here significantly

hit fibrosis which we think allows for
better contractions.

So we didn't have an acute effect but

we had this chronic effect which was
great but it made us want to think

maybe we could have done better
in the early time point as well.

And I'm going to switch gears a quick and
talk about this

early time point because what's good for
the goose is not good for the gander.

I showed you the specific temporal
constraints here and you can see here at

a later time points we have this
fibrosis but it really time points.

What you have is cardio my say that
Michelin about zero to seventy two hours.

You have a huge amount of cardio my
eyesight death which might be what's

limiting the function early
time points not fibrosis.

So our lab is also interested in reactive
oxygen species Emery has a wonderful core

for measuring reactive oxen species and
it's one of the top places in the world

for a reactive auction species as a way
of a little bit of background low.

The stress here simulates
growth you need to survive but

also to stress stimulates the Crocus
in addition to a whole host of

other negative things but the active
ox and species specifically super oxide

has been implicated in cardio my
sight in protest is in particular.

And you can see here you know low levels

similar growth we have trained
adaptation of these moderate levels but

these huge levels you just get
able to sustain the cosas.

Interestingly super oxide levels increase
in the inferred right after M.R.I.

and the R.N.A. levels of the in
Dodge Innes antioxidants such as So D.

sharply decrease after and why.

And there have been some
controversies in literature.

Referring to as thirty therapy so

S O D over expression protect the heart
from a senior profusion injury.

But and here are some therapies with the
actual protein as sodium catalyst which

takes a process of what they've been
attempted to for laboratories but

the results vary widely.

Some of them say it works great.

Others say they can never get it to work.

And one of the big issues is
unfavorable pharmacokinetic So

the half life of S O D
protein is about two hours.

So basically even if you can
get it to stay in there.

It's rapidly in one thousand aided by its
own product which is how your proc side

and it's also leads to an instability
of the protein so you get really

poor delivery kinetics also improper
delivery a very large active protein.

So you can't really get
proteins inside of cells

which is probably going
to hurt your little bit.

Lots of laboratories and
try to address these issues by attempting

different delivery
methods you can see here.

Peg couple a capsule.

Less lecithin eyes and
even couple of heparin binding domains.

I've actually tried that with other
proteins to get it to stick in the area

but really there hasn't been
a lot of work on micro.

Capsule ation of

days that we wanted to this wondering
if we can improve the delivery of this

protein by Cap's lading it with
these public he told particles.

So once again we use our friend here
P.C.T. K. This is little bit different

though because it's a protein we did a
double emulsion the first emotion is done

in a small volume of P.V.A.
here and we've done it different ways but

basically first emotion is to
protect the protein itself.

So you create these small little
particles either by on occasion or

by fast imagination and then you put that
into a second emotion container P.V.A.

to get your actual particles.

So you get these little tiny particles and
you get particles within particles.

And then the same delivery concept we're
going to hear which is hopefully psychosis

and intracellular delivery and you can see
here we did a little bit different this

time we used a skinny re profusion
which is when you tie off the L A D for

about thirty minutes late is the left
hand tear descending corner artery and

then you release it so

that actually we believe simulate
the clinical situation even better.

It's a very common technique that's used
but that's when you get that huge influx

of oxidative stress direct injection the
micro particles because it's an open chest

procedure we can put the micro
particles directly into the heart and

in this study go call did three
injections into the border zone.

And here the infarct core would be
the middle the borders on so obviously

around the borders which is where repro
fusion is thought to best occur and then

the thoughts come out of here again shows
pretty much what we're hoping is that

these particles will sit inside the tissue
between these mice say it's either going

to engage by macrophages or release the
product by itself and you can see here.

We did a lot of function this this study
instead of just measuring function at

seven twenty one days we.

Measure baseline function.

You can see how the animals were before
the surgery and then we measured echo

cardiogram each time point in time
points to look at function over time.

And we also did biochemical data like Axis
stress inside a common response to things

like that in vitro we
looked at macrophages and

you can see here we stimulated
macrophages with P.M.A.

just to make sure S.O.D. in this double
emotion procedure was still active.

So you can see here this is measuring
extracellular super oxide and this is with

H.P.L.C. So it's pretty quantitative and
we stimulate our macrophages P.M.A. you

get about a one point five fold increase
in super oxide production which is blocked

by extracellular a city this is the free
protein there's nothing special about it.

But it's what you'd expect.

It's a good control just to show
that that you can block with S.O.G.

if you look at are empty
particles you can see here.

There is no response.

These two blue bars here.

There is no effect of
empty particles on P.M.A.

and do super oxide simulation and
you see here this middle bar here.

But if you look at our P.K.S. India which
is their policy telling Catholicity to

very low doses point to five point
one to five milligrams per mil So

we're talking tiny tiny doses here you can
see a complete inhibition of extracellular

super oxide that is not really
cool that's just about what we

expected because super oxide can probably
fairly diffuse in and out anyway.

And this is outside the cell
there the interesting thing

is when we looked inside the cell.

You can see here there's much more of
a response inside the cell is about a four

fold increase in super outside production
inside the cell which is not blocked

by the free protein so
the proteins don't cross into cells so

we didn't really see much inhibition.

If you look at the particles once again
there was no effect of these empty

pollicie tells on
scavenging the super oxide.

But interestingly when you
look at our P.K. acid.

Particles.

You can see here you can at this point two
five dozen even a little bit one point

zero point one two five dose.

There was a significant scavenging

of super oxide her significant
inhibition of super oxide.

So what this means is that the macrophages
were able to fact as a toaster

engulf the particles and the active
protein was released inside the cell.

So not only do we think we have a way to
get sustained delivery of our protein but

we think we also have a way
to get inside the cells.

So I'll skip all
the exciting part of this.

So you that in vivo when you
look after a skin refused.

This is a dye that measures oxidative
stress this is bad advertising for

for an array and because we didn't use his
reactive action species but this is just

rather die hydro thirty M. which will
react with super oxide to form a thirty M.

bromide which anybody who works in D.N.A.
knows that it will bind to D.N.A.

and for us.

So you can see here
after profusion this is

heart tissue there is a lot lot
of super oxide being formed.

If you just give free day to
the animal nothing really happens but

you can see here.

R P K S E D completely prevents super
oxide formation in the my Cardium and

this led to acute changes so this graph
is little bit different on the Y.

axis is actually a change in function
rather than the actual function itself we

measured baseline and then measured at
three and twenty one days later and

you can see here this is that.

Three days this year profusion cost about
an eight to ten percent decrease in

function.

And that's percentage points I
should mention that percent so

that means if the animal had fifty percent
contraction on days hero it now at forty

percent contraction on day three.

You can see here.

A P K S N D significantly improve that.

Unfortunately at twenty one
days what we thought did not

really have much of a significant effect
anymore but we combined our P.K. S.O.G.

with our P thirty eight particles.

So if we attack both ends of it with.

And fibrosis.

We actually get a significant response.

This is suggests that there's not one
magic bullet going to really do it and

be really think more about spatial and

temporal delivery because there are
specific time courses to these things so

the P.K.S. a deal of work rate early
didn't work great very late and

the opposite was true for the P. thirty
eight inhibitor it didn't work great.

Karnak Labor didn't really have much
of an acute effect of this got us

to thinking is there a way we
can we can try to do both.

So this is bridgehead I'll just get into
this very quickly but this is data from in

do in the lab looking at cutting off
the super oxide I think at the heart.

So rather than just giving a protein
which may not last a long time is looking

at ways to knock down the enzymes
themselves that make the bad things.

So this is a public key to here with
our new particles in it and we can

complex R.N.A. with this molecule called
the tap which is just positively charged.

It's called ion pairing is just to get
get this into the particle because it

negatively charged piece of R.N.A. won't
really get into the particles well and

it also will be subject
to take medication.

So we paired up here we can
put it inside Palmers and

you can see here this is a little
schematic from the range.

Where you can have your public
you talk particle the S.

are in a get introduced to
the cell the cell will the fact

that the are unable to be released and
they can go to the nucleus.

So we looked at you can see here as they
are in a therapeutic have a lot of promise

because you can specifically target one
protein as opposed to a small molecule and

never returned to be a bit ubiquitous.

But deliver problems have really
not advanced the field very much.

And you can see here we
looked at Knox two so

this knocks two is a sub unit
of the enzyme knocks which.

Makes super oxides called and
indeed oxidase

it's one of the enzymes that is actually
response for making soup peroxide.

So rather than scavenge the superbugs that
after it's formed we try to cut it off at

the pass and you can see here this
is this really preliminary data but

here's an empty particles and this is
our scrambled as our new particles and

you can see here this is
normalized Knox two levels and

you can see here this is
after twenty four hours

you can see about a forty to fifty
percent decrease in knocks to expression.

So we're hoping that systemic delivery
of Knox USA are in a way to M.R.I.

or or perhaps local delivery with
the methods I've shown you in the past

few slides that we can actually stop the
source of the radicals before they even

come out so now I get switch
gears a little bit again and

talk about more exciting stuff
which is affinity delivery and

I might go through this little bit quicker
because there's a lot of background and

it's sort of unpublished but one of the
things that we saw from our previous life

here is that there's there's two molecules
which which improves the function one

early and one late and we actually had to
create two micro particles to do this so

we thought would it be great if you can
do it on a single micro particle to have

one micro particle that can release two
things with different time courses and

it just so happens that Jay was
digging through literature and

found this nickel affinity paper here.

So necklace finity columns
are used to purify proteins

which is normally done as you express your
protein in a cell with a histidine tag.

And then to purify your protein
from all the proteins that

the bacteria are used for making if
you run it over this middle calm.

Mickle N.T.A. column and
it will bind to histidine

tagged proteins right here and
we thought would it be great if

we could apply the same theory to a micro
particle and I said just so happened.

It was this molecule here called dogs and.

If you want to know what dog stands for
You can look it up.

It's a really long fatty acids and

this kind of very long name I
can't even begin to get into but

basically what it is is this is a long
fatty acid hydrophobia chain and

then you've got this hydrophilic
head group here which is trying to

see the acid and what happens here
is that what we have publicized

is that because this is hydrophobic in
this is hydrophilic if we put these into

our emulsion the hydrophobic part would
partition into the micro particle and

the hydrophilic part would stick outside
of the particle into the equities face and

it just so happened here.

That won't get into too much detail but
he has a schematic.

These are micro particle and
here's our nickel and

this is there and
here you combine it with nickel and

we're hoping that we combine
history proteins to the outside.

So you can get door deliver delivery of
something from the core that's hydrophobic

and something from the outside.

That's hydrophilic and
without getting too much into it.

We made these particles incubated
them with nickel chloride

to get nickel onto the N.T.A.
and then once these particles are made.

We can freeze dry them and
we can actually put them into an illusion

contain the protein so that we are protein
never has to see any organic solvents or

anything like that and even the great
thing about this is when you're done you

can spin it down and recover the unbound
protein but you can see here.

Jay given amounts of protein on
the outside and ran a quantitative

allies up to figure out the efficiency
of capture you can see here.

He learned on the X.
axis capture on the Y.

axis and the take home message here
is that this is about forty percent.

So we get about forty percent of
our protein down to the outside and

I should mention in our initial.

We were happy if we got five percent
of the protein inside the micro

particle because it's.

The motion the protein can seep out into
the phase of your motion here because

the entire thing is done in a solution
we get about forty percent binding.

And there is there is some saturation it's
dependent too on the amount of M.T.A.

You can see here one percent versus ten
percent and the binding changes but

the end result is you can get about fifty
four to fifty percent of your protein

bound to the outside of the particle.

And what does this allow for well you can
see here released from the outside This is

released study using G.F.P.
in our S.P. compound from inside data

you can see here as big compound
has a half life about seven days.

I mean about half of the the the
inhibitors release in about a week.

And it's probably mostly either diffusion
or particle hydrolysis whereas here

you can see the G.F.P. has Tell You on the
outside of the particle were released for

the half life of about two hours and

this was actually done in serum so
probably pushing up if we did in P.B.S.

might be able to different what this
shows is that we can encapsulate two

completely different proteins one
hundred phobic one hundred feel like and

get absolutely different release profiles
which is exciting for us because then we

can maybe go back and look at some of our
regional studies to see if we can get

the same results from
this one particle and

sort of from two separate particles
also at this level still as we came in.

A variety of different his type of
proteins on the outside of the polymer we

don't have to use just one protein and in
fact we're doing some collaboration's now

with Dr Young something in Emory
cardiology to see if we can generate

cells which is induced pluripotent stem
cells with a cocktail of factors on

the outside then I think this can be
used for as it can be used for targeting

I won't get too much into this because
this is probably just a small sampling and

we don't even choose the best protein but
instead of using an about.

Yes you can use targeting proteins.

If anybody has taken the I.B.S. class
knows that cat hearings are on the outside

of individual cells mainly these ve can
hear and vascular and feel can't hear and

they're used for cell cell communication
they bind to cat here and other cells and

their trans membrane proteins so
when they can hear and bind.

There is some calcium dependent process
sees the curious things that they form

homo dimmers with other ve cat hair and
so we have other sides this might be

a great way to try to target these micro
particles to enter the filial cells.

So if found to his stag ve had
here in fusion protein and

we put it on the outside of our
particle it and give it them and

the filial cells and you can see here.

J. did some can focal
Floyd into shows us so

the red is the particle Blue's nucleus and
these are in the cells in humans and

you can see here a little red particles
associated with the cells and

we did all sorts of different cells we did
hear that with the eco hearing particles

you can see about forty to fifty percent
as opposed to just cube X. This is

just nickel and T. a particle there's
no ve got here and also we do cardio.

Both with and without express the E.
can't hear.

And so you get about a two and
a half old increase in and targeting.

So this is exciting because now we might
believe these particles for things

like hypertension and atherosclerosis
which mainly involve filial cells.

Finally in terms of targeting this
is work that's been done by war and

there was a paper a few years ago that
showed you can use sugars to target cardio

my associates and Warren is no the good
part of a year trying to figure out

how to get the sugar which is and this you
know glucose I mean conjugated to a micro

particle and you can see here we employ
the same technique which is conjugating

the sugar to a big hydrophobic molecule.

So we can create this partition again and
then.

Hopefully we now have
these particles made and

labeled hopefully that they will by
intellect in something outside of

the card in my sights and
be internalised to carve my sights.

And this would be a huge leap in
the field because Carter my sights

are cells they don't eat particles they
don't do lots of things I just contract

my neuro friends always like to tell
me all the heart does this pump and

it's true all these kind of my
thoughts do this is contract.

So if we can figure out a way to
get proteins that part of my Sates

we can in fact give them either
positive contract out factors or

positive survival factors
directly after my.

So but the end of this tale here.

The public he does make excellent delivery
vehicles for treating M.R.I. we can get

release of multiple factors simultaneously
we can now get temporal control.

We can get spatial control here for
targeting different cell types.

We can deliver anything from protein
small molecules now we have S R N A.

And we can sustain the delivery for
several days and weeks.

So we're still doing
some pollicie ta work but

we have now got on to new new bio
materials because these are invasive

delivery techniques so I should mention
that in interim myocardial injection while

it's been done in humans still requires
a certain degree of technique you need

a trained physician who knows how to
use a catheter to go in there and

actually you have to put the catheter
in through the artery and

snake it all the way down into the left
ventricle which is a tricky procedure

sometimes and then you have to know where
you're injecting it because you don't want

to inject it randomly electrical
you want to inject it into an area.

So that's kind of the thinking you know
wouldn't be great if we could figure out

a way to target these directly to the
infant itself and I wish I could take all

the credit for this idea and maybe if
you weren't here I probably would.

Some of this came came through with
Mary Murphy who's doing this with cancer

therapeutics called autocatalytic
targeting and read we set down one day and

thought this might be a great a great
idea for informed delivery and

I'll get into this in a second.

So I mentioned earlier that cell death
follow the M.R.I. is a big problem.

You can see here all sorts of there's this
infarct own here there's the Parian fork

There's the border zone but what happens
here on the border zones in the infarct is

you get this you get on top of
the gene which is this totally

different creature but in the root
remote myocardial is almost no cell

death acutely there's crime and there's
delayed cell death you know after days and

weeks because of adaptive mechanisms but
acutely there's no cell death and

you can see here here's a picture
of the human heart with

that's that's had an M.R.I.
and you can see a lot of cell death.

And here's a little picture and
this one up which is good

because it shows the two processes
of a post this in the process.

I'm not going to call in
anybody that this for class and

asking which one is not process but

clearly the one on the the outside here
isn't across just because it's not

controlled it's releasing everything
into the extracellular environment

where you can see here since everything
is being packed into vessels and process.

What this means is that after a my you
get a huge increase in the process but

there's a lot of things being released
into the extra sailor environment

that shouldn't normally be there.

And one of those things is D.N.A.
So autocatalytic

target molecule targets D.N.A.
and it's for killing cancer cells but

we thought we could probably use this to
try to target D.N.A. infarction because in

healthy tissue D.N.A. shouldn't be there
and I like this because J. made it.

We're hoping that you can
inject it intravenously.

And it would all accumulate
just in the heart and

you can see in a little schematic
here it would accumulate over time.

Just in the inferred of the heart.

So in Fark specific target.

And like I said these are tales so

I'm going to show you use a hoaxed
molecule hoaxed is a D.N.A. binding agent.

It's been used in clinical trials for
measuring and you Genesis and even for

cancer cancer treatments but
it binds to the minor group of D.N.A. with

relatively high affinity and the great
thing is it can be it can be modified.

So what they did here you can see here's
the hoax what they did is they added

a polyethylene glycol linker so
this was anywhere from four to eleven peg

chains what this did is it made it cell
impermeable so hoaxed is cell permeable

made it completely cell impermeable so
it doesn't bind intracellular D.N.A. and

then you've got this reactive handle
here and the initial So we use this I.R.

seven hundred six die which is
an infrared dye which we can use

in vivo image or you think are low
tissue attenuation or imaging So

our theory here is that there
won't be any extracellular D.N.A.

unless there is an injury in the tissue
and that this doesn't bind because that's

how member and by intracellular D.N.A.
so I won't get into the whole story but

there's a lot of pictures and it doesn't
bind to cells so it is cells and

we don't see any nuclear fluorescence
until you permeable as the cells.

So we went ahead and we did this in vivo.

You see here.
Once again

we have this coronary artery ligation and
this time we close the chest and

move the leg a shin reproduce
the heart close the chest.

Just like you would see in a clinic
the patient gets reprovision happy sent

on his way two hours later
to twenty four hours later.

Jay and Milton injected the hoaxed I.R.

seventy six conjugate intravenously and
then after an hour sacrifice

animals refused the organs and
fixed it for imaging and

there's really a lot of pictures but
I'll only show you the ones that matter.

There wasn't really much
standing in the long as there was

a little bit in the liver.

As you would expect but it was not
specific staining it was just sort of some

diffuse standing in the liver and there
was no standing in the kidneys either.

So we're pretty certain to get through.

We wanted to go to when
we imaged the my car and

you can see here this is a sham operated
animal that received our seventy six and

here's an animal that got
the I R seventy six and

this is pretty It actually turned a little
better than we even hoped I took out

the one where the search was the base of
the sutures probably right about here.

So most of the damage
is below that mean L.V.

and you can see here we get a pretty
strong signal just in the Internet.

If you quantify that's about
a two fold increase in

two hours there wasn't really much change
of twenty four hours but I must say

that it's still not all that quantitative
because it's just looking at images

we're actually going to get some real
proteins and do some real connotation.

But the exciting thing is that when we
look at these under Plymouth trees so

when we dissected the tissue and we slice
them to see exactly where we were L.V.

you can see here here's the R.V.
over here there's no standing.

Here's the thing called
the remote my Cardium of the L.V.

there is no damage there.

It's all concentrated along this inverted
area of the V. Just like that picture and

sure if you took in forked
image of a real rat or human.

That's probably what you would see for
the dead tissue.

So pretty certain this is binding to the
dead tissue with a fairly high affinity

and there is almost no binding in
the remote tissue or other other tissues.

So pretty excited about this because if we
figure there's a way that you can treat

patients even before
they get to the hospital.

Maybe on their way to the hospital
with some kind of anti-inflammatory

agent right now they tell you to pop
an aspirin or some like that but

really once you until you get to the E.R.
there's really nothing else that you

can do so we thought it might be great if
you could have seen that you could inject

intravenously which would
attenuate cell death or at least.

Take away some of the information.

So like I said it's a lot of for targeting
of the damaged area and I the injection.

There's a lot of work
that needs to be done.

We can conjugate proteins
particles maybe even so.

To this using our elect in
the procedure something like that but

obviously there's a lot that needs to
be done to determine the economics.

How long it's there and if we can actually
use it to deliver anything meaningful and

right now or in collaboration with CA
to deliver veggie to see if we can

promote angiogenesis infarction so
I'll end the talk

here with a few more slides talking
about how we find new therapeutics and

I would really want to touch on everybody
else that we did work in the lab here.

So this is more about existing and find
new therapeutics or about more finding

more about existing therapeutics show you
know you can search the literature and

find cool drugs and it's fun to inject
biomaterials into the heart and play with

them and find new materials and things to
do but ultimately we need to know about

these things such as specificity timing
and function and we don't just guess it.

You know there's actually studies
that need to be done to figure out

what cells need to be targeted and when I
told you in the very beginning we started

by saying that micro functions
a very progressive disease and

takes weeks and days and weeks and
lots of different cell types are involved.

So everybody you know when I go to give
talks to places I say well how do we

actually figure out new therapeutics and
if you're got lots of money like

also that U.T. Southwestern you can
make a mouse every week that has a new

micro R.N.A.'s a new protein but we don't
have that kind of time energy or money.

So in the lab here we use
special mouse models and

cell culture and
to get back to the basics.

Just so people don't think we
only do materials in the lab we

try to figure out interesting new
things about cells specifically.

Stem cells and
to see if we can develop new therapeutics.

It's also we have transgender strategies
and we use this strategy to look at timing

and cell specificity I won't get into
data here but we've created mice

have the alpha mice and have a chamber
motor which is cardiac specific.

And then this mercury Merck which makes
sense from you an estrogen receptor so

it binds to something called Mock
sufficient to turn on this protein CRE so

your body doesn't have a lot of to mock
Safin you can imagine a male mouse doesn't

really have a lot of estrogen receptor and

what you can do that is you can take
create this lock screen technology and

you can use it to change
the genome of an animal.

So what we did in one study is we
have this G.F.P. and stop code on.

And then the human cattle of these genes.

So to talk too much about cattle.

But cattle is another antioxidant
that takes away Hydra proc side and

you can see here when you inject
Safin you will get create and

it will remove the stop code on and
allow you to get catalogs expression and

this will be just a minus sites and
using this approach we've been able

to look at the timing delivery so we can
say when should we deliver cattle that is

you can see here zero days minus
seven days and you fourteen days.

And this paper is acceptable.

What basically happens here is if you do
it acutely so this is this is animal that

got cattle just before surgery and
when they got it a week later you can see

there's almost no change depending on when
you give it acutely Earl week later would

suggest that maybe cattle isn't the best
protein to give acutely after M.R.I.

which you would know by date in the
literature because there really has been

a lot of work trying to figure out
why cattle it doesn't work early and

it's because we found that doesn't
really matter when you do it.

It doesn't change cell death.

It actually changes fibrosis over time
I won't get too much into that but

you can see here.

There's absolutely no difference
in delivering the timing.

You know I have macrophages
that over Express as well.

So we can look at cell specificity and

finally I do with some stem cells all
these different kinds of stem cells.

I'm sure you've all heard of
embryonic skeletal my glass they all

have benefits and they all have
downsides for the function of M.R.I.

we've really only looked at the Thiel
progenitors couple stem cells and

cardiac progenitors these are tissue
specific adult stem cells and

they've these three are really the only
ones that have had clinical trials done on

the McConnell trial show an acute
benefit but there's really

no long term benefit and moreover they
really have no idea why it works.

They just know that you inject cells and
magically the heart happens to heal

which is is good for
those patients for three months but

it's really helped them long term because
you can think of this character in effect.

There's making new my
safe there's new vascular

sation there's repairing the cells.

So these are these tissue
specific cells and

you can see these little
tiny cells in adult heart.

These are big my sites and these little
tiny stem cells and sit in the heart.

Some Our collaborators
have identified them and

we actually use magnetic beads to
pull them out of the heart and

we can study them in culture and
one of my things go cool looks

at what happens when you expose them
to super oxide and it turns out.

Unlike adult Cardium eyesight's when
you expose these cells to super oxide

they respond by acutely up
regulating their antioxidants and so

do you want to be two proxies and
they do this.

Probably what he thinks is
the activation of a Katie.

So by studying the pathways by
which this protective effect occurs

we can use it to try to direct
delivery of these proteins or

delivery of these factors in vivo or
we can module eight stem cell therapy

itself we can deliver stem cells
that have a K.T. activated in them.

OK To do this but but
oxygen stress regulates.

Angiotensin receptors which
is also really bad because

angiotensin two is critical for
inflammation hypertension.

When you expose these cells
one hundred proc side.

There and two receptors go way up which
will end up negatively in the cells and

preventing their differentiation and
preventing their survival.

So if you're going to inject the cells
into an area like the heart which is

rich in super oxide oxygen stress you
really have to understand what's going on.

But only if you look at his ankle stem
cells the complete opposite happens

when you treat stem cells with distress.

It actually drives or differentiation.

Almost themselves which have been treated
for a week with hydrogen peroxide we

measured some and affiliate markers and
you can see here that stress seems

to drive in the field differentiation
of the cells so I might show you this.

Well cell therapy is specific Emory we
have this thing called preventive care and

personalized medicine and

I think this is really going to
be one of the next forefront.

In medical research is personalized
medicine and when a patient comes in there

and you do autologous cell therapy
means you take their cells out.

Culture them and reject them back
into that you have to think about

what kind of cells are using them when
you're injecting them in there and

what they're going to be exposed to.

Because you need to understand how these
cells respond to their environment

before designing therapeutics and
you can see that we

found that progenitor cells acutely
activate protective mechanisms but

stress may drive differentiation
of missing from the stem cells.

So now we're going to go back and

we're going to see if we can module eight
these pathways in vivo with materials to

see if we can drive in dodginess and
exogamous repair.

So with that I think I was
bored enough this morning but

I want to thank all the people who did
the work I have a lot of great people in

the lab Milton Brown does all my animal
surgeries for the last four years and

probably about a thousand something.

Jay and

go cooler fifth year students about
to go on to bigger and better things.

Do Archon a worn car on
the lab lots of people too.

Also the three radical the M.R.I.
people that helped us out and

finally my collaborator Georgia Tech
Mary Murphy specifically in his lab

Stephen Yang and Scott Wilson hundreds
Garcia who helped with the Jeff study that

didn't show the history of Vegeta
livery but that's what we used for

our dog delivery and
we have more collaboration with him and

Melissa Kemp who helped us measure
inflammation response or particles.

So in our funding sources Thank you.

And don't be afraid.

You can ask anything.

Maybe maybe not.

We'll say

that

I said they were thought about you know
we're trying to figure out ways to slow

the release maybe by you know
I guess using different you

can maybe use different metal key
leaders or you know that's also

his tag G.F.P. So maybe larger proteins
might not to fuse away as quickly but

that is an issue we're trying to figure
out we haven't quite got the solution to

that and
I'm sure Jay's going to stick around for

another six months to figure out how
to get even more levels of release but

I was also under certain conditions so
I think if you do admit is all or

if it's his tag proteins in the in the
microenvironment those can also change or

kinetics in vivo so all.

Yes You know we've

looked at smooth muscle and we actually
see smooth muscle markers going down.

We look at cardiac markers and they don't
change and fiberglass markers go down so

it seems to be pretty specific for
driving and

to feel that we did not look at bone or
other markers or

venture out to look at that because
that can be the primary issue is it for

driving bone growth right there.

Thank you.