good afternoon everyone my pleasure to welcome you to a very special lecture
today this is the annual Margaret Pittman lecture in which we invite a
distinguished woman scientist to come and address the NIH community and
recognize this by the name that's attached to this lecture Margaret
Pittman she was NIH s first female lab chief Margaret Pittman made significant
contributions to microbiology and D vaccine development particularly in the
areas of pertussis and tetanus during a long career in the National Institute of
Allergy and Infectious Diseases from which she retired in 1971 in fact when
Margaret Pittman began working at NIH it wasn't here in Bethesda it was down
there in Washington and she moved with it back many decades ago but is
recognized as having really been a pioneer in both in science and
particularly in the place of women in science we at NIH feel this is a really
important issue and have a whole committee that looks at these issues
regularly and we're very happy to be able to highlight particularly today the
Margaret Pittman lecture by bringing in a spectacular speaker for you to listen to dr. Paula Hammond dr. Hammond got our
undergraduate degree in chemical engineering at MIT there'll be an MIT theme here she did
then get a master's degree in chemical engineering at Georgia Tech but then
returned to get her PhD at MIT and then joined the faculty where she has been
continuously since then beginning as assistant professor and now as the david
koch professor of engineering and chair of the department yeah in at MIT and
that's the Department of Chemical Engineering her research which you're
going to get to hear a lot about is groundbreaking in terms of its potential for medicine as well as a variety of
other applications because it is in this general area of nanotechnology and
particularly the self-assembly of polymeric nanometer
with a major emphasis on the use of electrostatics and other complementary
interactions to generate functional materials with lots of applications in
medicine that I think we will all be pretty excited to hear about dr. Hammond in addition to her academic achievements
is an elected member of the American Academy of Arts and Sciences is a
tellurian cancer innovator award a recipient has a TED talk which if you
enjoyed today you should go and listen to as well because it's very well done
presenting really quite challenging technology to a general audience and
doing a beautiful way of method of doing so so delighted that you are all here
welcome to all of those watching also remotely over the web and please join me
now in welcoming the pitman lecturer dr. Paula Hammond
thank you so much it's a real honor to be able to give this lecture today and
especially given the history of the Pittman lecture and I've had an
incredible time getting to know a number of old friends and meeting a number of
new friends and colleagues while I've been here I'm going to talk today and I think this is my pointer I'm going to
talk today about a layer-by-layer assembly approach which can be used to
generate new methods of delivering drugs and the nice thing about this approach
is that it can be used to deliver drugs on surfaces on almost every lane scale from nanometer Lane scale up to large
macroscopic millimeter and centimeter Lane scales this technique is very simple it involves the alternating
adsorption of charge species essentially you can start with substrate it can be
any substrate plastic that has been treated so that it has a negative charge
for example a metal or metal oxide which has its own native charge and you can
take that substrate and immerse it into a dilute aqueous solution which contains
something that is multiplied oppositely charged now when you do that you're
going to get adsorption of that charged polyvalent material onto the surface
until ultimately the surface charge gets reversed and at that point electrostatic
repulsion prevents any further deposition on the substrate so you have
something like a monolayer that is controlled it's a self-limiting adsorption and the thickness of that
layer is dependent on what you're absorbing if it's a polymer chain or a
nano particle for example and the pH and ionic strength of the solution that
you're absorbing from so you can make these films thicker or thinner based on
those kinds of conditions once you've absorbed to the point of reversal you
can rinse off anything that's not truly adsorbed on the substrate and immerse it
into another solution again oppositely charged and that material system will adsorb and we can continue this
alternating plus and minus to generate these multi-layered thin films now I
show these as green and blue stripes but in reality when they are polymeric
material systems they're highly interpenetrated however you can get
definition then plus or minus four layers which
means that if you create a series of layers which have one composition and
cap them with a series of layers of another composition you can create either gradients or our systems which
have two or more delivery systems combined now we can use hydrogen bonding
to generate these systems as well for example you can combine something that has a polyethylene oxide which is a
common biologically used polymer with some proteins so I showed you two
beakers but in reality in our lab we don't have students who have to move
back and forth with their wrists and generate these thin films very
fortunately we can use robotics to generate these films very effectively
and we can program those robots to generate any kind of film layer sequence that we want but even in that process it
can take anywhere from a few minutes to 15 or 20 minutes for each absorption
step which is wonderful for lab but it isn't great for manufacturing however
this is a very scalable process because you can actually use other methods and
here we're showing one method which is spray layer by layer that was an
automated form which was generated in our laboratory in which you can use
convection to spray dilute concentrations of these different systems onto a substrate directly spray
the rinse and then spray the other system and this is a actually a homemade
automated spray layer-by-layer system built by one of my former students who
later went on to start a company which made these micro sprayers and then went
on to generate large-scale layer by layer systems which can generate 50 feet
per minute of huge yards of layer by layer materials this demonstrates the
fact that this is scalable but spray layer by layer has also allowed us to KO
a broad range of substrates everything from very highly porous substrates to
flat ones here we can show a cartoon of an electro spun mat which has micron
scale fibers throughout and if we take a cross section we can see that we've
coated everything along the surfaces of these mats here you can see that there's
a nanometer scale coating on this fiber for example we can change the process
conditions to create content in films on these porous substrates as
well and this gives us a huge amount of versatility when we're generating
something like a tissue regenerative scaffold so I'm going to describe three
stories of how we use this layer-by-layer approach to generate thin
films for a range of different delivery applications but I'm going to first
start with why we're doing this in the first place the reason that layer by
layer is particularly compelling is because we're using water which means
that we can incorporate a range of drugs which are sensitive to solvent and
sensitive to heat biologic drugs proteins antibodies a range of si RNA
DNA mrna rot mod RNA a range of different systems which you normally
would not put in solvents and heat up on top of that if we think about a more
traditional polymer like poly lactic like colic acid as a carrier this
polymer would allow you to put in about one to two weight percent of a material
system like a drug then the rules of thermodynamics take over and you're
going to get phase separation because all polymeric systems can only contain small amounts of another material before
you get phase separation and if you get phase separation you're gonna get a
bolus release everything coming out at once on top of that if you put two three
or more drugs into a conventional codeine they're all going to come out at once on the other hand with layer by
layer we can select a degradable poly ion like this poly cation this is a
family of poly bait amino esters the main point here is that they have
positive charge and an ester group which allows them to break down and they're
biocompatible if we use something like this and layer it with a drug molecule
that is charged we can create thin films in which the drug is one of the components of the film now we've gone
from loadings that are one or two weight percent to loadings that are in the ten twenty thirty percent range and can be
as high as 50 weight percent this means that we can get a huge amount of the
drug into a very small film and this film is small enough thin enough that it
can coat a range of different substrates and fairly complex geometries on top of
that we compare each drug with a different polymer
and get a different release rate now we're actually tuning the release rates
of the drug if we're able to introduce separations between these drug layers we
can prevent inter diffusion of the drugs during the construction of the thumb and
with that we can get not only different release rates for each drug but a kind
of controlled stagger or stage-wise release of each drug and there are
several reasons why this is compelling one can imagine for example the desire
to release small molecules from an implant surface which may address pain
or infection followed by larger macro molecular systems like growth factors proteins that can allow or enable the
regeneration of tissue so with that I'm actually going to describe these three
different areas of work and the first one is about bone regeneration here
we're interested in being able to use this technique to essentially introduce
growth factors which normally are introduced in products in high
concentrations one of these is bmp-2 bmp-2 is osteo inductive it actually
recruits the native stem cells in bone marrow and allows them to differentiate
into osteoblasts what's nice about this protein is that it's potent it actually has a very high
osteo inductive effect what's been the downside of it is that when it is used
in the way that it's typically available a collagen sponge containing milligrams of BMP - there's a product for example
that allows a bolus release of this material you'll get a huge amount of it
released into the bloodstream and only a small amount that resides in the site at
the site of delivery so in these cases you end up flooding the bloodstream and
the body with BMP - and you can get ectopic bone growth you can activate
nascent tumorigenic cells and essentially activate cancer and there
are a number of other negative impacts of having large amounts of this molecule
and yet it's a very promising molecule for treatment so can we deliver this in
a physiologically relevant quantity and the time periods that are relevant not
in a few minutes or seconds but over an extended time period we're also
interested in growth factors that can support vascularization and some of
these include veggie is a direct angiogenic factor and plate the derived
growth factor PDGF which is mitogenic but also supports the generation of the
vascular system so these are proteins that are similar you notice that they
all have an isoelectric point of 8 and 1/2 that means that in water at pH 7 our
neutral pH their positively charged so we treat them as a positively charged
entity we adsorb them from very dilute solution onto these substrates and we
alternate them with a negatively charged species the negatively charged species
can be polyacrylic acid which is a very simple highly charged molecule or it can
be a glycosaminoglycan or a polysaccharide all of which are nicely
negatively charged and finally we're going to introduce a positively charged
degradable a biocompatible polymer here we've been using our poly beta amino
ester or P BAE layer and we call these poly 1 poly two we were running out of
names as you can tell in our lab and we named them but they give us an idea of
how long it takes for the materials to release in my first example I'm describing the work of my student in the
SARC sha and in this work the idea was to see if we could address large bone
defects these critical defects that cannot heal and replace what is the
known therapy which is to do an autologous bone harvest at a secondary
site usually the hip this secondary site often is the cause of morbidity
infection and a range of other problems on top of that a number of patients who
are suffering from either a large traumatic blast or shock or may be
experiencing some form of disease may not be able to donate very much bones so
there's a problem with this there are also problems with other approaches we
can look at other materials that can sort of cover or essentially protect
this area but then we have in issue with the material interface that
is generated as a result of that so we decided to investigate the release of Bro factors over very small with very
small doses over a very controlled time period from a scaffold that degrades and
to do this we started with PLGA which is a known fda-approved polymer and we generated porous membranes from them
using a very simple processing technique so these end up feeling like white soft
floppy films that you can cut out with a cookie cutter which was done here and in this case this was cut to the size of
our cranial defect model we then coated that PLGA which degrades over a reasonable timeframe of a couple of
months with a layer by layer film in here it's a little dark but you can see
that there's a layer by layer film sitting on top of this porous substrate
we can then allow release of the growth factor based on what we've done to
incorporate the material and in this particular example we built bmp-2
containing layers on the bottom and PDGF containing layers on the top if you
remember PDGF is going to help support vascular ization when we look at the end
vitro reliefs of this simple system we see that there's a slight delay of one
over the other so PDGF comes out over a period of the first week or so whereas
the bmp-2 is being released over a much more extended period which goes out to
about 30 to 32 days then we looked at what happens when we have single versus
dual growth factors and we also looked at dose here you can see micro CT of the
bone regeneration process here's our untreated control and you can see the critical defect is of course not closing
in this rat cranial defect model however if you look at the release of 0.2
micrograms of bmp-2 you can see that you are getting closure of the critical
defect and it seems to be fairly rapid with the 2 microgram dose we've
increased the dose by 10 but haven't really changed anything about the nature
of the closure of this wound over that time period which suggests that there is how much more can be impactful however
when we look at point two micrograms with bmp-2 and point two micrograms of
PDGF with micro CT we see perhaps a sign that there's somewhat more rapid healing
but we can't really tell what's going on with the bone until we look at the
histology and do mechanical measurements of the native bone and really examine
what what is being generated here so here you can see cross sections from
histology of the untreated defect in which we have this gap that remains and
the margins of the bone if you just have a PLGA membrane and nothing else that
you what you get a fibroblasts which crawl across the membrane and deposit
collagen you end up with this collagenous sort of scab like material but not bone if you look at 0.2
micrograms released from our scaffold what you can see is that you're going to
get bone generated and significant amounts certainly enough to bridge the
entire defect and this is taken all of these are taken at four weeks but you also see that the bone is somewhat
rough-looking and if we increase the dose by a factor of 10 we simply get a big lump of bone that has very little
relationship with the margins it's it's essentially a kind of cap of bone that's been formed however when we look at the
cascade of growth factors PDGF first followed by bmp-2
if you remember the time scales that PDGF is released
well more rapidly and over the first week and that bmp-2 is being released over this period of a month what we're
seeing is the result as a result is a more vascularized bone that appears to
be more like the margins but we're also seeing more complete remodeling rather than seeing a large number of defects
and bumpiness at the surface we see a surface that is very similar to the original cranial surface of the bone if
we examine the bone look at whether we have trabecular bone or not we see that
we do we generate very high molecular weight material I mean high mechanical
properties in these materials and we also are able to with mechanical
measurements compare them to Nate phone and see that they match them
native phone unlike what we've generated in the first two examples in which the bone is more brittle and breaks more
readily so we think that what we've done is introduced vascularization earlier in this phone so this bone is a
healthier bone it's much less likely to become necrotic but maybe as importantly
we've introduced pathways for a second wave of osteo class rather than osteoblasts in osteo class actually
remodeled bone and that bone remodeling is what is allowing us to achieve bone
which has the mechanical properties that we need now along with looking at the
cranial defect we've also been looking at ways in which we can integrate bone
onto implants and this is actually a growing and incredibly important problem
because it turns out that orthopedic implants and their use has increased
rapidly it's in fact because our population has grown older and is
healthier we have more of our population that has a whole joint implant shoulder
hip knee the fact that we're also a more active population adds to the
probability of seeing these implants in use more importantly there are more
revision arthroplasties that is the replacement of these implants after they
wear usually they last for about twelve or fifteen years and after that period
of time just the relative amount of loosening that can take place over time
and can require that the old implant needs to be replaced we want them to
last as long as possible but we're ultimately going to need to replace this
implant over the lifetime of many patients and what happens in that case
is that the infection rate soars from the primary case to the secondary this
revision case from lower than 1% - depending on which review article you
might read 10 to 20 percent there's a very real increase in infection so we
have a concern about this on top of that we would like to generate a very strong
bone interface the native bone and the implant the
better that interface the less likely that infection is going to return we are
interested in being able to accomplish this using a singular coating because
the common treatment right now is a two-step process in which you
essentially remove the implant from the patient you back fill the empty joint
area with these antibiotic beads which release over a period of six weeks or
more you brace the patient you wait and then you go back for a second surgery
clearly this to surgery extended hospitalization or a mobilization period is not only going to introduce more
stress on the patient but also more cost in the system so in our work we've been
looking at ways in which we could generate an osteoconductive base layer
because we're no longer working with a degradable membrane but with a stiff
rigid material like titanium or peak and we want to make sure that we have an
environment on which bone cells can grow and adhere so in our case that base
layer is going to be composed of a chi design wrapped hydroxyapatite
nanoparticle that has a net positive charge and will be alternated with
polyacrylic acid we just put down a few layers of this material we can think of
it as a system which doesn't degrade over the lifetime of our release film
but will be adsorbed or resorbs by cells over a longer time period now we have
this base layer that will allow deposition of bone and on top of that we're going to place layers of bmp-2
which can release over an extended period of time so this is our early
experiment we haven't introduced the anti-infective yet we'll get to that but we first had the ATS had to ask the
question of whether or not we can generate a strong bone material
interface so here we used a rat to be a puncture model which is demonstrated
here we create a defect in the rat tibia we take either a plastic or a titanium
implant coated or uncoated inserted in and then over time we are going to
observe the bone material in face using several metrics including
histology mechanical properties and just general measurement of the concentration
of growth factor and other factors in the area so here you can see the plastic implant that we used which was piqué and
piqué with holes drilled in it what we did was we coated these two examples
which represent a smooth implant and what we were trying to replicate as a
textured implant was coated with bmp-2 that was labeled with a fluorescent
protein so here we can actually see this release over time and it's a little bit
dark here but you can see that it's releasing over a period of 30 days if we
look at the radiant efficiency over time we can see that the release is actually
quite constant and it's taking place over this period of a month fairly
consistently we looked at different loadings 20 40 and 60 layers a representative of these three different
loadings and we look at the concentration of BMP T that B and p2
that is accumulated in this area of interest which is shown here by
excavating the tissue and determining how much bmp-2 is there and what's
interesting is the numbers here you can see over a period of 30 days for the
smooth versus the the porous or roughened implant that we can get a more
extended or sustained release when we have this texture on the surface or
roughening but what's also interesting is that at any given time the
concentrations don't exceed 200 nanograms per milliliter and we were
talking about the relevance of being able to control the amount of bmp-2
released here we're getting enough BMP to in the region to make a difference
physiologically without creating enough of a sink of BMP to to flood the
bloodstream with the growth factor we looked at histology and I'm only showing
you one example but we have an entire library of histological slices of the tissue this one is meant to show the
interface between a part of the implant this is the plastic and the bone and we
can see that the bone is very nicely arranged and oriented around this
plant we actually examined the nature of this phone and found that it was very
nicely oriented it had excellent mechanical properties on top of that we
did a pullout test in which we determined the amount of force required
to pull the implant out of bone and we did it at a period of one week two weeks
in four weeks here we can see the untreated control at the bottom and
these implants actually show that there's some bone that has grown near
the implant but it's not integrated into it and it's very easy to pull these out
but the pull out forces that we see with these different doses indicated by different numbers show that in the first
couple of weeks there's a dose dependence and a very strong increase in
the pull out force but by the time we reach four weeks
everything has leveled off and we see that even our lowest dose system is able
to provide a very highly integrated implant bone material interface and in
fact the strength of this bullet pullout force is about four times that that is
generated with polymethylmethacrylate bone cement which is commonly used we
can also note that when we pull these out at this point we see that the
fracture takes place in the bone the native bone and not at the bone material
interface so now we want to introduce an anti-infective and the way that we do
this is by introducing gentamicin sulfates turns out to be another charged
drug molecule and here my student Zhu how men came into the picture she began
to first model these systems and their release characteristics and then to
build them here she's using red to demonstrate the gentamicin sulfate and
green to represent the bmp-2 and she quickly began to construct
material systems in which there was no barrier and you could get some degree of
stagger but she also introduced barrier layers by using naturally occurring lappa night clay this is just a very
thin disc like nano clay they're used for a number of different applications and our case we chose the
system because the it exhibits good biocompatibility a
number of these laughing nights are generally regarded as safe and they're
very low-cost and biodegradable they are also potentially bioactive they can
actually enhance cell adhesion at surfaces and interfaces so what we're going to do is use them as a kind of
barrier layer in between the two drug layers the question here is can we get a
stager that we control on the Left what you can see is the thickness in the film as zhuhai built up differing numbers of
bmp-2 layers then she introduced these ultra-thin lappa night layers then
gentamicin sulphate and then in some cases she capped that off with another
lap a night layer the question was whether we could segregate the two drugs
into compartments and here you can see with electron microscopy and a method
called EDS that these dark regions are the drug containing layers these bright
regions are the lappa night capping and intermediary layers that separate them here we can see red as the scylla
containing silicon containing layers these are the Clay's that are
essentially separating our materials when we look at the reliefs between
these different systems with just the bmp-2 and gentamicin sulfate we get some nice results we see a rapid release of
gentamicin and we do want enough to eliminate an existing infection and
therefore this bolus release is not bad and we see that there is a delayed
release of bmp-2 however we see that there's a significant amount of bmp-2
that's coming out in the first four days where this is x-axis is in days and here
we're seeing a blow-up of that release behavior if we have this barrier layer
in between the two it turns out that with the bmp-2 we actually can decrease
this rate of release we get in fact a 14 times longer release period or half-life
and you can see this in the blow-up a little bit more easily you've sort of
flattened this curve over the first several days while you're getting rid of infection very rapidly with the
gentamicin sulfate and of course we can tune this even further with the capping
layer Juha was interested in this because she
wanted to be able to demonstrate the release of enough gentamicin sulfate in
the beginning to eliminate infection so you have this initial bolus
combined with the ability to maintain this seems this critical concentration
to prevent infection over longer periods of time so when we then move back to our rat to be a model now we've generated an
infected rat to be a model so juha generates a biofilm in this to be a defect cavity you can imagine this
is a rather challenging model so this was a nice little chunk of her PhD
thesis and she then is able to insert either an uncoded implant an implant
which contains gentamicin sulfate are one that contains the combination of both and what we've been observing is
that the combination of BMP two-engine termites and sulfate leads to a much more rapid regeneration of bone around
the implant furthermore if we look at the micro CT
we can see the resorption that occurs in the untreated sample we can see that
with just gentamicin sulfate bone is enabled it can begin to regenerate but
it's not quite as complete and with the combination we're getting a much more
solid slug of regenerated bone present in these systems and so now our hope is
to be able to continue to look at these compartmentalized systems but to use a
much more systematic approach to tuning the relative release of these systems
using barrier layers and also begin to introduce antibiotic combinations or
multi spectrum antibiotics that will give us a broad spectrum of attack
including addressing mersa all right I'm going to move to the second example
which is a soft tissue example here the idea is to try to address wound healing
processes that have gone awry and the idea is that a number of these wound
healing processes have over expression of a specific gene or some sort of
genetic dysregulation our earliest target was to target chronic wound healing and the idea was to correct the
over expression of certain genes that are overexpressed in hypoglycemic
patients during the wound healing so the idea is that we can try and
address some of these overexpressed proteins one of which is mmp9 this is a
protease that is of a natural part of the remodeling process that all wound
healing processes must go through but mmp-9 shows up earlier in the wound
healing phase in these diabetic patients and this has been demonstrated for a
number of different studies so the idea here is to knockdown mmp-9 for a delayed
for a small period of time and effectively enable the deposition of
granulation tissue or collagen which will then help to lead to wound closure
and I apologize it looks like there's a little disconnect with my computer in
the screen but what you're seeing here are the same components we talked about
before a degradable polymer poly 2 and here we use tyrosine which is positively charged and not degradable over short
time periods and dextran sulfate another natural sugar and the way we compose
these films was essentially to build an under layer of dextran sulfate with this
poly 2 and then on top of that layers si RNA in Qaida's an and in these studies
we actually used an unmodified version of Si RNA which is interesting because
we were definitely meeting the challenge si RNA degrades very rapidly in the
blood stream but we're going to put this in a wound cavity where there's a large
excess of proteases cytokines and other highly aggressive reactive molecules we
directly incorporated si RNA into these films and layered them onto a nylon Tegaderm bandage here si RNA is labeled
with this fluorescent markers so you can actually see it in compo Co we're
showing that we're coating all of the crevices we changed the relative number
of layers of the under layer to change the release rate and the top layers to
change the loading so here we're getting very significant loadings of Si RNA in
this range of 10 to 20 micrograms per centimeter squared we then used the
diabetic mouse model and we'll have a control wound in which we don't reduce any treatment we also have a
second control in which we're using a scrambled SI RNA doesn't encode for
anything and we're using our si mmp-9 treated system if you look over time at
the gene expression in these animals at one weekend at two weeks we and we were
essentially designing these systems to be active over a 14 day period what
we're seeing is a knockdown of mmp-9 in the wound of 60% at one week and 85% of
two weeks which is significant especially given that these are
unmodified si RNA we also see a corresponding drop in in mmp9 activity
if you look at mayson's trichrome staining we can look at the seven day
and the 14 day treated samples and here at the top we have the SI RNA treated
versus the seven day untreated and we have so have our 14 day treated and untreated and what you'll see is there's
a bridge of collagen in this stained blue sort of ribbon at the top here that
is bridging the wound at 7 days which we don't see in the untreated samples and
when we get to 14 days we see that there's a large amount of deposition of
collagen present compared to the untreated wound and here you can see a
close-up of that tissue so there's a stark contrast between the two and we
also see very real contrast in the way in the rate of wound closure in these
systems here we have a 7 day and 14 day and the dark bar is our treated sample
and we also see an improvement in the percent epithelial closure in these
animals this is really just the beginning there is more than one gene involved of course and these kinds of
wound healing processes and what we're interested in now is combining targets
with Si RNA and using growth factors in our combination systems to be able to
address this more effectively by replicating the wound healing process
another area of interest a real target for us is trying to address hypertrophic
scarring this is actually something that can
greatly influence those who are the victims of 3d I'm sorry of
third-degree burns those who have experienced large traumatic blasts a number of these patients when they heal
heal with large amounts of hypertrophic or highly fibrotic scarring and the scarring can actually reduce their
mobility there are a number of treatments that have been proposed but
most of them work primarily for small areas we're interested in being able to
address larger area kinds of wounds so in this work sort of summarized in this
slide we looked at B&B ball knockdown of one of the girl factors in the pathway
of fibrotic response this is connective tissue growth factor here we're trying to dress a different
part of the wound healing process and chronic wound healing
we had the difficulty of actually getting the wound healing process
started and we needed to get collagen production to begin here we have the
opposite problem a kind of over healing in this fibrotic stage the the deposition of collagen is accelerated
and we can't stop it so we're trying to knockdown CT gf so that we can lower
that fibrotic response and what we're looking for here is essentially this is
a third degree burn model that is carried out at the Shriners Hospital
Shriners Burn Center at Mass General Hospital with Martin Garmisch and in
this burn model you can suture the edges of the burn and then watch to see whether or not the burn contracts if
this tissue contracts the way it shows here this contractile wound healing is a
part of the fibrotic response in our system you can see that with just a
control we get the same kind of shrinkage of the wound but with the
knockdown of seat EGF in these cases we can see that there is a real reduction
in this kind of contraction what's interesting here is that we didn't quote
an entire bandage we only coated the suture we're actually literally coating
the thread that is used to sew up the animal and we're seeing that there is a
meaningful response to the SI RNA it's being released from that suture so we think that the application of this
approach is very promising because we can control the deposition of the our
multi layers on a number of different surfaces that might be used in this
wound healing application it's a little difficult to see here but we did look at some of the characteristics of scarring
and what we found was that for our si si TGF systems we increased vasculature and
it's a little hard to see but these are blood vessels where the black arrows are
compared to our scar tissue which shows very few if any blood vessels and we
also see an increase in the size of the blood vessels we also had a much rougher
epithelial surface and the indication of structures forming that are essentially
similar to what we would expect for the formation of hair follicles or sweat
glands so we think that this is the beginning of some very exciting work in
which we may be able to address fibrotic scarring using a very rational approach through the delivery of controlled
factors and SI RNA alright so we've moved through two examples in which
we've talked about wound healing and we've gone from bone which is you can
imagine something you can hold in your hand and imagine to these micro scale
meshes which we can cope very readily and I'm going to talk about coding
something on the nano scale and in this work we're working at the Koch Institute
with engineers and physicists and chemists biologists and clinicians to
address cancer and what's what's exciting about this work is that we can
use a very modular platform to integrate a rational approach to cancer the idea
here is that we can take a nanoparticle core and that core can be any core in
our case we're interested in vessels that have already been fda-approved
so it could be a PLGA nanoparticle it could be a lipids ohm that has a
negative charge and into that core we can incorporate a chemotherapy drug
we're very interested in highly aggressive forms of cancer
ricoeur or that are very resistant even on the first treatment to the
chemotherapy drug a number of these aggressive forms of cancer have
undergone the kinds of genetic modifications that allow them to encode
proteins that saved them from the impact of chemotherapy drug so the idea here is
that we can incorporate into the outer layers si RNA or perhaps an inhibitor
that will block that pathway for cell survival in the presence of the
chemotherapy drug finally we rely heavily on the fact that
we're using a poly electrolyte approach because this last layer that's put down
is a highly negatively charged layer and that's going to provide us with a nanoparticle that is much less likely to
engage with other cells when we introduce this into the bloodstream and
I'll talk a little bit more about the role of that outer layer so this is the
general idea making it enamel sized helps us because in cancer therapies
we're trying to take advantage of the fact that solid tumors or at least a
large number of solid tumors grow very rapidly in that rapid growth they
generate defective vasculature and the defects are holes in those blood vessels
provide us a route for nanoparticles to essentially extravasated to the tumor
tissue and give us an additional residence time in that tumor so you I showed you the slides with the beakers
you're probably thinking what are you gonna do with nanoparticles to get these
nano layers on the surfaces in our work we can use an alternating titration
adsorption step with a centrifugation step we're beginning to replace the
central fugacious step with a whole fiber filtration step which is turning
out to be very fruitful for us and also speeds up the rate of generation of
these nanoparticles significantly I have very happy students right now because of
that now this is showing you a quantum dot a lead sulfide quantum dot that had
a net negative charge and what happens is we begin to put layers down on that
quantum dot we chose this because it's small it's 20 nanometers and you can
actually see the impact of the of each layer on the
the nanoparticle and you're seeing that you're adding anywhere from five to ten
nanometers perception stop you can also see the charge going from plus to minus
as you build up the layer by layer film in these nanoparticles when we first began to look at this work we chose some
very simple polymers to cope with we used poly l-lysine and poly l-arginine
as friendly polypeptides and we used a number of the glycosaminoglycan family
as our negatively charged system and i'm describing some of the work of Stephen
Morton who was a graduate student in my lab when we began to examine the
behavior of these systems we we made several interesting observations the
fact that this is a layer by layer film gives us an advantage because we can
layer a film which has a very stable nature to it at pH 7.4
however with hyaluronic acid it turns out this particle built at pH 7 point 4 gives us the strong negative charge
which begins to disappear if we move it to slightly more acidic environments
because what's happening is we're beginning to protonate those carboxylic
acid groups and as we do that we're beginning to erase the negative charge
then that impact of that is twofold one of them is that if you erase the
negative charge you're erasing some of the electrostatic interactions and the
film's cross-linking decreases and it begins to swell now rather than this
compact negatively charged halo we have this big fat but rather soft swollen
layer around the nanoparticle the second thing that changes is the net charge it goes from minus 30 millivolts to almost
plus 4 millivolts its charge shift is significant because we were relying on
the fact that it was strongly negatively charged as one of the mechanisms to avoid interactions for cells on top of
that we were relying on the fact that hyaluronic acid is highly hydrated so we
believe that these nanoparticles have a large amount of bound water around them
and it's that bound water shower which is helping us to repel the adsorption of
proteins in the bloodstream and that gives us a long residence time in blood so the interesting thing here is that
with these two changes we see a huge increase in cell uptake nonspecific cell
uptake this means that if we're able to enter a tumor that is hypoxic we may be
able to enhance the uptake in hypoxic tumors based solely on the physical
chemical properties of the layer-by-layer film so we tested this
and we found that when we injected animals with solid tumors and looked at
where those nail particles localized they Co localized strongly with hypoxic
regions of the tumor here we used hip 1-alpha as a way of labeling these
hypoxic regions the nanoparticles are labeled red and you can see this overlap
in this yellow zone here however getting into the tumor is a good thing and
encouraging uptake through these nonspecific interactions can also be
good but we can't rely only on hypoxia tumors are highly heterogeneous we need
a way to ensure that these nanoparticles are taken up by tumor cells and in this
case we decided to use molecular targeting in our systems we can attach a
range of different ligands from folate to very specific binding peptides that
bind to receptors that are overexpressed on cancer cells and we have indeed done
that with a number of our layer-by-layer systems but it turned out that because
we were focused on ovarian high-grade ovarian cancer triple negative breast
cancer and some of the advanced lung cancers all of those cancers over
expressed the cd44 receptor which happens to be the higher Doronin receptor as it turns out that high
affinity for cd44 allowed us to use just the hyaluronic acid outer layer as the
targeting molecule so once again we did a series of experiments to show that we're getting targeting based on cd44
interactions with hyaluronic acid and I'm describing the work of my postdoc Eric Dryden and this work we looked at
xeno grass of a triple negative breast cancer and we did show that these
overexpress cd44 when we look at the uptake of these nanoparticles labeled
red in this image here we see a huge amount of uptake about ten times that of a layer-by-layer
particle that contains dextran sulfate similar charge no hyaluronic acid and we
can also tie treat out the interaction by adding excess hyaluronic acid and
here you can see that almost completely eliminates the uptake when we do a tumor section in this case and label cd44 with
red and the nanoparticles with green again we can see a huge overlap between
the localization of our nanoparticles and the localization of the tumor cells and what we see in this outer region are
the stromal cells that surround that tumor which suggests that we have a mode
of triple targeting we're using nano particle size we're using the tumor
microenvironment or hypoxia and we're using the cell receptor interaction to ensure that these nanoparticles are
taken up now that we have a system that can do some form of targeting we can
load the inner core of the nanoparticle of a chemotherapy drug and we can begin
to layer our si RNA we actually found that the positively charged poly-cotton
that we use in that layering process greatly impacts whether the s RNA gets
both into the cell and out into the cytosol it turns out that poly
l-arginine is a polymer that undergoes a great deal of buffering and therefore
enables these nanoparticles to essentially compromise the endosomal
membrane and that allows SI RNA to get out so what we began to develop then was
a four layer nanoparticle we would start with a corm nanoparticle and then we
layer poly l-arginine SI RNA Polly l-arginine and hyaluronic acid plus
minus plus minus and that would be our outer layer system we found that when we
generated these nanoparticles we got very nice blood half-lives this is key
because we need a long residence time in the blood stream to be able to allow nanoparticles to accumulate in the tumor
in this case we found that we had a 28 hour half-life which is actually quite
extended for these kinds of nanoparticles the size of these nanoparticles are 100
nanometres I should also mention that we didn't observe any kind of amino or
saddle toxicity we did of course use a fairly simple panel of cytokines to
examine that if you look at the release characteristics of the nanoparticle once
again we see the staggered effect we can release si RNA over a more rapid time period of about 24 hours in this case we
load our lipids ohm with doxorubicin so we have this Co delivery we're using a
negatively charged phospholipid LIPA's ohm doxorubicin is water-soluble and
goes into the interior of this lipid zone and si RNA goes on the outside and
we get our stagger our s RNA was si RNA against multi resistance protein one our
MRP one which is a drug pump that pumps out doxorubicin even as it had arrived
at the cell membrane surface thus preventing it from interacting or
engaging the nucleus and killing tumor cells in this work by Jason Dane who was
a postdoc in my lab we looked at a series of injections in these animals which contain xenografts of a triple
negative breast cancer we found that we were able to get significant knock-down
after treatment here we're looking at tumor sections that were staying and
here we're looking at PCR which shows that there is roughly 75 to 80 percent
knockdown of mr p1 in these tumors when we examine the using a it's a xenograft
model tumor remediation in these animals we find it in the Saline control we're
getting of course this rapid growth of the triple negative breast cancer if we
look at doxorubicin with the scrambled SI RNA we see that we are reducing the
growth of these tumors but not their size they're still essentially doubling
in size however if we treat these with the combination system we do see that
the tumor size is getting smaller and we're getting some real regression so
we're very excited about these results and we began to move to a more
challenging model Tyler jacks at the Koch Institute has developed a model in
which these mice are essentially genetically modified so that they have
nations inkay wrasse and in p53 and I won't have time to go into the details
but these are two of the highly known oncogenes inhibition of K rats therefore
is highly desirable but small molecules have not been developed to effectively
inhibit this K rash gene it's also known that p53 which is one of the Guardian
genes that prevents the kinds of tumorigenic activity that we see in cancer cells is also lost so we're going
to introduce a micro RNA to restore the p53 function in these cells again we're
going to build up a four layer nanoparticle as shown here but this time
with cisplatin and the two different RNAs si RNA against the K wrath uncle gene and micro RNA Mir 34 and here we're
just describing this orthotopic tier ass mutant model I won't go into the details
but what it does allow is for us to activate tumors in mice and these tumors are small tumors spread all the way
across the lungs we looked at the act of targeting of our nanoparticle and
healthy mice so here we do at L vein injection and we see that the nanoparticle resides throughout the
mouse's body but there's no particular localization versus mice which exhibited
these lung cancers these mini tumors we saw that over a period of time we got a
large accumulation of the nanoparticles in the tumor lungs and just to see the relative differences here you can see
the lungs of the healthy mice versus the ones that contain these tumors and here
you can see the relative difference in recovered fluorescence and we're looking
at orders of magnitude difference in the amount of accumulation of these
nanoparticles in the lung and this is due to the fact that again cd44 is
highly over expressed in this one cell lung cancer type in our in vivo
treatment we used again a this orthotopic model so
a little bit hard to see the tiny tumors on the screen however you can look at
the fold change in tumor volume here and see the vehicle versus RNA cisplatin and
the combination and we see that there is a very meaningful difference when we
look at the combination therapy both in survivability and in fold change of
tumor volume we also can check and see whether or not we have modified the pathways we think we're modifying and we
see from this series of staining against Peter and a range of other proteins that
we have down regulated the krad pathway that we have reinstituted the p53
pathways and that we have enabled cell death through caspase-3 so I'm coming to
the end of my talk I'm going to give you a very short example this is a
microsecond extension of our nanoparticle project and I'm bringing it
up because one of the exciting things about working at MIT and at the Koch
Institute is being able to work across disciplines a lot of the work I
described previously has benefited from the collaborations of Michael Yaffe who's a systems biologist and a range of
clinical clinical researchers at Dana Farber and Mass General Hospital I also
have a collaborator who's another engineer Angie Beltre and she engineers
a range of different inorganic molecular systems using biological methods she has
a mom of her innovations been looking at ways to develop nanoparticles that adsorb in the second window near-ir
this is a window of near-ir we haven't taken advantage of as well but would
allow us to get much deeper penetration for imaging of tumors and in this work
my postdoc Lee and Angie's purse pasta Shannon have worked together to see if
we can develop an imaging nanoparticle in which the core contains one of
Angie's down-conversion nanoparticles and the outer layers contain an LDL film
which could ostensibly contain si RNA plus a range of inhibitors or
chemotherapy agents so we actually looked at a range of different layer by
layer systems to compare with Angie's quantum dot and down-conversion nanoparticle and
this is just showing some of those characteristics and images of those particles and what we found is that by
using Angie system in comparison with the others we can completely escape a
range of autofluorescence that is considered a tween swith dye molecules
and single wall nanotubes and actually see what's going on inside of these
animals without that autofluorescence using the down-conversion nanoparticle in quantum dot in fact with the
down-conversion nanoparticle we can see fine structure of blood vessels muscular
skeletal structure and even the beating of the heart is much more visible so
we've been able to apply this to the imaging of ovarian cancer tumors by
using that hyaluronic acid binding capability and here we've shown in an
orthotopic ovarian cancer model that we get a huge amount of sort of
concentration of these nanoparticles in the regions where we have the tumor and
here you can see just images that are sections in which we compare the
nanoparticle accumulated in tumor versus healthy tissue and you can get down to
sub millimeter scale resolution with these systems and actually see and
differentiate the tissue between tumor and pancreas gut liver and of course we
get a very nice view of solid tumor tissues so right now we're developing this technology to see if we can
generate these kinds of theranostics systems in which you can monitor ovarian
tumor suppression at the same time that we're delivering therapies so in
conclusion I talked about the fact that we can use this very modular water based
approach to incorporate a range of different molecules and that they can be
used for very significant wound healing applications including the cranial
defect example I didn't talk about but we've also had collaborations with
members of the Koch Institute including Darrell Ervin on the incorp raishin of layer-by-layer films for
vaccines and i talked about how we can use these kinds of layer-by-layer films
nano films on the nano scale and generate very effective targeting
nanoparticles I think this is a very modular approach and this is why we're
excited about it it can be applied to a very broad range of surfaces and it can
also be adapted for use with a range of other kinds of tools for health
applications so what we're showing is that electrostatics is a very critical
tool that can be used for controlled release and that this approach can
introduce functionality that can lead to very promising biomedical applications
I'd like to thank the contributors to the work I highlighted my students and
postdocs in bold whose work I highlighted here as well as my
collaborators Myron Spector and a lot of our bone work Michael Yaffe who's the
biologist at the Koch Institute we've worked with Angela Belcher right now
we're working with Mike beer at Mass General Hospital to address some of our
ovarian cancer applications and I'd like to acknowledge the NIH for funding a
number of these early discoveries in our lab and for continuing to support our
work and I'd like to thank the al Varian Cancer Research Program and Research
Foundation and welcome any questions that you may have thank you
thanks very much for a fascinating romp through a lot of applications and please if people have questions come to the
microphone so those watching can hear and please start here thank you very
much for a wonderful presentation what I was interested in understand is how
consistent is the loading of your experimental your actual therapeutic
agents into either your films or the nanoparticles and what level of
variation you tolerate and as we really want to see this moving forward into a clinical what do we think the FDA is
gonna say about that how consistent do they need to be excellent actually they're extremely consistent one of the
nice things about this polyelectrolyte approach is that charge is very
predictable so if you assemble these systems under the same conditions you end up with the same amount adsorbed
if we look at our you saw several curves in which we were building films and you
see these linear curves that linearity is beautiful and it's always there so we can go back to the same conditions and
generate the same films this has been shown by the fact that for example in
the bone work we've passed on this formulation from across three students and a postdoc different hands same
loading and and I think this will help us with the FDA that we can actually use this as a very reproducible effect you
have to do something quite intentional to disrupt the the sort of electrostatic
adsorption behavior of these systems so that's called a biological replicate
when it's more than one postdoc doing this what happens to these nanoparticles that you've put into the body where do
they go how do they ever disappear very good question although they have a very
long half-life they will accumulate in the liver and the kidney and this is why we've been measured trying to measure
signs of distress we measure body weight and we also measure cytokines to see if
there's any inflammatory response or any other kind of response we've also taken
out the liver and resected it to see if there's any long-term accumulation in
the liver while we find is that they pass out of the liver so if you wait
roughly 24 hours to 36 hours later you're not seeing these nanoparticles in
the liver anymore so over a period of time they're actually somehow breaking
down and getting out so they are biodegradable there they are now each of that is a good point each of the systems
that we choose for the nanoparticle applications contains degradable
components so we're using a phospholipid lipids oum and then layering it with
these hyaluronic acid containing systems so we try to maintain a system that's
going to either be resorbed or broken-down congratulations for pushing
the difficult frontiers of nano medicine so in one of the studies is certainly
showed that the talc powder causes cervical cancer in fact it was recently settled so as you are putting the layers
he also have to be careful which might be the acceptable model versus which
could cut into you into trouble so in terms of multi-layered particles I was
looking at it you have the layer of hyaluronic acid is there any speculation to prevent phagocytic removal of the
nanoparticles excellent we we actually considered peg versus the hyaluronic
outer layer and there's a couple of papers that you'll find of ours in which
we capped off the final layer with a peg brush we actually find that the half-lives are very similar or better
for the hyaluronic system and there are there reasons for this and in fact there
are other research labs who have used hyaluronic acid as well and they're
beginning to use it more frequently and in fact Shawn Chen is one of the labs
that has looked at this to some extent but we think there are several reasons
one of them is that hyaluronic acid itself has a great relationship of water
and for that reason that bound water is helping us with the issue of both steric
repulsion it provides some steric repulsion but it also provides a bit of
a cloaking layer behavior it prevents optimization so proteins tend not to
absorb as readily on these surfaces but we also think that the presence of the layer by layer film has an influence so
when the seduction takes place the poly electrolyte is absorbing on a
positively charged underlying surface but it's not a flat surface it's another coiled polymer chain and we end up
getting these loopy structures that adsorb so the molecular conformation of
the polymer chain is a little bit different we think that we have this
brush like structure formed from these loops which is actually providing a much
denser cloud of water and associated ions then you would get if you had just
an extended linear chain extending from the surface of the palm of the nanoparticle it's very interesting talk
polysaccharides you know is a very common vaccine component they are at
least 23 different part in pneumococcus they are 23 and they are licensed
meningococcal therefore and Hamas influenza there are mostly
negatively-charged you probably know have you thought of using any of them the vaccines as you're coding or Seward
a year ah very interesting that it would be you know like delivering the vaccine
as well as for other purposes - excellent in fact I had a very
interesting conversation today with I Joe bars bars it was someone who is
actually working on glycol glycolic materials and he talked about the fact
that he can create glycol peptides that are highly immuno genic this was actually very interesting to us because
we think we can combine those kinds of interactions with our layer by layer
system initiate in immune response and then release an antigen and and have
control of the rate of that presentation of antigen and that's interesting to us
we have a collaborator of Darrell Ervin and we've been begun to talk about using
our layer by layer approach for immunotherapies for cancer and we have
our first student who's going to be working on this so these kinds of ideas
are very helpful at this stage I do think it's interesting what we've
learned from the micro needle work I didn't talk as much about was that we
can a nucleic acid that encodes for these or
a protein onto these layer-by-layer films get a very slow release and for
various reasons getting the slower release of the antigen seems to give us
a more sustained memory t-cell effect and that may be something that we can use for immunotherapy against cancer if
we can get this more sustained release yeah the licensed vaccine can be coming
two forms one is the party sacra alone and the other one is a chemical conjugate into the protein I have
another question that is the one of the most common negatively charged party electrolyte is nucleic acid have you
ever thought of using poly nucleic acids could you say just one party nucleic
acids Oh poly nucleic acids right yes yeah yeah regularly negatively charged
we have so ironically we are coincidentally we have a project in
which we have been generating very large
hairpin shaped RNA essentially it's it's a kind of multi periodic RNA structure
and that work was really directed toward a different part or a different approach
to si RNA therapy because these break down in the presence of Dicer into
hairpins that can be used for delivery but we're discovering that those
structures are activating the in Kappa beta
mekka mechanism in cells and we're actually getting an inflammatory
response that seems specific or at least highly amplified in cancer cells so what you're saying is I think what we're
beginning to observe with these molecules so recognizing that we are a
bit after the hour and I know people have things they need to do maybe others who have questions can pose them to our
speaker as we all migrate towards the library for a reception with some coffee
and cookies but please join me in thanking dr. Hammond again