Bioadhesive Nanoparticles for Skin Cancer Treatment and Prevention

October 14, 2021

Information

Yale Cancer Center Grand Rounds | September 21, 2021

Drs. Michael Girardi and W. Mark Saltzman

ID7043

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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.