Bioadhesive Nanoparticles for Skin Cancer Treatment and Prevention

Transcript

• 00:002 Long term colleagues and friends from Yale.
• 00:05Today one is there actually speaking on
• 00:08the same topic which is by which he said
• 00:11degradable nanoparticles for skin cancer.
• 00:13So it's great to have them here today.
• 00:17And one second left to find some notes right?
• 00:20OK, so our first speaker is Mark Salzman.
• 00:23He's with Cela foundation professor,
• 00:25biomedical engineering and professor
• 00:27cellular molecular Physiology.
• 00:29He focuses on trying to develop
• 00:31methods for disease prevention and to
• 00:34effectively deliver drugs to cells,
• 00:35particularly to deliver
• 00:36chemotherapy to brain tumors.
• 00:38He's interested in controlled drug
• 00:40delivery to brain bow polymers,
• 00:42to supplement or stimulate immune function.
• 00:45Still, interactions with polymers.
• 00:47In tissue engineering and in fact he's
• 00:49developed what is now the standard care for
• 00:51treating some brain tumors is very exciting.
• 00:53He will be joined today by
• 00:55another colleague and friend,
• 00:57Mark Mike Gerardi,
• 00:58who's a professor of dermatology.
• 01:00He received his MD degree
• 01:01here as many of you know,
• 01:02and also his clinical training here.
• 01:05His principal focus of research has
• 01:07been on the relationship between
• 01:09the immune system and cancer
• 01:10with clinical expertise in areas
• 01:12including cutaneous T cell lymphoma,
• 01:14squamous cell carcinoma in the and
• 01:18extracorporeal photochemotherapy.
• 01:20He's credited with major contributions
• 01:22to understanding skin biology,
• 01:24immunology and skin cancer development,
• 01:27and has actually foot co-founder
• 01:29of two Yale startup companies to
• 01:31exploit some of these discoveries.
• 01:33So today they're going to talk on a
• 01:36collaboration looking at Bio case of
• 01:38nanoparticles with long interest of
• 01:40Doctor Salzman to treat skin cancer
• 01:42along interests of Doctor Gerardi.
• 01:44And so I think it will be very exciting.
• 01:47Talk about a new approach that's
• 01:49an alternative to fit too.
• 01:50Surgery so will start out with Mark.
• 01:55Great thank you. Thank you Dan.
• 01:56It's a pleasure for me to to to be
• 01:59here and to speak in this forum again.
• 02:03I I was. I was hopeful that that this
• 02:05month we'd be back to meeting in the
• 02:07usual way where I actually have to
• 02:09stand up to give a talk but but we
• 02:12will do it this way and I look forward
• 02:14to talking to you today about this
• 02:17work that that Michael Gerardi and I
• 02:19have been collaborating on over the
• 02:21past over the past several years.
• 02:24First some.
• 02:25Financial disclosures the most
• 02:27important one here is the top one.
• 02:31Mike and I have our Co founders of a
• 02:34company called Stratified Biosciences
• 02:36which is licensed intellectual property
• 02:39to the technologies that we'll be
• 02:40talking about today and we receive
• 02:42some research funding from them.
• 02:44Next so I'll start with just a
• 02:49general introduction to both.
• 02:51Health care products that are
• 02:53collaborations of physicians and
• 02:55engineers and then to some biomaterials
• 02:57and then to the particular technology
• 02:59that we've used in this project.
• 03:01And so you know,
• 03:03general statement that you probably
• 03:04all know many of the products that
• 03:06make modern healthcare effective
• 03:08are innovations that came from
• 03:10collaborations between physicians
• 03:11and engineers and the first one I
• 03:14show here is is hemodialysis for
• 03:16treating end stage kidney disease.
• 03:19This is a medical device with
• 03:20a specially designed material.
• 03:22This is.
• 03:22Responsible for his most important function.
• 03:24In this case, it's a.
• 03:25It's a polymer hollow fiber that allows
• 03:28for separation of waste products from blood.
• 03:31The second is shown here on the on
• 03:34the right is drug eluting stent.
• 03:36This is one that's made all of polymers.
• 03:38Stents have made remarkable progress
• 03:42for treating. A vascular disease.
• 03:45This is again a medical device
• 03:47with a special function.
• 03:48In this case.
• 03:49Here there's a coating on this
• 03:51stent that slowly releases drug to
• 03:53prevent re stenosis of vessels.
• 03:55And last is a an orthopedic product.
• 03:59Another medical device.
• 03:59This one formed of two different
• 04:01kinds of materials.
• 04:02It's an artificial hip affirmative
• 04:04metal strong material so
• 04:05it can support your weight.
• 04:07But there's a polymer involved
• 04:09and you can see that as the white
• 04:12replacement for the acetabular cup,
• 04:13which provides lubrication between
• 04:16the two components of the artificial
• 04:18hip and other medical device with
• 04:21who that uses a material that's
• 04:24specially designed.
• 04:25And responsible for its most important
• 04:28function which is replacement
• 04:30of mobility in the hip.
• 04:32Now the and these products that were
• 04:34the collaborative works of teams
• 04:36of physicians and engineers have
• 04:37had a huge impact on health care,
• 04:39and you can see some evidence for that here.
• 04:44We're going to talk about using degradable
• 04:46polymers as a basis of drug delivery
• 04:49systems and the degradable polymers
• 04:51have a long history of use in medicine.
• 04:54This is an example of one that's
• 04:55been used for a long time.
• 04:56A product of ethicon's called vicryl sutures
• 04:59made of a copolymer of lactide and glycolide,
• 05:03and it's a material that has mechanical
• 05:06strength, so you can use it as a
• 05:08suture as you see on the bottom here.
• 05:10You can also use it in orthopedic
• 05:12applications by forming this
• 05:14polymer into a bone screw.
• 05:15And it remains mechanically
• 05:17strong for some period,
• 05:19typically weeks or months,
• 05:21and then it slowly degrades
• 05:23down to safe components.
• 05:25Lactic acid and glycolic acid.
• 05:28Next So what we've done?
• 05:31We and others have done over the
• 05:33last twenty years or so is is is.
• 05:35Figure out how to make these degradable
• 05:38polymers into tiny particles,
• 05:41and that's shown in this scanning
• 05:43electron micrograph here.
• 05:44These are spherical particles that
• 05:46are about 100 nanometers in diameter,
• 05:48so that's about the same diameter as a virus,
• 05:52but they're made of all synthetic
• 05:54components in this case.
• 05:56This picture is of pure plga.
• 05:59Nanoparticles,
• 05:59but you can load them with agents like
• 06:03chemotherapy agents or or others,
• 06:05and make them into pharmacologically
• 06:08active particles.
• 06:10Next, and they have some features
• 06:13which make them interesting.
• 06:15One is that if made of the
• 06:17right materials like Plga,
• 06:18which I just showed you, they're non toxic.
• 06:20If you add them to into cell cultures
• 06:23or you inject them into animals and
• 06:25in fact you can deliver very high
• 06:28doses of these into animals and people
• 06:31without any significant side effects.
• 06:33If the particles are loaded with drugs,
• 06:36then if they're engineered in the right way,
• 06:37the drugs are slowly released
• 06:39from the particles.
• 06:40Into in this case, into an aqueous medium,
• 06:43but also released into the body
• 06:45if they're deployed that way.
• 06:47Sometimes,
• 06:48and when it's shown in the bottom
• 06:51left panel here,
• 06:53this is when we added different
• 06:55concentrations of camp to Thiessen
• 06:57loaded nanoparticles to cells in
• 06:59culture that the loaded particles are
• 07:02actually more effective at killing
• 07:04these tumor cells than the drug is
• 07:06when it's delivered on its own.
• 07:08And so there's some.
• 07:09There's some property of the
• 07:11particles which makes the drugs
• 07:12more active and as a result you
• 07:14can inject these particles into
• 07:16tumors and is shown in the bottom.
• 07:19Right diagram here,
• 07:21in this case,
• 07:22injected into a tumor in the flank of a rat.
• 07:26You can arrest the growth of the tumor
• 07:28with a single injection of nanoparticles,
• 07:30and these features of nanoparticles
• 07:32seem to be related to the fact
• 07:34that the particles themselves.
• 07:36Can be highly loaded with drugs
• 07:38and they're much smaller than
• 07:39tumor cells that we're using to
• 07:41treat them in these examples,
• 07:43and so the particles get
• 07:44internalized into tumor cells as
• 07:46shown in this confocal image.
• 07:47Here you can see the green
• 07:50nanoparticles are inside of these
• 07:53tumor cells in culture,
• 07:55and they surround the nucleus and
• 07:57they're releasing their their active
• 07:59ingredients very close to the target
• 08:01of action from many anti cancer drugs.
• 08:06The technology that we've developed
• 08:08for this collaborative project
• 08:10is shown schematically here.
• 08:11It involves a block copolymer,
• 08:14so there's so there's two polymers
• 08:16that are covalently coupled together.
• 08:18One is lactic acid and that's shown
• 08:21as as the blue in this diagram,
• 08:24and the second is hyperbranched polyglycerol,
• 08:27which is shown as the green with
• 08:30red pendant branches coming off of
• 08:32the surface of the nanoparticle,
• 08:35so the core is this degradable.
• 08:36Poly lactic acid polymer that can be
• 08:39loaded with drugs or active ingredients
• 08:41and that's shown by the white dots here.
• 08:43And because it's a block copolymer
• 08:45that's assembled in a particular way,
• 08:48you have this degradable core
• 08:49surrounded by a green sort of corona
• 08:53of Hyperbranched polyglycerol.
• 08:54And it's that hyperbranched polyglycerol,
• 08:56which gives the nanoparticles
• 08:59certain surface properties which
• 09:01we've wanted to exploit.
• 09:05And one of the interesting things
• 09:08about Hyperbranched polyglycerol is
• 09:09that in its native state it has a
• 09:12lot of hydroxyls at the end of the
• 09:14end of the branched polymer chain,
• 09:16so this would be a a particle is
• 09:18shown on the left here that we
• 09:21call a non adhesive nanoparticle
• 09:22that has hydroxyl rich surface,
• 09:25and so it doesn't adhere very
• 09:28well to to proteins or to cells
• 09:30has a property of stealth.
• 09:32But I'll show you in just a few moments,
• 09:34but you can.
• 09:35Convert this particle into
• 09:36a different form by a brief
• 09:38exposure to sodium per iodate,
• 09:39which convert which converts the
• 09:41vicinal dials on the surface of
• 09:44the nanoparticle into aldehydes,
• 09:46and it then becomes a very adhesive
• 09:49particle adhesive.
• 09:51Because the aldehydes that are
• 09:52now covering the surface of the
• 09:54nanoparticle can react with amines
• 09:56in proteins or means on a a cell
• 09:59surface and they'll form a shift base
• 10:01covalent attachment which allows the
• 10:03nanoparticle to adhere to the cell.
• 10:06Or a matrix of very strongly.
• 10:11So this shows two of the typical properties
• 10:14of our non adhesive nanoparticles,
• 10:17NPS or bio hesive nanoparticles BMPS.
• 10:21The non adhesive particles because
• 10:23they have very little interaction
• 10:25with biological cells and tissues,
• 10:27will circulate for a long time if
• 10:30you inject them intravenously.
• 10:32They avoid uptake in most organs and
• 10:34that results in long circulation.
• 10:37You can see here the blue dots
• 10:40show a circulation half type.
• 10:42Time of about 10 hours compared
• 10:44to a conventional nanoparticle,
• 10:46which has a half life of of
• 10:48much less than an hour.
• 10:50And so that gives you the opportunity
• 10:53to to deliver nanoparticles to
• 10:55highly dispersed regions of the body.
• 10:58On the other hand,
• 11:00the bio adhesive nanoparticles are BMPS
• 11:02because they'll adhere to a tissue surface,
• 11:06can be made into very local
• 11:08drug delivery systems,
• 11:09and we show you this here in the
• 11:12diagram on the right which shows BNP
• 11:16adhesion to the outside surface of skin.
• 11:19So in this example,
• 11:20the red fluorescent nanoparticles were
• 11:23just added in solution on top of the
• 11:25skin on the side of the stratum cornea,
• 11:27and you can see that even after extensive.
• 11:29Washing those particles not only for
• 11:32mcconn formal coating on the on,
• 11:35the stratum corny AM,
• 11:36but they they are abundant on the surface
• 11:38as well and very difficult to wash off.
• 11:43So we want to talk about using
• 11:45these kinds of materials in two
• 11:47different but related applications.
• 11:49One for prevention of skin cancer,
• 11:52and in this case we'd like to convert
• 11:56the nanoparticles into a sunscreen
• 11:59by incorporating FDA approved UV
• 12:02absorbing agents into the nanoparticles.
• 12:05And we think that will
• 12:06have several advantages.
• 12:07Safety, because the adhesive
• 12:08nanoparticles don't enter the skin,
• 12:10and so they'll keep these
• 12:12chemicals outside of your body.
• 12:13But they'll still provide long lasting
• 12:15protection because of the adhesion
• 12:17and presumably increased efficacy.
• 12:19And then secondly want to talk about using
• 12:21these same materials to treat tumors,
• 12:23and we're going to give some examples
• 12:26of different tumors in animal models,
• 12:28but our focus here is on treating
• 12:30skin cancer and the advantages of the
• 12:32approach here is that you can load
• 12:34chemotherapy agents that are slowly
• 12:36released from the nanoparticles because
• 12:38of their bioadhesive properties,
• 12:39they are get retained in the tumor
• 12:41microenvironment, and they said that.
• 12:43That bio adhesion also facilitates
• 12:45uptake into tumor cells,
• 12:47and you can create a localized treatment
• 12:49that that reduces systemic toxicity.
• 12:54I think my friend and colleague
• 12:56is going to take over from here.
• 12:58Yes, thank you Mark.
• 13:00So sunscreens are something we use all
• 13:02the time and may take it for granted
• 13:05what we're putting on our skins.
• 13:07In particular, these multi benzene
• 13:10ring structures that form what are
• 13:13called the chemical types of actives
• 13:16within sunscreens and and as such being
• 13:19so hydrophobic they penetrate into
• 13:21and through the skin right into the
• 13:24bloodstream and deposit in your fat.
• 13:25There are concerns about off target effects,
• 13:27in particular estrogen and
• 13:30progesterone receptors,
• 13:32and another major effect is as
• 13:34they absorb this UV energy and and
• 13:38help protect against UV exposure,
• 13:41they are prone to give off reactive
• 13:44oxygen species and that is a major
• 13:46focus of something we're trying
• 13:48to prevent with this technology.
• 13:50On the other hand,
• 13:51we can use some of the physical sunscreens,
• 13:53in particular zinc oxide and
• 13:55titanium dioxide.
• 13:56They have limited penetration
• 13:57really through the skin.
• 13:59They will kind of work their way through hair
• 14:02follicles and through broken areas of skin.
• 14:04Even micro breaks a major concern
• 14:06about their use in general,
• 14:08as their aesthetic appearance,
• 14:10but they are major producers of Ross.
• 14:12If they do get into cells,
• 14:14even though they're less likely to.
• 14:16They're not just physical blockers,
• 14:18they clearly will generate Ross as well.
• 14:20And here you can see why they don't have
• 14:23some of the appeal of a views otherwise.
• 14:27So this is a confocal we made of the skin.
• 14:30You know,
• 14:31we're studying some of the
• 14:32relationship of cells here,
• 14:33but I want to point to one thing
• 14:35this is towards the top of the
• 14:36skin and you see longer hansel's.
• 14:38These dendritic cells are
• 14:39populate the epidermis,
• 14:41extend their dendrites really right up
• 14:44through these claudin tight junctions to
• 14:47really be samplers of the environment.
• 14:50And people.
• 14:51Think of, you know,
• 14:52skin as an impenetrable barrier
• 14:55with its stratum, cornea, minutes,
• 14:56lipid.
• 14:57A protective components but in
• 14:59point of fact it is very interactive
• 15:02with the environment.
• 15:04In many ways oops circulate that for
• 15:07you a little bit and you can see how
• 15:11they can bring potential agents down
• 15:13into the deeper layers of the epidermis,
• 15:16and they will actually navigate
• 15:18from there through the dermis,
• 15:20into lymphatics and lymph nodes too.
• 15:24So another kind of spark on the
• 15:26controversy of of sunscreen usage
• 15:28came about a year and a half ago
• 15:31when FDA was studying the plasma
• 15:35concentrations within folks that
• 15:37frequently applied these sunscreens
• 15:39and noted that they achieved these
• 15:42levels of concentration that are known
• 15:45to have a special designation by the
• 15:50FDA as requiring toxicology studies,
• 15:52which.
• 15:53Of course,
• 15:54have never really been done
• 15:55by the sunscreen industry,
• 15:56but are taking place now after that study.
• 16:02So the bioadhesive nanoparticle
• 16:04technology really allows for us to
• 16:08develop nonpenetrating sunscreen
• 16:10and avoid some of these concerns
• 16:13about these agents getting in.
• 16:14In particular,
• 16:15these hydrophobic chemical agents.
• 16:18If you apply just on the surface,
• 16:20it doesn't just sit there.
• 16:21There are a lot of film formers
• 16:23and technologies that
• 16:24the industry tries to use,
• 16:25but they work only to some degree,
• 16:27as the FDA showed.
• 16:29But if we're able to
• 16:32encapsulate those within BMP's,
• 16:34we can keep these agents on the
• 16:36surface bound to the stratum corny AM.
• 16:38Otherwise, if they penetrate
• 16:40within after photo exposure,
• 16:42you'll see very high levels of Ros
• 16:45generation directly attributable
• 16:47to those sunscreen agents that
• 16:49are supposed to be protecting.
• 16:51Here's what it looks like when we use
• 16:54fluorescent loaded BMP nanoparticles
• 16:56on the on the skin surface,
• 16:59and you can almost form a a confluent.
• 17:03Blanket as a as the sun might see it.
• 17:10So this affords several major advantages.
• 17:14One of them is this
• 17:16durability after application.
• 17:17This is a covalent bond.
• 17:19It's a shift based bonding that takes
• 17:21place with the aldehydes on the on,
• 17:23the bioadhesive nanoparticles
• 17:25and in particular affords it
• 17:27a a waterproofing protection,
• 17:30water resistance and so that can
• 17:32be tested in these animals that
• 17:34can be tested on other surfaces.
• 17:36And it can be applied to industry standards.
• 17:40Like to wash off these agents
• 17:42and see how protective they are.
• 17:45Current sunscreen formulations
• 17:46require reapplication every two hours.
• 17:49You don't see anything that
• 17:50lasts longer than that,
• 17:51but we can see these sticking around
• 17:54for much longer than a couple hours.
• 17:59The other thing that clearly helpful by
• 18:03using BMP to incorporate these agents
• 18:06within is that we don't see penetration
• 18:09of the active sunscreen agents to the
• 18:12point that with free sunscreen we might
• 18:15generate endproducts of Ross damage.
• 18:18For example gamma H2X or recruited proteins
• 18:21to sites of DNA damage due to Ross,
• 18:24but if the agent is incorporated within
• 18:27the BMP's, we don't see that damage.
• 18:29After UV exposure,
• 18:30we've already applied these to human skin.
• 18:33We don't see we.
• 18:35We see a nice physical appearance to him.
• 18:38We see the capacity for them to protect
• 18:41against what's called minimal or THEMA doses,
• 18:44and we can do SPF testing for example
• 18:48with them and see their performance
• 18:50and their aesthetic advantages.
• 18:54But if we really want to kind
• 18:57of vigorously studies and.
• 19:00And according to industry standards,
• 19:02we use materials such as vitro skin.
• 19:04This is a proprietary material
• 19:06that has the means within it,
• 19:09which is actually quite good for us to
• 19:11look at and study this bio adhesion.
• 19:14This is evil Ben Zona,
• 19:16very active in the UV spectrum.
• 19:19Agent incorporated into NMPS.
• 19:20So you see how that looks on a pre
• 19:24wash and you see after it's exposed to
• 19:27washing for three hours in a water bath.
• 19:30What happens to the the
• 19:32PHOTOPROTECTIVE Spectra?
• 19:34And you can see that just deteriorates
• 19:36immediately and in contrast to Eva
• 19:39Benzon incorporated within BMP's,
• 19:41which maintain quite nicely there.
• 19:44The photoprotective capacity
• 19:45across the full spectrum of the
• 19:48performance of evil Benzon.
• 19:50We've done it with other agents,
• 19:51including Juvenil A to see that continued
• 19:56protection even clearly after three hours.
• 20:01And longer.
• 20:04We've taken this to the next level
• 20:07of using poor sign skin and really
• 20:11trying to vigorously wash that off,
• 20:15wrapping up the revolution per minute
• 20:17and the time constraints and then
• 20:20using HPLC in a very quantitative way
• 20:22to see how much evil benzon we were
• 20:24able to keep it here to the skin.
• 20:27Here it is at 150 RPM for 20 minutes.
• 20:31This is the industry standard
• 20:33for waterproof measurements and
• 20:35MPs will come off at a 60% lost.
• 20:38The BMP's will adhere quite nicely.
• 20:41Stayed here through all of that
• 20:43at greater than 95% retained and
• 20:45then we start to Rev it up too.
• 20:49Way past industry standards 450 RPM's
• 20:52three hours and see that you know we
• 20:55get the same relationship and the the
• 20:58full adherence of BMP's upwards of about 80%.
• 21:01After three hours at that level.
• 21:06We were quite surprised to actually see
• 21:09that BMP's that gave us another advantage,
• 21:11and that is the capacity to
• 21:13prevent photodegradation of a
• 21:15quality called photostability.
• 21:17This is very important in sunscreen
• 21:20formulation, able benzon in particular
• 21:23as being really the main UV,
• 21:26a protector active agent.
• 21:27It's a major concern 'cause it's
• 21:30so susceptible to photodegradation.
• 21:32You could see that here after.
• 21:35An industry standard dose of UV.
• 21:37What happens to the performance
• 21:39of evil Benzon?
• 21:41So you imagine you put it on.
• 21:43You get exposed to ultraviolet
• 21:45light and it just degrades.
• 21:46So if you incorporate it within BMP's.
• 21:52And we're not completely sure of
• 21:54exactly how this is happening,
• 21:55but obviously within the PLA there's
• 21:59a protective millou that help
• 22:01prevent some of that degradation
• 22:03from the Eva benzon quite nicely.
• 22:08So Octocrylene is a nice partner for
• 22:11able benzon because it's a UV absorber,
• 22:15so it complements it in that way,
• 22:16but also because it itself is a photo
• 22:20degradation stabilizer for able benzon.
• 22:23So we were very interested if we
• 22:25just incorporated able benzon.
• 22:26We can see a rate of
• 22:29degradation photodegradation,
• 22:30but if we come incorporated with
• 22:33octocrylene we were hoping to maintain a
• 22:36photostability at very high levels of UV.
• 22:38Exposure upwards of three hours
• 22:41and we were able to do that by Co,
• 22:43incorporating those agents and and
• 22:46found an optimal ratio for those also,
• 22:49but we were very surprised to see
• 22:51if we incorporated them separately
• 22:53that we still had that capacity for
• 22:56protection against photodegradation.
• 22:58Again, not something we completely
• 23:00understand as relationship
• 23:01between particles where agents
• 23:03are individually incorporated.
• 23:07And then one more surprise from incorporation
• 23:11came about when we measured reflectance.
• 23:13So if you look at zinc oxide,
• 23:15so-called physical blocker as we
• 23:17described before, you're going to see a
• 23:20lot of of reflectance that helps in its
• 23:23performance and protection against UV.
• 23:25But it also gives it some of this shiny,
• 23:29sometimes even purplish
• 23:31whitish hue to people skin.
• 23:34Whereas if you just use 3.
• 23:37Able benzon and octocrylene.
• 23:38You don't really get much of any reflectance
• 23:41from those chemical sunscreen agents,
• 23:44but within bpce for whatever
• 23:48reason able benzo not crawling do
• 23:51provide provide some reflective or
• 23:54extra protection from UV exposure,
• 23:57probably because of the state
• 24:00that they're in.
• 24:02Something that we might refer to as kind
• 24:05of a hydrophobic crystal if you will.
• 24:09If you can imagine as opposed to being in
• 24:13a a more of an oily millou or emulsion.
• 24:17Empty BMP's don't do that,
• 24:19so this is really about the
• 24:21actives within the PLA.
• 24:25And then we can do some in vitro SPF
• 24:29measurements using some industry
• 24:31standard spectrophotometry and and
• 24:34see that we can gain a level of
• 24:37performance that would be predicted
• 24:39to be above the active ingredients.
• 24:41In addition, we can see that we can sprinkle
• 24:44in some of the physical blockers here,
• 24:46in this case titanium dioxide at
• 24:481% or 5% and get levels of SPF
• 24:52protection with that combination that.
• 24:55Kind of speaks to where we're
• 24:57heading with a prototype for this,
• 25:00use as a as a novel sunscreen formulation.
• 25:06I want to just come use this slide to talk
• 25:10about our other major collaborator here.
• 25:12Douglas Brash, who is a really a
• 25:16pioneer in understanding triplet state.
• 25:20Species that get generated after UV
• 25:23exposure and how they do damage DNA
• 25:26even well after the lights are out.
• 25:30We are also working with a with the
• 25:33Center for molecular discovery here at
• 25:35Yale to screen a bunch of compounds.
• 25:37In this case a about 1000 natural found
• 25:41in nature compounds and and looking
• 25:43for their capacity to be photostable UV
• 25:47absorbers and then looking at their capacity.
• 25:51To not be so toxic to the skin and then
• 25:55not generate Ros after UV exposure
• 25:58and using this series of steps,
• 26:01we've really come down to a handful
• 26:04of major candidates that we're really
• 26:07excited about moving forward.
• 26:08With that we might use.
• 26:11For example,
• 26:12if we deem them safer than current
• 26:15agents outside of the particles.
• 26:17If there needs to be protection,
• 26:19we can put them inside the particles.
• 26:21So this is something that we think
• 26:23that we can will be very complementary
• 26:26to to what we're working on.
• 26:30Mark
• 26:35going to change gears for
• 26:37the for the rest of the
• 26:38talk slightly and talk about using these
• 26:41bpce for therapeutic drug delivery.
• 26:43So this slide just sort of reminds you
• 26:46of the potential for the particles that
• 26:49are converted into the bioadhesive state.
• 26:52BMP's to interact with
• 26:54the proteins or any any.
• 26:58Amine containing group by because
• 27:01the aldehyde that's on the surface of
• 27:04the BMP will form a shift base which
• 27:07leads to this covalent attachment,
• 27:09and so we think there's potential
• 27:12advantages for particles that
• 27:13work by this mechanism to deliver
• 27:16therapeutics locally.
• 27:17And in addition,
• 27:19because the core of the particle is Poly,
• 27:22lactic acid and pretty hydrophobic
• 27:24polymer that's really compatible with
• 27:27drugs that have low solubility's.
• 27:29In in in aqueous media,
• 27:30so you can so you can use drugs that are
• 27:33difficult to formulate in conventional ways,
• 27:36but you can load them highly inside
• 27:38the particles and that allows you to
• 27:41have controlled release overtime at
• 27:42the site of action and hopefully limit
• 27:45systemic exposure to the toxic compounds.
• 27:50So here's one example of using these.
• 27:55Biodiesel nanoparticles to
• 27:57treat tumors in animals,
• 27:59and this is a collaboration with
• 28:01Alessandro Santin in OB GYN.
• 28:03And here, what we did was deliver
• 28:07the particles intraperitoneally.
• 28:08So these in the in the panel be shown
• 28:11here shows you the retention of either
• 28:14NNPS which are on the left or bppe
• 28:18Switcher on the right you see if you
• 28:20inject them intraperitoneally and animals.
• 28:22After five minutes they distributed widely
• 28:24throughout the intraperitoneal space.
• 28:26After four hours,
• 28:27the concentration of of NPS and not
• 28:30easy particles dropped substantially,
• 28:32while the BMP concentration and
• 28:34distribution remains pretty much
• 28:35the same after one day.
• 28:37Still a lot of BMP's in the IP
• 28:39space where most of the NPS are
• 28:41gone and we even see persistence in
• 28:42the IP space for up to five days.
• 28:45So this this kind of data convinced
• 28:47us that maybe you could treat.
• 28:51My peritoneal carcinomatosis with
• 28:53these kinds of nanoparticles by
• 28:55injecting them IP and and exploiting
• 28:58the mechanism where the bioadhesive
• 29:01nanoparticles would associate with the
• 29:03tumor cells or tumor nodules that are
• 29:06distributed throughout the peritoneum.
• 29:07We tested this with a drug
• 29:09called a path alone B.
• 29:11You can see that when it's loaded in
• 29:12the nanoparticles and panel see here,
• 29:14it comes out.
• 29:16Relatively slowly overtime,
• 29:17although most of it comes out
• 29:19over the first 12 hours and then
• 29:21it sort of leaks out after that.
• 29:22This is an in vitro release,
• 29:24very difficult to measure.
• 29:26The corresponding release once
• 29:27it's deployed in the animal,
• 29:29but you see the the most
• 29:31impressive result up in panel a.
• 29:33These are animals that that got
• 29:36intraperitoneal injections of a of a
• 29:40uterine serous carcinoma cell line that
• 29:43doctor Ellis Dr Stanton had developed.
• 29:45If you don't treat them,
• 29:47they die within about 60 days.
• 29:49If you treat them with EB alone,
• 29:50it's it's.
• 29:51It's difficult to find a dose that
• 29:53doesn't cause early toxicity and still
• 29:56provide some increase in survival.
• 29:57You can see that by the black line here,
• 29:59but if you put the EB inside
• 30:01the biodiesel nanoparticles,
• 30:02we see no toxicity and a dramatic
• 30:05improvement in survival.
• 30:09A similar example, but now we're
• 30:11treating locally in the brain.
• 30:13Here we're infusing the nanoparticles
• 30:15by convection enhanced delivery
• 30:17into the brain of animals that
• 30:20have intracranial tumors.
• 30:21This is work by Yazi Wang in my laboratory
• 30:24in collaboration with Raymond Hall
• 30:26at at the University of Connecticut.
• 30:29And here we put into the into the
• 30:33nanoparticles and anti mirror.
• 30:35Actually two anti mirrors,
• 30:36anti mirror 21 and anti mere 10B.
• 30:39These are two micro RNA's that have
• 30:42been highly associated with gliomas,
• 30:45so we do in the animals.
• 30:46One infusion we introduce the tumor as
• 30:48you can see on the timeline at the top,
• 30:50at day zero, at day six.
• 30:53At the tumor is growing,
• 30:54we infuse the nano particles that
• 30:57contain these anti mirrors and then
• 30:59one day later we given IP dose of
• 31:02Tim's Olamide and so the the hope is
• 31:05that the anti mirror activity will
• 31:07sensitize the tumor cells to Tim's
• 31:08Olamide and so it will be active.
• 31:10At low doses and you can see
• 31:12the result down here,
• 31:13which is pretty dramatic animals
• 31:15without any treatment dead by 50 days.
• 31:17If you just treat them with the bio
• 31:19adhesive nanoparticles with the anti mirrors,
• 31:20you see some prolongation in survival.
• 31:22That's the green line if you
• 31:24just treat them with TMZ,
• 31:25you see some prolongation and survival.
• 31:26That's the red line.
• 31:28If you treat them with both we see
• 31:30100% survival out to 120 days here,
• 31:32which is pretty remarkable.
• 31:35Next and you can deliver other
• 31:37agents to other tissues as well,
• 31:39so this is an example of
• 31:41delivering to mucosal surface.
• 31:42These were nanoparticles that were
• 31:44delivered intravaginally in mice,
• 31:46either NPS or BMP'S.
• 31:48You see the same sort of effect
• 31:50on sustained retention of the
• 31:52BMP's in the up to 24 hours,
• 31:56and these these particles were delivering.
• 32:00Antiretroviral drugs to
• 32:03the reproductive tract.
• 32:05And you can see if you take that
• 32:07issue and you dissociate it and
• 32:09look for cells that express CD 45
• 32:12or cells that express epithelial
• 32:13markers that with the bioadhesive
• 32:15nanoparticles the majority of the
• 32:18cells are are have nanoparticles
• 32:21within them and nanoparticles
• 32:23that contain the active drug.
• 32:30So. You know the the burden of human
• 32:35skin cancer is most striking when we
• 32:39consider volumes, numbers of cases per
• 32:41year at 5.5 million in EU. S. Uhm?
• 32:45You know more more cases of skin cancer
• 32:48than all other cancers combined and this.
• 32:52Though most of them in particular basil cell
• 32:56not and squamous cell a little bit Melanoma.
• 32:59Much more can result in death Accumulatively
• 33:03it's about 15,000 per year in EU.
• 33:06S and it's just a burden
• 33:08on the health care system.
• 33:10Tremendous burden on treating all
• 33:12of these cases of skin cancer
• 33:14multiple on a lot of patients in
• 33:16particular transplant patients.
• 33:18Fair skinned individuals,
• 33:19multiple scars that can run into.
• 33:23Each other and cause other complications
• 33:27from destructive and surgical procedures.
• 33:31So there's really an unmet need for
• 33:35non-surgical options for patients.
• 33:37Those that may not be
• 33:38great surgical candidates,
• 33:40or those who would like something a little
• 33:42more simpler and less cost dependent.
• 33:47So a minimally invasive local alternative
• 33:49would be ideal for patients who might have.
• 33:53Superficial or minimally invasive lesions,
• 33:55so numerous simple ones they may have
• 33:58locally advanced cancers where you want
• 34:00to come in with something local and
• 34:03that could be used in in conjunction.
• 34:06For example with a with a systemic
• 34:10agent or combination,
• 34:11that could be an immunotherapeutic.
• 34:13Agents such as checkpoint inhibitors
• 34:16for example.
• 34:17Or there may be some that you really
• 34:19have really deep ones and you
• 34:21want to minimize the side effects
• 34:22of providing a systemic.
• 34:24Chemotherapeutic agent and how you
• 34:25might deliver it locally and in those
• 34:28cases it could be a targeted therapy.
• 34:29It could be a chemotherapeutic agent.
• 34:31The point is you're going to
• 34:33maintain high concentrations of the
• 34:35actives where you put the particles.
• 34:39So here's a model that I've worked with.
• 34:41Uhm. For many years of keratinocyte
• 34:46tumor squamous cell carcinoma,
• 34:49it's a set up quite simply by
• 34:53transplantable injection and it
• 34:54grows over a course of about
• 34:57four weeks and forms a nice big
• 34:59nodular blue ball of cells.
• 35:02It's very aggressive.
• 35:04But if we treat it with BMPS with
• 35:06camp to thicken incorporated as
• 35:09the chemotherapeutic active agent,
• 35:11we can get complete clinical and
• 35:14histologic resolution and those
• 35:16pathologists in the audience can
• 35:18appreciate the tumor destruction
• 35:20and amorphis changes that that we
• 35:23see here after after resolution.
• 35:30So I'm trying to understand.
• 35:33Process here and so that we can maybe
• 35:37potentially leverage some of that.
• 35:40We can look at how the particles,
• 35:42for example, die Incorporated BMPS.
• 35:47Might interact with the tumor cells
• 35:48and Mark alluded to some of the
• 35:50interactions with other tumor cells,
• 35:51but we were studying here in in
• 35:54skin cancer squamous cell carcinoma
• 35:56PDB cells and you can see that
• 35:58the NPS barely will stick to the
• 36:01cells and barely getting side.
• 36:02But you can just see this
• 36:04tremendous adhesion to cell surface,
• 36:06which of course that is a protein
• 36:09rich environment and that further
• 36:12facilitates and triggers.
• 36:14And we've broken down the mechanism
• 36:16a little bit of micro Pinot cytosis a
• 36:19passive internalization that occurs
• 36:21to bring these particles and their
• 36:24payloads right within the tumor cells.
• 36:30And we can really get very
• 36:33quantitative with this interaction,
• 36:34and we can use dyes that are
• 36:37bound covalently to the PLA.
• 36:38Or we can do in ones that are loosely
• 36:41within the appeal doesn't matter,
• 36:43they they will readily get incorporated
• 36:46with into the tumor cells taken up.
• 36:50Very readily over the course of three days.
• 36:56Relative to BMP's that don't have that
• 36:59bio adherent surface component to him.
• 37:04We can also create kind of a an in vitro
• 37:07tumor matrix where we put use Poly L
• 37:10lysine as a tumor rich environment and
• 37:13adhered adjacent to cells and show that
• 37:17our BMP's are the ones that are going
• 37:20to provide a kill because they will bind
• 37:22not just to cell surface but just to
• 37:25this tumor matrix and MP's don't do that.
• 37:27So we don't see that tumor kill
• 37:29and we don't see it with CPT.
• 37:31These were our with a washout.
• 37:33From the tumor matrix.
• 37:35But the BMP's in here,
• 37:36there and then are readily available
• 37:38to the tumor cells to kill,
• 37:40so we think there's two.
• 37:43Mechanisms that work together there
• 37:45one where the BMP's with their payloads
• 37:48or binding to the tumor rich matrix
• 37:51of tumors as well as readily being
• 37:54internalized by the tumor cells themselves.
• 37:57We can move to in vivo established tumors,
• 38:00inject our bpce with with Die
• 38:04or MPs for comparison,
• 38:07and see what kind of distribution
• 38:09we get through the tumor cells
• 38:12and what kind of staying.
• 38:14Power we might get.
• 38:17Uhm, in fact, we can measure that over days
• 38:20and we can do that by harvesting the tumors,
• 38:23pulverising them and extracting and doing
• 38:26HPLC quantification on the drug levels.
• 38:29And you can see here this is intralipid with
• 38:31the capital seeking chemotherapeutic agent.
• 38:33We just don't detect it after
• 38:35day zero if it's in any piece,
• 38:37there is a little bit of detection today too,
• 38:39but that pales in comparison to what
• 38:43BMP's due to keeping drug present.
• 38:45Again, there may be released.
• 38:48From the particles, but it's there.
• 38:51Maybe particles that contain
• 38:52depost more slowly release.
• 38:54They may do that in the Peri
• 38:56tumoral area of the tumor matrix.
• 38:57They may do that within
• 38:59tumor cells themselves.
• 39:04And then we can look at the therapeutic
• 39:06efficacy of using for example,
• 39:08camp to thicken incorporated within
• 39:10BMP'S to treat establish screen or
• 39:14cell carcinomas injected here at day
• 39:17four we can measure tumor size and and
• 39:21and see what we do to tomb of growth.
• 39:24We can also harvest at the end and
• 39:26do histological analysis for the
• 39:28presence of any residual tumors.
• 39:30We do get an inflammation with the BNP CPT.
• 39:34As you might expect,
• 39:35we do with both arms of the CPT alone.
• 39:41So that. You know,
• 39:44clinical tumor measurements are are not
• 39:46as definitive as the histologic ones,
• 39:48but both the clinical tumor growth
• 39:52curves were showed protection with
• 39:55btes relative to CPT alone at the same
• 39:58dose of drug and in histologically we
• 40:02saw a 62% tumor free rate with the BNP
• 40:06skeds that was impressive in a parallel
• 40:10experiment at at four weeks out.
• 40:15So we were really interested in whether
• 40:18this localized treatment could be combined
• 40:22with with immunotherapeutic strategies,
• 40:24and the first thing we did was to go local.
• 40:27We are designing experiments for
• 40:29checkpoint inhibitors which might
• 40:31be on the minds of several people.
• 40:33We're working with Marcus Bosenberg
• 40:35on what that might look like,
• 40:37for example with a localized.
• 40:42Invasive Melanoma or metastatic
• 40:44nodule of Melanoma,
• 40:46but in this case this is our our BMP
• 40:49screen PDV squamous cell carcinoma
• 40:51again and we looked at again the
• 40:55capacity for BMP's to incorporate
• 40:58CPT but be combined with a local
• 41:02immunotherapeutic agent in this case.
• 41:05Kcse people familiar with
• 41:06that know this this is a TLR.
• 41:08Nine login, so we're kind of creating a.
• 41:12Kill and thrill strategy,
• 41:15where we're not just killing tumor cells,
• 41:19but help trying to harness local
• 41:21immunity to help clean up residual ones.
• 41:25Maybe some of that tumor debris,
• 41:27tumor antigen rich material,
• 41:29and immunostimulation might
• 41:31create an in vivo.
• 41:33Vaccination effect when we compare
• 41:35it to just intralipid CPT with that
• 41:38with that same immunostimulatory
• 41:39agent we just do not get the level
• 41:42of protection we can get by pushing
• 41:44the system hard on the tumor side.
• 41:49This might be a little bit more easy to see,
• 41:53and when we look at individual tumor
• 41:55growths and you see the the the
• 41:57shutting down of a lot of those tumors
• 42:00that were treated with combination.
• 42:07Mark
• 42:10just to finish up.
• 42:11Just remind you of the of the
• 42:14two classes of nanoparticles.
• 42:16We've worked here really the
• 42:18same when they're synthesized and
• 42:20converted from NPS into BMP's.
• 42:21We can load agents into the into
• 42:24the PLA polylactic acid shell,
• 42:27and then we manipulate the hyperbranched
• 42:29polyglycerol in the order to either
• 42:31make stealthy particles and NPS,
• 42:32or adhesive particles BMPS.
• 42:36So Polly PLA is made from L.
• 42:40Lactide is a monomer that costs
• 42:42about $5000 per 10 kilograms.
• 42:44It's been going up over time
• 42:46because of worldwide demand,
• 42:47for for for lactide based polymers.
• 42:51There are some alternates that have
• 42:52been used quite a lot in medicine like
• 42:54caprolactone or or Penta deco lactone loser,
• 42:56shown here.
• 42:57They're they're cheaper, but not.
• 42:58But but but maybe by a factor of two,
• 43:02but we focused on ethylene brassil 8,
• 43:03which is also a a lactone.
• 43:06But it it,
• 43:07but it's much cheaper 10
• 43:09times cheaper than L lactide,
• 43:11which makes a big difference in
• 43:14terms of manufacturing costs.
• 43:15Another advantage of ethylene brassil 8
• 43:17is that it's produced in large quantities.
• 43:19It's been used it a lot in
• 43:21the fragrance industry,
• 43:22so it's been put on lots of people
• 43:24skin and its properties are known.
• 43:26It's a sustainable product 'cause it's
• 43:27'cause it's produced from Castor oil.
• 43:29It's not made from petroleum like
• 43:31those other like those other polymers
• 43:33are an it we knew going into this
• 43:36that that others had made these
• 43:38polymers and you could make them with
• 43:41similar mechanical properties to play.
• 43:43So can you make them into bio adhesive?
• 43:46Nanoparticles,
• 43:46the answer is yes,
• 43:48and post auction all put up Pythia
• 43:52and graduate student Alex Johnson,
• 43:53which have shown that in the
• 43:56next slide that there's these are
• 43:59particles that were made variety,
• 44:00different conditions,
• 44:01which shown in the graph here,
• 44:02but you can see some of the particles,
• 44:04but by scandal around micrographs.
• 44:07In this scanning electron micrograph,
• 44:08so we're encouraged that there this
• 44:10is something that we can accomplish,
• 44:11not just with the material we've shown here.
• 44:14We certainly have proof of principle that
• 44:15that material works in a variety of settings,
• 44:17but one can innovate on
• 44:18the material side as well,
• 44:20and potentially make things
• 44:21that are that are better.
• 44:27Alright, I'll I'll summarize our.
• 44:30Joint efforts and skin cancer
• 44:33prevention and treatment.
• 44:35So we've worked on in formulating
• 44:38a prototype for our sunscreen that
• 44:40shows this bio adhesion advantage,
• 44:42photostability advantage anti permeation
• 44:45advantage and SPF optimization advantages.
• 44:50We're working now on preclinical modeling.
• 44:52For that, this is the MC1RE mouse,
• 44:55so it has the same defect as fair skin red
• 45:00haired people with freckles to look at
• 45:03both acute and chronic kind of modeling.
• 45:06With that to really try to optimize
• 45:09our performance prior to moving
• 45:11to the clinical spectrum.
• 45:12All in addition,
• 45:14we're also looking at protecting
• 45:16specifically against both squamous
• 45:18cell carcinoma and Melanoma mutations.
• 45:21Over chronic exposure protocols.
• 45:23With that as part of the sport and
• 45:27and then you heard about some further
• 45:29BMP bio engineering improvements
• 45:31that we're working on.
• 45:33In addition,
• 45:34you heard about our efforts on
• 45:37localized therapy for skin cancer
• 45:40as a nonsurgical alternative,
• 45:42the advantage of matrix bio adhesion,
• 45:44tumor cell binding and uptake advantages,
• 45:47and how this translates into
• 45:49a drug retention advantages,
• 45:51efficient drug delivery and tumor
• 45:54elimination when delivered locally
• 45:56decrease systemic toxicity levels
• 45:58which we had didn't show here
• 46:00compatibility with immunotherapy.
• 46:01Which I to me is very,
• 46:04very exciting for the potential to
• 46:07use a localized therapy in combination
• 46:09with a systemic immunotherapy
• 46:11or localized immunotherapy.
• 46:15And that is. Are ping pong
• 46:19tag team talk for the day?
• 46:21UM, obviously a lot of people
• 46:23working in in both our labs,
• 46:26in particular, Julie Lewis,
• 46:28Sholud Komar, and Amanda Zoo
• 46:31contributed extensively to to data
• 46:33you saw on the skin cancer side and
• 46:37mark highlighted people in his lab,
• 46:39but in particular he wants to has
• 46:43been the tremendous link between
• 46:46our two labs to bring up the.
• 46:49By many engineering component to skin
• 46:52cancer and skin cancer prevention
• 46:54modeling and Doug crashes are also
• 46:58our partner and developing other
• 47:01strategies on skin cancer prevention.
• 47:04Who's also been very much involved
• 47:05in in in in how we try to make these
• 47:09formulations that might ultimately
• 47:10also prevent some of the oxidative
• 47:12damage that we talked about.
• 47:14Marcus Bosenberg and Harriet Kluger
• 47:16in particular as part of the.
• 47:18Or have been tremendously supportive
• 47:21of our work and Ruth Taliban runs
• 47:25a core here that has provided us
• 47:28with numerous human skin samples
• 47:31and they were very appreciative,
• 47:32especially Antonella as part of that.
• 47:35And of course,
• 47:37funding sources include the cancer spore,
• 47:40but other grants from NCI, NIAMS, and IEHS.