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>> GOOD AFTERNOON, EVERYONE MY PLEASURE TO WELCOME YOU TO A VERY SPECIAL LECTURE TODAY THIS IS THE ANNUAL MARGARET PITTMAN LECTURE IN WHICH WE INVITE A DISTINGUISHED WOMAN SCIENTIST TO COME AND ADDRESS THE NIH COMMUNITY AND RECOGNIZE THIS BY THE NAME THAT’S ATTACHED TO THIS LECTURE MARGARET PITTMAN SHE WAS NIH’S FIRST FEMALE LAB CHIEF MARGARET PITTMANMADE SIGNIFICANT CONTRIBUTIONS TO MICRO BIOLOGY AND VACCINE DEVELOPMENT PARTICULARLY IN THE AREA OF PERTUSSIS AND TETNUS, FROM THE IN FIAIA, AND WHEN MARGARET BEGAN WORKING AT NIH, IT WAS BT HERE IN BETH ESTIMATE THADA DAILY BASIS, IT WAS IN WASHINGTON AND SHE MOVED WITH IT MANY DECADES AGO, BUT HAS BEEN RECOGNIZED AS A PILOT PROJECTEE NEAR PARTICULARLY WOMEN IN SCIENCE AND WE AT NIH FEEL THIS IS A IMPORTANT ISSUE AND WE HAVE A WHOLE COMMITTEE THAT LOOKS AT THESE ISSUES REGULARLY AND HAPPY TO HIGHLIGHT PARTICULARLY TODAY, THE MARGARET PITTMAN LECTURE BY BRING NOTHING A SPECTACULAR SPEAKER FOR YOU TO LISTEN TO, DR. PAULA HAMMOND SHE GOT HER UNDERGRADUATE HAT M I.T. AND THEN GOT A MASTERS DEGREE AT GEORGIA TECH AND THEN RETURNED TO GET HER Ph.D. AT M. I.T. AND JOINED THE FACULTY WHERE SHE’S BEEN SINCE THEN BEGINNING AS ASSISTANT PROFESSOR AND NOW AS THE DAVID COKE PROFESSOR OF ENGINEERING AND CHAIR OF THE DEPARTMENT AT M I.T AND THAT’S THE DEPARTMENT OF CHECKICAL ENGINEERING HER RESEARCH WHICH YOU’RE GOING TO GET TO HEAR A LOT ABOUT IS GROUND BREAKING IN TERMS OF ITS POTENTIAL FOR MEDICINE AS WELL AS A VARIETY OF OTHER APPLICATIONS BECAUSE IT IS IN THIS GENERAL AREA OF NANO TECHNOLOGY, AND PARTICULARLY THE SELF-ASSEMBLY OF POLYMERIC NANO MATERIALS WITH A MAJOR EMPHASIZE ON THE USE OF EXPECTATIONS –ELECTROSTATIC S AND OTHER INTERACTIONS TO GENERATE MATERIALS WITH LOTS OF APPLICATIONS IN GENERAL MEDICINE THAT WE WILL ALL BE EXCITED TO HEAR ABOUT DR. HAMMON IN ADDITION TO HER ACADEMICS ACHIEVEMENTS IS AN INNOVATOR, OVARIAN CANCER ADVANCES AND HAS DONE A TED TRIMBLE TALK WHICH IS TELLING ABOUT TECHNOLOGY TO A GENERAL AUDIENCE AND A BEAUTIFUL WAY OF DOING SO DELIGHTED YOU ARE HERE, AND DELIGHTED TO THOSE WATCHES OVER THE WEB AND PLEASE HELP ME WELCOME THE PITTMAN LECTURER DR. PAULA HAMMOND >> THANK YOU SO MUCH IT’S A REAL HONOR TO GIVE THIS LECTURE TODAY ESPECIALLY GIVEN THE HISTORY OF THE PITTMAN LECTURE AND I’VE HAD AN INCREDIBLE TIME GETTING TO KNOW A NUMBER OF OLD FRIENDS AND MEETING A NUMBER OF NEW FRIENDS AND COLLEAGUES WHILE I’VE BEEN HERE I WILL TALK TODAY AND I GUESS THIS IS MY POINTER I WILL TALK TODAY ABOUT A LAYER BY LAYER ASSEMBLY APPROACH WHICH CAN BE USED TO GENERATE NEW METHODS OF DELIVERING DRUGS AND THE NICE THING ABOUT THIS APPROACH IS THAT IT CAN BE USED TO DELIVER DRUGS ON SURFACES ON ALMOST EVERY LANE SCALE FROM NANOMETER LANE SCALE UP TO LARGE MACROSCOPIC MILLIMETER AND LANE SCALES THIS TECHNIQUE IS SIMPLE IT INVOLVES THE ALTERNATING A–BITS SORPTION OF SPECIES, ESSENTIALLY IT CAN BE A SUBSTRATE, ANY SUBSTRATE, CLASSIC HAS BEEN TREATED SO THAT IT HAS A CHARGE, A METAL OR METAL OXIDE WHICH HAS ITS OWN NATIVE CHARGE AND YOU CAN TAKE THAT SUBSTRATE AND IMMERSE IT INTO AQUEOUS SOLUTION AND IT CONTAINS SOMETHING THAT IS CHARGED NOW WHEN YOU DO THAT YOU WILL GET THAT CHARGED POLYVALIENT MATERIAL UNTIL THE SURFACE CHARGE GETS REVERSED AND AT THAT POINT ELECTROSTATIC PROPOSITION HAS SOMETHING ON THE SUBSTRATE IT’S A SELF-LIMITINGLY ABSORPTION AND THE THICKNESS OF THAT LAYER IS DEPEND EPT ON WHAT YOU’RE ABSORPTIONING, IF IT’S A POLYMER CHANGE OR NANO PARTICLE FOR EXAMPLE AND THE Ph AND IONIC STRENGTH OF THE SOLUTION

THAT YOU’RE ABSORBING FROM SO IT CAN MAKE THESE FILMS THICKER OR THINNER BASED ON THESE KINDS OF CONDITIONS ONCE YOU ABSORB TO THE POINT OF REVERSAL, CAN YOU RINSE OFF ANYTHING THAT’S NOT TRULY ABSORBED INTO THE SUBSTRATE, AND AGAIN OPPOSITELY CHARGED AND THAT MATERIAL SYSTEM WILL ABSORB AND AND WE CAN CONTINUE TO ALTASD MATING PLUS AND MINUS TO DO THESE MULTILAYER FILMS NOW I SHOW THIS IN GREEN AND BLUE STRIPES BUT WHEN THEY’RE POLYMERIC SYSTEMS THEY’RE HIGHLY INFILTRATED HOWEVER, YOU CAN GET THAT DEFINITION TWO WITHIN PLUS OUR MINUS FOUR LAYERS AND IF YOU HAVE ONE COMPETITION AND CAP THEM WITH ANOTHER COMPETITION CISION, CAN YOU CREATE GRADIENT SYSTEMS WHICH HAVE TWO OR MORE DELIVERY SYSTEMS COMBINED NOW WE CAN USE HYDROGEN BONDING FOR THIS SEIZES AS WELL, SO CAN YOU COMBINE A POLYETHYLENE OXIDE WHICH IS A USED POLYMER WITH PROTEINS SO I SHOWED YOU TWO BEAKERS BUT IN REALITY IN OUR LAB WE DON’T HAVE STUDENTS WHO HAVE TO MOVE BACK AND FORTH WITH THESE AND GENERATE THE FILM, FORTUNATELY WE CAN USE ROBOTICS TO GENERATE THESE FILMS EFFECTIVELY AND WE CAN PROGRAM THEM TO GENERATE ANY KIND OF FILM LAYER SEQUENCE WE WANT BUT ANYWHERE IN THAT PROCESS IT CAN TAKE FROM A FEW MINUTES TO ABSORPTION AND HOWEVER THIS, IS A SCALABLE PROCESS BECAUSE YOU CAN ACTUALLY USE OTHER METHODS AND HERE WE’RE SHOWING ONE METHOD WHICH IS SPRAYED LAYER BY LAYER WHICH IS GENERATED IN OUR LABORATORY WHERE YOU CAN USE CONVECTIONS TO SPRAY THESE SYSTEMS ON TO A SUBSTRATE DIRECTLY AND SPRAY THE RINSE AND THEN SPRAY THE OTHER SYSTEM AND THIS IS ACTUALLY A HOME PLATE AUTOMATED STRAY LAYER BY LAYER SYSTEM BUILT BY ONE OF MY FORMER STUDENTS WHO LATER WENT ON TO START A COMPANY WHICH MADE THESE MICROSPRAYERS AND WENT ON TO GENERATE LARGE SCALE LAYER BY LAYER SYSTEMS WHICH CAN GENERATE 50 FEET PER MINUTE OF HUGE YARDS OF LAYER BY LAYER MATERIALS THIS DEMONSTRATES THE FACT THAT’S SCALABLE AND ALLOWED US A BROAD RANGE OF SUBSTRATES, HIGH SUBSTRATES TO FLAT ONES HERE WE CAN SHOW A CARTOON OF AN ELECTROSPUN MAT WHICH HAS FIBERS THROUGHOUT AND IF WE TAKE A CROSS SECTION, WE CAN SEE THAT WE COATED EVERYTHING ALONG THE SURFACES OF THESE MATS, HERE YOU CAN SEE THAT THEREY A NANOMETER SCALE COATING ON THIS FIBER FOR EXAMPLE, BUT WE CAN CHANGE THE THESE ON THE POROUS SUBSTRATES AS WELL THIS GIVES US VERSATILITY WHEN WE’RE REGENERATING SOMETHING LIKE TISSUE REGENRATIVE SCAFFOLD SO I WILL DESCRIBE THREE STORIES OF HOW WE USE THIS LAYER BY LAYER APPROACH TO GENERATE ZONES FOR A LAYER BY LAYER APPLICATION THE REASON WE’RE DOING THIS IS AND WHY IT’S PARTICULARLY COMPELLING IS BECAUSE WE USE WATER, THIS IS SENSITIVE TO SOLVENT AND HEAT, BIOLOGIC DRUGS, PROTEINS, ANTIBODIES A RANGE OF SiRNA, MRNA, RODRNA, A RANGE OF SYSTEMS WHICH YOU NORMALLY WOULD NOT PUT IN SOLVENTS AND HEAT UP ON TOP OF THAT IF WE THINK ABOUT A MORE TRADITIONAL POLYMER LIKE POLYLACTIC GLYCOLLIC AX SID AS A CARRIER, THIS WOULD ALLOW YOU TO PUT IN ONE OR TWO WEIGHT PERCENT OF A MATERIAL LIKE A DRUG THEN THE RULES OF THERMODYNAMICS WILL TAKE OVER BECAUSE YOU WILL GET BASE SEPARATION BECAUSE ALL SYSTEMS CAN ONLY CONTAIN SMALL AMOUNTS OF THE SEPARATION, IF IF YOU GET BASE SEPARATION, YOU WILL GET A BOLUS RELEASE, EVERYTHING AT ONCE, IF YOU PUT TWO ORIGINAL THREE DRUGS INTO A CONVENTIONAL CODING THEY WILL COME OUT AT ONCE ON THE OTHER HAND WITH LAYER BY LAYER WE CAN SELECT THE POLYION WITH THIS POLYCAT ION, THIS IS A POLYBETTA AMINO ESTHERS, THE MAIN POINT IS WE HAVE A CHARGE AND A GROUP THAT ALLOWS THEM TO BREAK DOWN AND THEY’RE BIOCOMPATIBLE IF WE USE SOMETHING LIKE THIS, AND LAYER IT, WITH A DRUG MOLECULE THAT IS CHARGED, WE CAN CREATE STEM CELLS IN WHICH THE DRUG IS THE COMPONENTS OF THE FOAM NOW WE’VE GONE FROM LOADING FROM ONE OR TWO% TO LOADINGS THAT ARE IN THE 10, 20, 30% RANGE AND BE AS HIGH AS 50 WEIGHT% WE CAN GET A HUGE AMOUNT OF A DRUG INTO A VERY SMALL FILM AND THE FILM IS THIN ENOUGH IT CAN COAT A RANGE OF DIFFERENT SUBSTRATES AND FAIRLY COMPLEX GEOMETRY, ON TOP OF THAT WE COMPARE EACH DRUG WITH A DIFFERENT POLYMER AND GET A DIFFERENT RELEASE RATE

NOW WE’RE ACTUALLY TUNING THE RELEASE RATES OF THE DRUG IF WE’RE ABLE TOW INTRODUCE SEPARATIONS BETWEEN THOSE DRUG LAYERS, WE CAN PREVENT INTERFUSION OF THE DRUGS DURING THE CONSTRUCTION OF THE FILM AND WITH THAT WE CAN GET NOT ONLY DIFFERENT RELEASE RATES FOR EACH DRUG BUT A KIND OF CONTROLLED STAGGER OR STAGE WISE RELEASE OF EACH DRUG AND THERE ARE SEVERAL REASONS WHY THIS IS COMPELLING ONE CAN IMAGINE FOR EXAMPLE, THE DESIRE TO RESMALL MOLECULES FROM AN IMPLANT SURFACE WHICH MAY CONTAIN INFECTION, FOLLOWED BY MOLECULAR SYSTEMS LIKE PROTEINS THAT CAN ALLOW OR ENABLE THE REGENERATION OF TISSUE SO WITH THAT I AM ACTUALLY GOING TO DESCRIBE THESE THREE DIFFERENT AREAS OF WORK AND THE FIRST ONE IS ABOUT BONE REGENERATION HERE WE’RE INTERESTED IN BEING ABLE TO USE THIS TECHNIQUE TO INTRODUCE GROWTH FACTORS WHICH NORMALLY ARE INTRODUCED IN A PRODUCT AND HIGH CONCENTRATION ONE OF THESE IS THE BMP-TWO, IT’S OSTEOINDUCTIVE IT RECRUITS THE NATIVE STEM CELLS AND BONE MARROW, AND ALLOWS THEM TO DIFFERENTIATE INTO OSTEOBLASTS WHAT’S NICE ABOUT THIS PROTEIN IS THAT IT’S POTENT IT ACTUALLY HAS A VERY HIGH OSTEOINDUCTIVE EFFECT WHAT’S BEEN THE DOWN SIDE OF IT IS THAT WHEN IT’S USED IN THE WAY THAT IS TYPICALLY AVAILABLE, A COA GEN SPONGE CONTAINING MILLIGRAMS OF THE BMP TWO, A PRODUCT FOR EXAMPLE TAKEN–THEY ALLOWS A BOLUS RELEASE OF THIS MATERIAL, YOU WILL GET A HUGE REMOUNT RELEASED INTO THE BLOOD STREAM AND AND A SMALL AMOUNT THAT RESIDES IN THE SITE AND AT THE SITE OF DELIVERY SO IN THESE CASES, YOU END UP FLOODING THE BLOOD STREAM AND THE BODY WITH BMP TWO AND YOU CAN GET ECTOPIC BONE GROWTH YOU CAN ACTIVATE TUMOR GENIC CELLS AND ESSENTIALLY ACTIVATE CANCER AND THERE ARE A NUMBER OF NEGATIVE IMPACTS OF HAVING THIS LARGE MOLECULE AND YET IT’S A PROMISING MOLECULE FOR TREATMENT SO CAN WE DELIVER THIS IN A PHYSIOLOGICAL RELEVANT QUANTITY AND OVER THE TIME PERIODS THAT ARE RELEVANT NOT IN A FEW MINUTES OR SECONDS BUT IN AN EXTENDED TIME PERIOD WE’RE ALSO INTERESTED IN GROWTH FACTORS THAT CAN SUPPORT VASCULARIZATION AND SOME OF THESE INCLUDE VEG F WITH IS A DIRECT ANGIOGENIC FACTOR AND THEN PDGF WHICH ALSO SUPPORTS THE GENERATION OF THE VASCULAR SYSTEM SO THESE ARE PROTEINS THAT ARE SIMILAR YOU NOTICE THEY HAVE A NICE ELECTRIC POINTS OF EIGHT AND HALF, THAT MEANS IN WATER AT PH SEVEN OR NEUTRAL Ph, THEY’RE POSITIVELY CHARGED SO WE TREAT THEM AS A POSITIVELY CHARGEDDENTITY AND WE ABSORB THEM FROM VERY DILUTE SOLUTIONS ON TO THESE SUBSTRATES AND WE ALTERNATE THEM WITH A NEGATIVELY CHARGED SPECYINGS THE NEGATIVELY CHARGED SPECIES WITH BE POLYKRILLIC ACID WHICH A SIMPLE HIGHLY CHARGED MOLECULE OR IT CAN BE A POLYSACCHARIDE ALL OF WHICH ARE NICELY NEGATIVELY CHARGED AND FINALLY WE GET A BIODEGRADABLE, BIOCOMPATIBLE AND POLYESTHER OR PBACE LAYER AND WE CALL THESE POLYONE AND POLYTWO, WE’RE RUNNING OUT OF NAMES AS YOU CAN TELL WHEN WE NAMED THEM BUT THEY GIVE US AN IDEA OF HOW LONG IT TAKES FOR THE MATERIALS TO RELEASE IN MY FIRST EXAMPLE I’M DESCRIBING THE WORK OF MY STUDENT NISARG SHAH, WE’RE DESCRIBING BONE DEFECTS THAT DO NOT HE’LL AND REPLACE THE KNOWN THERAPY WHICH IS TO DO A BONEAR VEST AT A SECONDARY SITE, USUALLY THE HIP THE SECONDARY SITE OFTEN IS THE CAUSE OF MORBIDITY, INFECTION AND A RANGE OF OTHER PROBLEMS ON TOP OF THAT A NUMBER OF PATIENTS WHO ARE SUFFERING FROM EITHER A LARGE TRAUMATIC BLAST OR SHOCK OR MAY BE EXPERIENCING SOME FORM OF DISEASE MAY NOT BE ABLE TO DONATE VERY MUCH BONE SO THERE’S A PROBLEM WITH THIS THERE ARE ALSO PROBLEMS AND LOOK AT OTHER MATERIALS THAT SORT OF COVER OR ESSENTIALLY PROTECT THIS AREA, BUT THEN WE HAVE AN ISSUE WITH THE MATERIAL INTERFACE THAT IS GENERATED AS A RESULT OF THAT SO WE DECIDED TO INVESTIGATE THE RELEASE OF GROWTH FACTORS OVER SMALL DOSES OR VERY CONTROLLED TIME PERIOD FROM A SCAFFOLD THAT DEGRADES AND TO DO THIS WE START IT WITH PLGA WHICH IS A KNOWN FDA APPROVED POLYMER AND WE GENERATED POROUS MEMBRANES FROM THEM USING A SIMPLE PROCESSING TECHNIQUE SO THIS IS LIKE WHITE SLOPPY FILMS YOU CAN CUT OUT WITH A COOKIE CUTTER WHICH WAS DONE HERE AND IN THIS CASE, THIS WAS CUT TO THE SIDES OF OUR CRANIAL

DEFECT MODEL WE THEN CODED THAT PLDA WITH WHICH OCCURS OVER A COUPLE OF MONTHS WITH A LAYER BY LAYER FILM AND CAN YOU SEE IT’S SITTING ON TOP OF THIS POROUS SUBSTRATE WE CAN THEN ALLOW RELEASE OF THE GROWTH FACTOR BASED ON WHAT WE’VE DONE TO INCORPORATE THE MATERIAL AND IN THIS PARTICULAR EXAMPLE WE WOULD BMP CONTAINING LAYERS ON THE BOTTOM AND PDGF WILL HELP SUPPORT VASCULARIZATION WHEN WE LOOK AT THE RELEASE OF THIS SIMPLE SYSTEM, WE KNOW THERE’S A SLIGHT DELAY OF ONE OVER THE ORGT SO PDGF COMES OUT OVER THE PERIOD OF THE FIRST WEEK OR SO WHEREAS THE BMP TWO IS MUCH MORE EXTENDED PERIOD WHICH GOES OUT FOR 30-32 DAYS AND THEN WE LOOKED AT WHAT HAPPENS WHEN WE HAVE SINGLE VERSUS DUAL GROWTH FACTORS AND HERE WE LOOK AT THOSE HERE WE CAN SEE CT OF THE BONE REGENERATION PROCESS, HERE’S OUR UNTREATED CONTROL AND YOU CAN SEE THE CRITICAL DEFECT IS OF COURSE NOT CLOSING IN THE RAT CRANIAL DEFECT MODEL HOWEVER IF YOU LOOK AT THE RELEASE OF 0.2 MICROGRAMS OF BMP TWO YOU CAN SEE THAT YOU ARE GETTING CLOSURE OF THE CRITICAL DEFECT AND IT SEEMS TO BE FAIRLY RAPID WITH THE TWO MICROGRAM DOSE, WE’VE INCREASED THE DOSE BUT HAVEN’T CHANGED ANYTHING ABOUT THE NATURE OF THE CLOSURE OF THIS WOUND OF THIS TIME PERIOD WHICH SUGGESTS THAT THERE IS A LIMIT TO HOW MUCH MORE CAN BE IMPACTFUL HOWEVER WHEN WE LOOK AT .2S OF BMP TWO AND .2 OF PDGF, WE SEE SOMEWHAT MORE RAPID HEALING BUT WE CAN’T TELL WHAT’S GOING ON WITH THE BONE UNTIL WE LOOK AT HISTOLOGY AND DO MECHANICAL MEASUREMENTS OF THE NATIVE BONE AND SEE WHAT’S GENERATED HERE SO HERE YOU SEE CROSS SECTIONS OF THE UNTREATED DEFECT IN WHICH WE HAVE A GAP THAT REMAINS AND THE MARGIN OF THE BONE IF IF YOU JUST HAVE A PLGA MEMBRANE AND NOTHING ELSE, YOU GET FIBROBLASTS AND END UP WITH THE COLLAGE NOWS SCAB LIKE MATERIAL BUT NOT BONE IF YOU LOOK AT .2 MICROGRAMS RELEASED FROM OUR SCAFFOLD, WHAT YOU CAN SEE IS THAT YOU’RE GOING TO GET BONE GENERATED AND SIGNIFICANT AMOUNTS, CERTAINLY ENOUGH TO BRIDGE THE ENTIRE DEFECT AND ALL OF THESE ARE TAKEN AT FOUR WEEKS BUT YOU ALSO SEE THAT THE BONE IS SOMEWHAT ROUGH LOOKING AND IF WE INCREASE THE DOSE BY A FACTOR OF 10, WE SIMPLY GET A BIG LUMP OF BONE THAT HAS VERY LITTLE RELATIONSHIP WITH THE MARGINS IT’S ESSENTIALLY A KIND OF CAP OF BONE THAT’S BEEN FORMED: HOWEVER, WHEN WE LOOK AT THE CASCADE OF GROWTH FACTORS, PDGF FIRST FOLLOWED BY BMP TWO, IF YOU REMEMBER THE TIME SCALES PDGF RELEASED MORE RAPIDLY OVER THE FIRST WEEK AND BMP TWO IS RELEASED OVER A PERIOD OF A MONTH, WHAT WE’RE SEEING AS A RESULT IS A MORE VASCULARIZED BONE THAT APPEARS TO BE MORE LIKE THE MARGINS BUT WE’RE ALSO SEEING MORE COMPLETE REMODELING, RATHER THAN SEEING A LARGE NUMBER OF DEFECTS AND BUMPINESS AT THE SURFACE, WE SEE A SURFACE THAT IS VERY SIMILAR TO THE ORIGINAL CRANIAL SURFACE OF THE BONE IF YOU LOOK AT THE BONE WE SEE THAT WE DO WE GENERATE VERY HIGH MOLECULAR WEIGHT MATERIAL, HIGH MECHANICAL PROPERTIES IN THESE MATERIALS AND WE’RE ALSO ABLE TO WITH MECHANICAL MEASUREMENTS COMPARE THAT THEY MATCH THE NATIVE BONE UNLIKE WHAT WAS GENERATED IN AT THIS TIME FIRST TWO EXAMPLES IN WHICH THE BONE IS MORE BRITTLE AND BREAKS MORE READILY SO WE THINK THAT WHAT WE’VE DONE IS INTRODUCE VASCULARIZATION EARLIER IN THIS BONE, SO THIS BONE IS A HEALTHIER BONE, IT’S MUCH LESS LIKELY TO BECOME THE PRODUCT BUT MAYBE AS IMPORTANTLY WE’VE INTRODUCED PATHWAYS FOR SECOND WAVE OF OSTEOCLASS RATHER THAN OSTEOBLASTS AFTIO CLASS REMODEL BONES AND THAT BONE REMODELING IS WHAT IS ALLOWING US TO ACHIEVE BONE WHICH HAS MECHANICAL PROPERTIES THAT WE NEED NOW ALONG WITH LOOKING AT CRANIAL DEFECT, WEEP BEEN LOOKING AT WEIGHS IN WHICH WE CAN INTEGRATE BONE ON TO IMPLANTS AND THIS IS A GROWING AND INCREDIBLY IMPORTANT PROBLEM BECAUSE IT TURNS OUT THAT ORTHOPEDIC IMPLANTS AND THEIR USE HAS INCREASED DRAMATICALLY BECAUSE OUR POPULATION HAS GROWN OLDER AND IS HEALTHIER WE HAVE MORE OF OUR POPULATION THAT HAS A WHOLE JOINT IMPLANT, SHOULDER, HIP, KNEE, THE FACT THAT WE’RE ALSO A MORE ACTIVE POPULATIONADS

TO THE PROBABILITY OF SEEING THESE IMPLANTS IN USE MORE IMPORTANTLY THERE ARE MORE REVISIONEDART ROUGH ATOM PLASTYS THAT IS THE ENVISION OF THESE AFTER THEY LAST FOR DWELVE OR 15 YEARS AND AFTER THAT PERIOD OF TIME THE RELATIVE AMOUNT OF LOOSENING THAT CAN TAKE PLACE OVER TIME CAN REQUIRE THAT THE OLD IMPLANT NEEDS TO BE REPLACED WE WANT THEM TO LAST AS LONG AS POSSIBLE BUT WE’RE ULTIMATELY GOING TO NEED TO REPLACE THIS IMPLANT OVER THE LIFETIME OF MANY PATIENTS AND IN THAT CASE, THE INFECTION RATE SOARS FROM LOWER THAN ONE% DEPENDING ON WHICH REVIEW ARTICLE YOU MIGHT READ, 10-20% THERE’S A VERY REAL INCREASE IN INFECTION SO WE HAVE A CONCERN ABOUT THIS: ON TOP OF THAT WE WOULD LIKE TO GENERATE A VERY STRONG BONE INTERFACE WITH THE NATIVE BONE AND THE IMPLANT THE BETTER THAT FACE, THE LESS LIKELY THAT INFECTION WILL HAVE TO RETURN WE’RE INTERESTED IN BEING ABLE TO ACCOMPLISH THIS USING A SINGULAR CODING BECAUSE THE COMMON TREATMENT RIGHT NOW IS A TWO STEP PROCESS IN WHICH YOU ESSENTIALLY REMOVE THE IMPLANT FROM THE PATIENT YOU BACK FILL THE EMPTY JOINT AREA WITH THESE ANTIBIOTIC BEADS WHICH RELEASE OVER A PERIOD OF SIX WEEKS OR MORE, YOU WILL BRACE THE PATIENT, WAIT AND GO BACK FOR A SECOND SURGERY THIS TWO SURGERY EXTENDED HOSPITALIZATION OR IMMOBILIZATION PERIOD IS NOT ONLY GOING TO INTRODUCE MORE STRESSOT PATIENT BUT ALSO MORE COST IN THE SYSTEM SO IN OUR WORK WE’VE BEEN LOOKING AT WAYS IN WHICH WE COULD GENERATE AN OSTEOCONDUCTIVE BASE LAYER BECAUSE WE’RE NO LONGER WORKING WITH A DEGRADABLE MEMBRANE BUT WITH A STIFF RIGID MATERIAL LIKE TITANIUM OR PEEK AND WE HAVE AN ENVIRONMENT IN THIS WHICH BONE CELLS CAN GROW AND ADHERE SO IN OUR CASE THAT BASE LAYER IS GOING TO BE COMPOSED OF A KITAS AN WRAPPED NANO PARTICLE AND WILL BE LAYERED WITH A POLYACRYLIC ACID WE PUT DOWN THIS MATERIAL, WE CAN THINK OF IT AS A SYSTEM THAT DOESN’T DEGRADE OVER THE LIFETIME OF OUR ZONE, BUT WILL BE REARBSORBED BY PLAYERS OVER A TIME PERIOD NOW WE HAVE THAT POSITION OF BONE AND ON TOP OF THAT, WE WILL PLACE LAYERS OF BMP TWO, WHICH CAN RELEASE OVER AN EXTENDED PERIOD OF TIME SO THIS IS OUR EARLY EXPERIMENT, WE HAVEN’T INTRODUCED THE ANTIINFECTIVE YET, WE WILL GET TO THAT BUT WE FIRST HAD TO ASK THE QUESTION OF WHETHER OR NOT WE CAN GENERATE A STRONG BONE MATERIAL INTERFACE SO HERE WE USE A RAT TO BE A PUNCTURE MODEL WHICH IS DEMONSTRATED HERE AND WE CREATE A DEFECT IN THE RAT TIBIA AND WE TAKE A PLASTIC OR TITANIUM IMPLANT COATED OR UNCOATED AND INSERT IT IN AND THEN OVERTIME WE WILL OBSERVE THE BONE MATERIAL INTERFACE USING SEVERAL METRICS INCLUDING HISTOLOGY, MECHANICAL PROPERTIES AND JUST GENERAL MEASUREMENT OF THE CONCENTRATION OF GROWTH FACTOR AND OTHER FACTORS IN THIS THE AREA SO HERE YOU CAN SEE, THE PLASTIC IMPLANT WE USED WHICH WAS PEEK AND PEEK WITH HOLES DRILLED IN IT WE COATED THESE TWO EXAMPLES WHICH REPRESENT A SMOOTH IMPLANT AND THE WHAT WE WERE REPLICATING AS A TEXTURED IMPLANT WAS COAT WIDE BMP TWO WAS THAT LABELED WITH FLUORESCENT PROTEIN SO HERE WE CAN ACTUALLY SEE THIS RELEASE OVER TIME AND IT’S A LITTLE BIT DARK HERE BUT CAN YOU SEE THAT IT’S RELEASED OVER A PERIOD OF 30 DAYS IF WE LOOK AT RADIANT EFFICIENT, WE CAN SEE THE RELEASE IS QUITE CONSTANT AND IT’S TAKING PLACE OVER THIS PERIOD OF A MONTH, FAIRLY CONSIST EPTLY WE LOOKED AT DIFFERENT LOADINGS, 20, 40, 60 LAYERS ARE REPRESENTATIVE OF THESE THREE DIFFERENT LOADINGS AND WE LOOK AT THE CONCENTRATION OF BMP THREE, THAT BMP TWO THAT IS ACCUMULATED IN THIS AREA OF INTEREST WHICH IS SHOWN HERE BY EXCAVATING THE TISSUE AND DETERMINING HOW MUCH BMP TWO IS THERE AND WHAT’S INTERESTING IS THE NUMBERS HERE, CAN YOU SEE OVER A PERIOD OF 30 DAYS FOR THE SMOOTH VERSUS THE PORUS OR ROUGHENED IMPLANT WHERE WE CAN GET A STAINED RELEASE ON THE SURFACE OR ROUGHENING, BUT AT ANY GIVEN TIME THE CONCENTRATIONS DON’T EXCEED 200 NANO GRAMS PER MILLI LITER AND WE WERE CONTROLLING THE AMOUNT OF BMP TWO RELEASE

HERE WE’RE GETTING ENOUGH IN THE REGION TO MAKE A DIFFERENCE PHYSIOLOGICALLY WITHOUT CREATING ENOUGH OF SYNCH OF BMP TWO WITHOUT FLOODING THE BLOOD STREAM WITH THE GROWTH FACTOR WE LOOKED AT HISTOLOGY BUT WE HAVE AN ENTIRE LIBRARY OF HISTOLOGICAL SLICES OF THE TISSUE THIS ONE IS MEANT TO SHOW THE INTERFACE BETWEEN PART OF THE IMPLANT THIS IS THE PLASTIC AND THE BONE AND WE CAN SEE THAT THE BONE IS VERY NICELY ARRANGED AND ORIENTED AROUND THIS IMPLANT WE ACTUALLY EXAMINED THE NATURE OF THIS BONE AND FOUND IT WAS VERY NICELY ORIENTED IT HAD EXCELLENT MECHANICAL PROPERTIES ON TOP OF THAT WE DID A PULL OUT TEST IN WHICH WE DETERMINED THE AMOUNT OF FORCE REQUIRED TO PULL THE IMPLANT OUT OF BONE AND WE DID IT AT PERIODS OF ONE WEEK, TWO WEEKS AND FOUR WEEKS HERE WE CAN SEE THE UNTREATED CONTROL AT THE BOTTOM AND THESE IMPLANTS SHOW THAT IT’S GROWN NEAR THE IMPLANT BUT IT’S NOT INTEGRATED INTO IT AND IT’S VERY EASY TO PULL THESE OUT BUT THE PULL OUT COURSES WE SEE WAS THESE DIFFERENT DOSES INDICATED BY DIFFERENT NUMBERS SHOW THAT IN THE FIRST COUPLE OF WEEKS THERE’S DOSE DEPENDENT AND A VERY STRONG INCREASE IN THE PULL OUT FORCE BUT BY THE TIME WE REACH FOUR WEEKS EVERYTHING HAS LEVELED OFF AND WE SEE THAT EVEN OUR LOWEST DOSE SYSTEM IS ABLE TO PROVIDE A HIGH LIE INTEGRATED IMPLANT BONE MATERIAL INTERPHASE AND THE STRENGTH OF THIS IS FOUR TIMES THAT OF WHAT IS GENERATED WITH POLYSACRAMENT WHICH IS BONE USE WHEN WE PULL THESE OUT WE SEE THE FRACTURE TAKES PLACE AT THE NATIVE BONE MATERIAL AND NOT AT THE INTERFACE NOW WE WANT TO LOOK AT ANOTHER WAY TO DO THIS, WE USED IT GENETICALLIER MY O SIGN SULFATE, AND JUAHA MIN USED THESE SAYSS AND SHE USED RETINAL LOCATION TO GENERATE THE SULF EIGHT AND GREEN TO REPRESENT BMP TWO AND SHE BEGAN TO CONSTRUCT THE SYSTEMS IN WHICH THERE WAS NO DEGREE OF BARRIER AND SHE ALSO INTRODUCED BARRIER LAYERS BY USING NATURALLY OCCURRING FLOW AND THIS IS JUST LIKE NANO PLAY IT’S USED FOR A NUMBER OF APPLICATIONS AND IN OUR CASE WE CHOSE THIS SYSTEM BECAUSE IT EXHIBITS GOOD BIOCOMPATIBILITY AND THIS IS GENERALLY REGUARDED AS SAFE AND THEY’RE LOW COST AND BY O DEGRADABLE THEY’RE BIOACTIVE WILL THEY CAN ENHANCE CELL ADHESION AT SURFACES AND INTERPHASES SO WE WILL USE THEM AS A BARRIER LAYER BETWEEN THE TWO DRUG LAYERS, THE QUESTION HERE IS CAN WE GET A STAGGER THAT WE CAN CONTROL ON THE LEFT WHAT YOU CAN SEE IS THE THICKNESS IN THE FILM AS JUOHA BUILT UP THESE LAYERS THEN INTRODUCED THESE TWO LAYERS AND THEN SULFATE AND THEN SOME CASES SHE CAPPED THEM OFF WITH ANOTHER LAYER THE QUESTION IS COULD WE SEGGREGATE THE TWO DRUGS WITH COMPARTMENTS AND HERE CAN YOU SEE WITH ELECTRON MICROSCOPY AND A METHOD CALLED EDS, THESE DARK REGIONS ARE THE LAYERS AND THE BRIGHT REGIONS ARE THE INTERCAPILLARY LAYERS THAT SEPARATE THEM HERE WE SEE THE RED SILICONE LAYERS AND THESE ARE THE CLAY THAT ARE SEPARATING OUR MATERIALS WHEN WE LOOK AT THE RELEASE BETWEEN THESE SYSTEMS, WITH JUST THE MICEIN AND THE SULFATE, WE GET NICE RESULTS WE GET A RAPID RELEASE OF THE GENETICALLY O MICEIN AND WE DO WANT TO ELIMINATE AN EXISTING INFECTION AND THIS BULLET RELEASE IS NOT BAD AND WE SEE THERE IS A DELAYED RELEASE OF THE MP TWO AND WE SEE IF THERE’S A SIGNIFICANT AMOUNT IN THE FIRST FOUR DAYS AND THE X AXIS IN THE DAYS AND HERE WE’RE SEEING A BLOW UP OF THAT RELEASE BEHAVIOR IF WE HAVE THIS BARRIER LAYER IN BETWEEN THE TWO, IT TURNS OUT THAT WITH THE BMP TWO WE ACTUALLY CAN DECREASE THIS RATE OF RELEASE, WE GET IN FACT A 14 TIMES LONGER RELIEVE PERIOD OR HALF LIFE AND YOU CAN SEE THIS IN THE BLOW UP MORE EASILY AND YOU FLATTEN THIS CURVE OVER THE FIRST SEVERAL DAYS WHILE YOU’RE GETTING RID OF INFECTION RAPIDLY WITH THE GENETICALLY O MICEIN SULF EIGHT AND WE CAN TUNE THIS FURTHER AND SHE WANTED TO BE ABLE TO DEMONSTRATE RELEASE OF ENOUGH IN THE BEGINNING TO ELIMINATE INFECTION SO HAVE YOU THIS INITIAL EBOWL US COMBINED WITH THE ABILITY TO MAINTAIN

THIS CRITICAL CONCENTRATION TO PREVENT INFECTION OVER LONGER PERIODS OF TIME SO THEN WHEN WE MOVE BACK TO OUR RAT MODEL WE GENERATED AN INFECTED RAT TIBIA MODEL SO SHE INFECTS THIS TIBIA KAFITY, IT’S A RATHER CHALLENGING MODEL SO THIS IS A NICE CHUNK AND SHE THEN IS ABLE TO INSERT EITHER AN UNCOATED IMPLANT SUSPICIOUS AN IMPLANT WHICH CONTAINS GENT O MICEIN SULFATE OR ONE THAT CONTAINS BOTH AND WE’VE BEEN OBSERVING THAT THE BMPTWO DOES THE GENETICALLY O MICEIN SULFATE LEADS TO REGENERATION OF BONE AROUND THE IMPLANT FURTHER MORE IF WE LOOK AT THE MICRO CT, WE SEE THE ABSORPTION THAT OCCURS IN THE SAMPLE AND WE CAN SEE THAT JUST WITH THE SULFATE, BONE IS ENABLED IT CAN BEGIN TO REGENERATE BUT IT’S NOT AS COMPLETE AND WITH THE COMBINATION WE’RE GETTING A MUCH MORE SOLID SLUG OF REGENERATED BONE PRESENT IN THE SYSTEM SO OUR HOPE IT TO CONTINUE TO LOOK AT THESE COMPARTMENTALIZED SYSTEM BUT USE A SYSTEMATIC APPROACH TO TUNING THE RELATIVE RELEASE OF THESE SYSTEMS USING BARRIER LAYERS AND ALSO BEGIN TO INTRODUCE ANTIBIOTIC COMBINATIONS OR MULTISPECTRUM ANTIBIOTICS THAT WILL GIVE US A BROAD SPECTRUM OF ATTACK INCLUDING ADDRESSING MRSA ALL RIGHT, I’M GOING TO MOVE TO THE SECOND EXAMPLE WHICH IS A SOFT TISSUE EXAMPLE HERE THE IDEA IS TO TRY TO ADDRESS WOUND HEALING PROCESSES THAT HAVE GONE AWRY THE IDEA IS THAT A NUMBER OF THESE WOUND HEALING PROCESSES HAVE OVEREXPRESSION OF A SPECIFIC GENE OR SOME SORT OF GENETIC DISREGULATION OUR EARLIEST TARGET WAS TO TARGET CHRONIC WOUND HEALING AND THE IDEA WAS TO CORRECT THE OVEREXPRESSION OF CERTAIN GENES THAT ARE OVEREXPRESSED IN HYPOGLYCEMIC PATIENTS DURING THE WOUND HEALING PROCESS SO THE IDEA IS THAT WE CAN TRY AND ADDRESS SOME OF THESE OVEREXPRESSED PROTEINS, ONE OF WHICH IS MMP NINE THIS IS A PROTEASE THAT IS A NATURAL PART OF THE REMODELING PROCESS THAT ALL HEALING PROCESSES MUST GO THROUGH AND THIS IS EARLIER IN THE WOUND HEALING FACE IN THESE DIABETIC PATIENTS AND THIS HAS BEEN DEMONSTRATED FOR A NUMBER OF DIFFERENT STUDIES SO THE IDEA HERE IS TO KNOCK DOWN MMP NINE FOR A DELAYED–FOR A SMALL PERIOD OF TIME AND EFFECTIVELY ENABLE THE DEPOSITION OF GRANULATION TISSUE OR COLLAGEN WHICH WILL THEN LEAD TO HELP TO WOUND CLOSURE AND I APOLOGIZE, IT LOOKS LIKE THERE’S A LITTLE DISCONNECT WITH MY COMPUTER AND THE SCREEN BUT WHAT YOU’RE SEEING HERE ARE THE SAME COMPONENTS BEFORE, A DEGRADABLE POLYMER, POLYTWO AND HERE WE USE CHITOSAN, AND DEXTRAN SULFATE, AND WITH A LAYER OF SUGAR AND SO WE BUILT IT WITH THE DEXTRAN SULFATE AND THEN THE POLYTWO, AND THEN THE CHITOSAN, AND THIS IS INTERESTING BECAUSE WE WERE MEETING THE CHALLENGE SiRNA DEGRADES QUICKLY IN THE BLOOD STREAM BUT WE’RE GOING TO PUT IN THIS A WOUND CAVITY WHERE THERE’S LARGE EXCESS PROTEASES AND CYTOKINES AND REACTIVE MOLECULES WE DIRECTLY INCORNERATED SiRNA INTO A LAYER THEM WITH A BANDAGE HERE CAN YOU SEE IT IN CONFOCAL WE ARE SHOWING THAT WE ARE COATING ALL OF THE CREVICES, WE CHANGED THE NUMBER OF THE LAYER TO CHANGE THE RELEASE RATE AND THE TOP LAYERS TO CHANGE THE LOADING SO WE’RE WE’RE GETTING SIGNIFICANT LOADINGS WITH SiRNA IN THE RANGE OF 10-20 MICROGRAMS PER CENTIMETER SQUARED WE THEN USE A DIABETIC MOUSE MODEL AND WE’LL HAVE A CONTROL WOUND IN THIS WHICH WE DON’T INTRODUCE ANY TREATMENT WE ALSO HAVE SECOND CONTROL IN WHICH WE USE A SCRAMBLED SiRNA, DOESN’T ENCODE FOR ANYTHING AND WE’RE USING THE SIMMPI TREATED SYSTEM IF YOU LOOK OVER TIME AT THE GENE EXPRESSION IN THESE ANIMALS AT ONE WEEK AND AT TWO WEEKS WE WERE DESIGNING THESE SYSTEMS TO BE ACTIVE OVER A FOUND DAY TOWARD–14 DAY PERIOD WE SAW A IMOWK DOWN IN THE WOUND OF 60% AT ONE WEEK AND 85% AT TWO WEEKS WHICH IS SIGNIFICANT GIB THESE ARE UNMODDIFIED SiRNAs WE SEE A CORRESPONDING DROP IN

MMP NINE ACTIVITY IF YOU LOOK AT MASON’S TRI CHROME STAINING WE CAN LOOK AT THE 17 DAY AND 14 DAY TREATED SAMPLES AND HERE AT THE TOP WE HAVE THE SiRNA TREATED VERSUS THE SEVEN DAY UNTREATED AND WE ALSO HAVE OUR 14 DAY TREATED ASK UNTREATED AND WHAT YOU’LL SEE IS THERE’S A BRIDGE OF COLLAGEN IN THIS STAINED BLUE RIBBON AT THE TOP HERE THAT IS BRIDGING THE WOUND AT SEVEN DAYS WHICH WE DON’T SEE ON THE UNTREATED SAMPLES AND WHEN WE GET TO 14 DAYS WE SEE THERE’S A LARGE AMOUNT PRESENT COMPARED TO THE UNTREATED WOUND AND HERE YOU CAN SEE A CLOSE UP OF THAT TISSUE SO THERE’S A STARK CONTRAST BETWEEN THE TWO AND WE SEE CONTRAST IN THE RATE OF WOUND CLOSURE IN THE SYSTEM, HERE WE HAVE SEVEN DAY, 14 DAY AND THE DARK BAR IS OUR TREATED SAMPLE AND WE ALSO SEE IN IMPROVEMENT IN THE EPITHELIAL CLOSURE IN THESE ANIMALS THIS IS JUST THE BEGINNING THERE IS MORE THAN ONE GENE INVOLVED OF COURSE AND THESE KINDS OF WOUND HEALING PROCESSES AND WHAT WE’RE INTERESTED IN NOW IS COMBINING TARGETS WITH SiRNA AND USING GROWTH FACTORS IN OUR COMBINATION SYSTEM TO BE ABLE TO ADDRESS THIS MORE EFFECTIVELY BY REPLICATING THE WOUND HEALING PROCESS ANOTHER AREA OF INTEREST, A REAL TARGET FOR US IS TRYING TO ADDRESS HYPER TROPEIC SCARRING THIS IS ACTUALLY SOMETHING THAT CAN GREATLY INFLUENCE THOSE WHO ARE THE VICTIMS OF 3D, I’M SORRY OF THIRD DEGREE BURNS THOSE WHO HAVE EXPERIENCED LARGE TRAUMATIC BLASTS, A NUMBER OF THESE PATIENTS WHEN THEY HEAL, HEAL WITH LARGE AMOUNTS OF HYPER TROPIC OR HIGHLY FIB ROTTIC SCARRING AND THE SCARRING CAN REDUCE THEIR MOBILITY THERE’S A NUMBER OF TREATMENTS PROPOSED BUT MOST OF THEM WORK PRIMARILY FOR SMALL AREAS WE’RE INTERESTED IN BEING ABLE TO ADDRESS LARGER AREA KINDS OF WOUNDS SO IN THIS WORK, SORT OF SUMMARIZE IN THIS SLIDE, WE LOOK AT THE IN VIVO KNOCK DOWN OF ONE OF THE GROWTH FACTORS IN THE PATHWAY OF FIB ROTTIC RESPONSE THIS, IS CONNECTIVE TISSUE GROWTH FACTOR HERE WE’RE TRYING TO ADDRESS A DIFFERENT PART OF WOUND HEALING PROCESS IN CHRONIC WOUND HEALING WE HAVE THE DIFFICULTY OF GETTING THE PROCESS STARTED AND WE NEED TO GET COLLAGEN PRODUCTION TO BEGIN HERE WE HAVE AN OPPOSITE PROBLEM A KIND OF HEALING HERE IN THE STAGE THE DEPOSITION IS COLLAGEN IS ACCELERATED AND WE CAN’T STOP IT SO WE’RE TRYING TO KNOCK DOWN CTGF SO WE CAN LOWER THAT RESPONSE SO WE’RE LOOKING FOR, THIS IS A THIRD DEGREE BURN MODEL THAT IS CARRIED OUT AT THE SHINER’S BURN CENTER AT MASS GENTLEMEN HOSPITAL WITH MARTIN YARMISH, AND IN THIS BURN MODEL YOU CAN SUTURE THE EDGES OF THE BURN AND WATCH TO SEE WHETHER OR NOT THE BURN CONTRACTS IF THIS TISSUE CONTRACTS THE WAY IT SHOWS HERE, CONTRACTILE WOUND HEAL SUGGEST PART OF THE FIB ROTTIC RESPONSE IN OUR SYSTEM YOU CAN SEE THAT WITH JUST A CONTROL, YOU GET THE SAME KIND OF SHRINKAGE WITH THE WOUND BUT WITH THE KNOCK DOWN OF THE CTGF IF THESE CASES, WE AND SEE THERE’S A REAL REDUCTION IN THIS CONTRACTION FELT WHAT’S INTERESTING HERE IS WE DIDN’T COAT AN ENTIRE BANDAGE WE ONLY COATED THE SUTURE, COATING THE THREAD THAT IS USED TO SO UP THE ANIMAL AND WE’RE SEEING THERE IS A MEANINGFUL RESPONSE TO THE SiRNA THAT IS RELEASED FROM THAT SUTURE SO WE THINK THAT THE APPLICATION OF THIS,A PROACH IS VERY PROMISING BECAUSE WE CAN CONTROL THE DEPOSITION OF THE MULTILAYERS ON A NUMBER OF DIFFERENT SURFACES THAT MIGHT BE USED IN THE WOUND HEALING APPLICATION IT’S DIFFICULT TO SEA HERE BUT WE DID LOOK AT SOME OF THE CHARACTERISTICS OF CARING AND WE FOUND THAT FOR OUR SICTGF SYSTEMS WE INCREASED VASCULATURE AND IT’S HARD TO SEE BUT THESE ARE BLOOD VESSELS WHERE THE BLACK ARROWS ARE COMPARED TO SCAR TISSUE WHICH SHOWS VERY FEW, IF IF ANY BLOOD VESSELS AND WE ALSO SEE AN INCREASE IN THE SIZE OF THE BLOOD VESSELS WE ALSO HAD A MUCH ROUGHER EPITHELIAL SURFACE AND THE INDICATION OF STRUCTURES FORMING THAT ARE ESSENTIALLY FORMING TO WHAT WE COULD EXPECT FOR THE FORMATION OF FOLLICLES OR SWEAT GLANDS SO WE THINK THIS IS THE BEGINNING OF EXCITING WORK IN WHICH YOU MAY ADDRESS FIB ROTTIC SCARRING USING THE RATIONAL APPROACH THROUGH THE DELIVERY OF

CONTROLLED FACTORS AND SiRNA ALL RIGHT SO WE MOVED THROUGH TWO EXAMPLES WHERE WE TALK ABOUT WOUND HEALING AND WE TALK ABOUT BONE SOMETHING YOU CAN HOLD IN YOUR HAND AND IMAGINE TO THESE MICROSAIL MESHES WHICH WE CAN COAT READILY NOW I’M GOING TO TALK ABOUT COATING SOMETHING ON THE NANO SCALE IN THIS WORK WE’RE WORKING AT THE COKE INSTITUTE WITH ENGINEERS AND PHYSICIST AND CHEMISTS BIOLOGIST AND CLINICIANS TO ADDRESS CANCER: WHAT’S EXCITING ABOUT THIS WORK IS THAT WE CAN USE A VERY MODULAR PLATFORM TO INTEGRATE A RATIONAL APPROACH TO CANCER THE IDEA HERE IS THAT WE CAN TAKE AWE NOW PARTICLE CORE AND THAT CORE CAN BE ANY CORE IN OUR CASE WE’RE INTERESTED IN THIS VESSELS THAT HAVE ALREADY BEEN FDA APPROVED SO IT COULD BE A PLGA NANO PARTICLE, A LIP O STUDIES OF MULTIPLE ENDOCRINE THAT HAS A NEGATIVE CHARGE AND INTO THAT CORE WE CAN INCORPORATE A CHEMO THERAPY DRUG WE’RE INTERESTED IN HIGHLY AGGRESSIVE FORMS OF CANCER THAT RECUR OR THAT ARE VERY RESISTANT EVEN ON THE FIRST TREATMENT TO THE CHEMO THERAPY DRUG A NUMBER OF THEE FORMS OF CANCER HAVE UNDERGONE THE KINDS OF GENETIC MODIFICATIONS THAT ALLOW THEM TO ENCODE PROTEINS THAT SAVE THEM FROM THE IMPACT OF CHEMO THERAPY DRUG SO THE IDEA HERE IS THAT WE CAN INCORPORATE INTO OUTER LAYERS SiRNA ORIGINAL PERHAPS AN INHIBITOR THAT WILL BLOCK THAT PATHWAY FOR CELL SURVIVAL IN THE PRESENCE OF THE CHEMO THERAPY DRUG TYPEALLY WE RELY HEAVILYOT FACT THAT WE USE A POLYELECTROLIGHT, PROACH BECAUSE WE PUT DOWN A NEGATIVELY CHARGED LAYER AND THAT WILL PROVIDE US WITH A NANO PARTICLE THAT MUCH LESS LIKELY TO ENGAGE WITH OTHER CELLS WHEN WE INTRODUCE THIS INTO THE BLOOD STREAM AND I’LL TALK MORE ABOUT THE RULE OF THAT LAYER THIS IS THE GENERAL IDEA, MAKING IT NANO SIZED HELPS US BECAUSE IN CANCER THERAPIES WE’RE TRYING TO TAKE ADVANTAGE OF THE FACT THAT SOLID TUMORS GROW RAPIDLY IN THIS THE RAPID GROWTH AND THEY GENERATE VASCULATURE AND THE DEFECTS ARE HOLES IN THOSE BLOOD VESSELS PROVIDE US A ROUTE FOR NANO PARTICLES TO ESSENTIALLY ENTER ARE THE TUMOR TISSUE AND GIVE US ADDITIONAL REZONING DENSE TIME IN THAT TUMOR SO I SHOWED THE YOU THE SLIDE WITH THERS, YOU ARE PROBABLY THINKING WHAT ARE YOU GOING TO DO TO GET THE NANO LAYERS ON THE SURFACES IN OUR WORK WE CAN USE AN ALTERNATING TITRATION STEP WITH THE [INDISCERNIBLE] STEP WE ARE REPLACING THE TRI FIGGATION ACCEPT, I HAVE VERY HAPPY STUDENTS BECAUSE OF THESE PARTICLES NOW THIS IS SHOWING AWE QUANTUM DOT, A LEAD SULFATE QUANTUM DOT THAT HAS A NEGATIVE CHARGE, AND WE PUT LAYERS DOWN ON THE QUANTUM DOT WE CHOSE IT BECAUSE IT’S SMALL, 20-NANOMETER AND YOU CAN SEE THE IMPAC OF EACH LAYEROT NANO PARTICLE AND YOU’RE SEEING THAT YOU ADD ANYWHERE FROM FIVE TO 10-NANOMETERS FOR ABSORPTION AND YOU CAN SEE THE CHARGE GOING FROM PLUS TO MINUS AS YOU BUILD UP THE LAYER BY LAYER FILM IN THESE NANO PARTICLES WHEN WE FIRST CHOSE THIS WORK, POLYMERS COATED, WE USE POLYL LYSINE AND POLYR AND POLYPEPTIDES ANDY WE USED A NUMBER OF THE GLUE MARIOUSICOSA MINE FOR OUR SYSTEM AND I’M DESCRIBING SOME OF THE WORK OF STEVEN MORTON WHO IS A GRADUATE IN MY LAB WHEN WE BEGAN TO EXAMINE THE BEHAVIOR, WE MAKE SEVERAL INTERESTING OBSERVATIONS, FACT THAT THIS IS A LAYER BY LAYER FILM GIVES US AN ADVANTAGE BECAUSE WE CAN LAYER A FILM WHICH HAS A VERY STABLE NATURE TO IT AT PH 7.4 HOWEVER WITH HIGHA LUREONIC ACID THIS GIVES A STRONG CHARGE WHICH DISAPPEARS IF WE MOVE IT TO MORE ACIDIC ENVIRONMENTS BECAUSE WE’RE BEGINNING TO PROTEINATE THOSE GROUPS AND WE’RE BEGINNING TO ERASE THE NEGATIVE CHARGE THE NETIMPACT OF THAT IS TWO FOLD ONE OF THEM IS IF YOU ERASE THE NEGATIVE CHARGE, YOU ARE ERASING ELECTROSTATIC INTERACTIONINGS AND THE FILM’S CROSS LINKING DECREASES AND ABOUT IT BEGINS TO SWELL NOW RATHER THAN THE HALO WE HAVE THIS BIG FAT BUT RATHER SOFT SWOLLEN LAYER AROUND THE NANO PARTICLE THE SECTIONAL ANALYSIS THING IS THE CHARGE IT GOES FROM .4, AND

WE WERE LE LYING ON THE FACT THAT IT WAS STRONGLY CHARGED TO,A VOID INTERACTION WITH CELLS ON TOP OF THAT WE WERE RELYING ON THE FACT THAT HYALLUREONIC ACID IS CHARGED SO WE BELIEVE THESE ANIMAL VS THE BOUND WATER AROUND THEM AND IT IS THAT SHELL THAT’S HELPING US REPEL THE PROTEINS IN THE BLOOD STREAM AND THAT GIVES US A LONG RESIDENCE TIME IN BLOOD SO THE INTERESTING THING HERE IS THAT WITH THESE TWO CHANGES WE SEE A HUGE INCREASE IN CELL UPTAKE NONSPECIFIC CELL UPTAKE THIS MEANS THAT IF WE’RE ABLE TO ENTER A TUMOR THAT IS HIPOXIC WE CAN ENHANCE THE UPTAKE IN THE TUMORS BASED SOLELY ON THE PHYSICAL CHEMICAL PROPERTIES IN IN LAYER BY LAYER FILM SO WE TESTED THIS AND WE FOUND THAT WHEN WE INJECTED ANIMALS WITH SOLID TUMORS AND LOOKED AT WHERE THOSE NOW PARTICLES LOCALIZE, THEY CO LEGALIZE STRONGLY WITH HIPOXIC REGIONS OF THE TUMOR, THIS IS A WAY OF LABELING THESE HIGHWAY POXIC REGIONS, THESE ARE LABELED RED AND YOU CAN SEE THE OVERLAP IN THE YELLOW ZONE HERE HOWEVER, GETTING INTO THE TUMOR IS A GOOD THING AND ENCOURAGING THESE ACTIONS CAPTAIN BE GOOD, SO WE CAN’T RELY ON HYPOXIA LET TUMORS ARE HETEROGENEOUS, SO WE NEED A WAY TO INSURE THESE ARE TAKEN UP BY TUMOR CELLS SO IN THIS CASE WE DECIDE TO USE MOLECULAR TARGETING AND IN OUR SYSTEMS WE CAN ATTACH A RANGE OF DIFFERENT LIGANDS FROM FOALATE TO BINDING PEPTIDES THAT BIND TO RECEPTORS THAT OVEREXPRESSED IN CANCER CELLS AND WE HAVE DONE THAT WITH A NUMBER OF LAYER BY LAYER SYSTEMS AND IT TURNED OUT THAT BECAUSE WE’RE FOCUSED ON OVARIAN CANCER, TRIPLE NEGFIVE BREAST CANCER AND ADVANCED LUNG CANCERS ALL OF THOSE CANCERS OVER EXPRESS THE CD44 RECEPTOR WHICH HAPPENS TO BE THE HIGHER RECEPTOR, AS IT TURNS OUT, THAT HIGH AFFINITY FOR CD44 ALLOWED US TO USE JUST THE HYALLUREONIC LAYER AS THE TARGETING TARGETING MOLECULE SO ONCE AGAIN WE DID EXPERIMENTS TO SHOW WE’RE GETTING TARGETING ON CD44 WITH THE HYALLUREONIC ACID AND I’M SHOWING ERIK DREADEN, IN MY LAB WHEN HE LOOK AT THE PARTICLES LABELED RED HERE, WE SEE A HUGE AMOUNT OF UPTAKE, ABOUT 15 TIMES THAT OF A LAYER BY LAYER PARTICLE THAT CONTAINS DEXTRAN SULFATE, SIMILAR CHARGE, NO HYALLUREONIC ACID AND WE CAN TITRATE IT IN THE ACID AND HERE CAN YOU SEE THAT ALMOST COMPLETELY ELIMINATES THE UPTAKE WHEN WE DO A TUMOR SECTION IN THIS CASE AND LABEL CD44, WITH RED, IN THE NOW PARTICLES OF GREEN, AGAIN WE CAN SEE A HUGE OVERLAP BETWEEN THE LOCALIZATION OF OUR NANO PARTICLES AND THE LOCALIZATION OF THE TUMOR CELLS AND WHAT WE SEE ARE THE STROMAL CELLS THAT SURROUND THAT TUMOR: WHICH SUGGEST THAT WE HAVE A MOTE OF TRIPLE TARGETING WE’RE NOW USING TUMOR SIZE, WE’RE USING THE MICRO ENVIRONMENT OR HYPOXIA AND USING THE CELL RECEPTOR INTERACTION TO INSURE THAT THESE NANO PARTICLES ARE TAKEN UP NOW THAT WE HAVE A SYSTEM THAT CAN DO SOME FORM OF TARGETING WE CAN LOAD THE INNER CORE OF THE NANO PARTICLE WITH THE CHEMO THERAPY DRUG AND WE CAN BEGIN TO LAYER SiRNA WE ACTUALLY FOUND THAT THE POSITIVELY CHARGED PALY PEPTIDE IMPACTED WHETHER THE SiRNA GETS INTO THE CELL OR OUTSIDE THE CYTOSOL, THE POLYL UNDERGOES GREAT DEAL OF BUFFERING AND ENABLES THESE TO COMPROMISE THE ENDOSOMAL MEMBRANE AND THAT ALLOWS SiRNA TO GET OUT SO WE START WIDE A–WOULD BE OUR OUTER LAYER SYSTEM, WE FOUND THAT WE GENERATED THESE PARTICLES THOUGHT VERY NICE PLOD HALF LIVES THIS IS KEY BECAUSE WE FOUND IN THE BLOOD STREAM TO ACCUMULATE IN THE TUMOR AND IN THIS CASE, WE FOUND THAT WE HAD A 28 HOUR HALF LIFE WHICH IS ACTUALLY QUITE EXTENDED FOR THESE NANO PARTICLES THE SIZE OF THESE NANO PARTICLES ARE 120-NANOMETERS I SHOULD ALSO MENTION WE DIDN’T OBSERVE ANY KIND OF AMINO OR SUBTLE TOXICITY WE DID USE A PANEL OF CYTOKINES

TO EXAMINE NA: IF WE LOOK AT THE RELEASE CHARACTER ONCE AGAIN WE SEE THE STAGGERED EFFECT WE CAN RELEASE SiRNA OF A RAPID PERIOD OVER 24 HOURS AND THAL CASE WE LOAD OUR RIB SO STUDIES OF MULTIPLE ENDOCRINE CAN DOXORUBICIN SO WE USED A NEGATIVELY CHARGED LIP I STUDIES OF MULTIPLE ENDOCRINE AND SiRNA GOES ON THE OUTSIDE AND WE GET OUR STAGGER OUR SiRNA WAS SiRNA AGAINST PROTEIN ONE, MRP ONE WHICH IS A DRUG PUMP THAT PUMPS OUT DOXORUBICIN AS IT ARRIVES AT THE CELL MEMBRANE SURFACE THUS PRESENTING IT FROM INTERACTING OR ENGAGING THE NUCLEUS AND KILLING TUMOR CELLS IN THIS WORK BY JASON DENG, WE LOOKED AT SCRAMBLES HERE WE’RE LOOKING AT TUMOR SECTIONS THAT WERE STAINED AND HERE WE’RE LOOKING AT PC R WHICH SHOWS THAT THERE’S ROUGHLY 85-FLINT PRK KNOCK DOWN IN THESE TUMORS WHEN WE EXAMINE USING A XENOGRAFT MODEL, IF WE LOOK AT DOXORUBICIN WITH THE SCRAMBLED SiRNA WE SEE WE ARE REDUCING THE GROWTH OF THESE TUMORS BUT NOT THEIR SIZE THEY’RE DOUBLING IN SIZE HOWEVER, IF WE TREAT THESE WITH THE COMBINATION SYSTEM WE DO SEE THAT THE TUMOR SIZE IS GETTING SMALLER AND WE’RE GETTING REAL REGRESSION SO WE’RE VERY EXCITED ABOUT THESE RESULTS AND WE BEGAN TO MOVE TO A MORE CHALLENGING MODEL THE WE HAVE DEVELOPED A MODEL IN WHICH THESE MICE ARE ESSENTIALLY GENETICALLY MODIFIED SO THEY HAVE MUTATIONS IN KRASE MODEL AND IN P53 AND I WON’T HAVE TIME TO GO INTO THE DETAILS, BUT THESE ARE TWO OF THE HIGHLY KNOWN ONCOGENES IN ADDITION TO KRASE MODEL IS HIGHLY DESIRABLE BUT SMALL MOLECULE VS NOT BEEN DEVELOPED TO EFFECTIVELY INHIBIT THIS KRASE MODEL GENE IT’S ALSO KNOWN THAT P53 COME IS ONE OF THE GUARDIAN JEEPS THAT PREVENTS THE KINDS OF TUMOR GENIC ACTIVITY WE SEE IN CANCER CELLS IS ALSO LOST SO WE’RE GOING TO INTRODUCE A MICRORNA TO RESTORE THE P53 FUNCTION IN THESE CELLS AGAIN WE’RE GOING TO BUILD UP A FOUR LAYER NANO PARTICLE AS SHOWN HERE, BUT THIS TIME WITH SIS PLATIN AND THE TWO DIFFERENT RNAs, SiRNA AGAINST THE KRASE MODEL, ONCA GENE AND MICRORNA, MIR34 AND HERE WE’RE DESCRIBING THE MUTANT MODEL, I WON’T GO INTO DETAILS BUT IT ALLOWS FOR US TO ACTIVATE TUMORS MICE AND THESE ARE SMALL TUMORS SPREAD ALL THE WAY ACROSS THE LUNGS WE LOOKED AT THE ACT OF TARGETING OF OUR NANO PARTICLE IN HEALTHY MICE SO HERE WE DO AN INJECTION BUT THERE’S NO PARTICULAR LOCALIZATION VERSUS MICE WHICH EXHIBITED THESE LUNG CANCERS, MANY TUMORS, WE SAW THAT OVER A PERIOD OF TIME WE GOT A LARGE ACCUMULATION OF THE NANOGIE PARTICLES IN THE TUMORRED LUNGS AND JUST TO SEE THE RELATIVE DIFFERENCES HERE, YOU CAN SEE THE LUNGS OF THE HEALTHY MICE VERSUSS LUNGS THAT CONTAIN THESE TOMB OARS AND HERE CAN YOU SEE THE RELATIVE DIFFERENCE IN RECOVER FLUORESCENCE AND LOOK AT ORDERS OF MAGNITUDE DEFICIENCY IN THE AMOUNT OF ACCUMULATION OF THE NANO PARTICLES IN THE LUNG AND THIS IS DUE TO THE FACT THAT AGAIN CD44 IS HIGHLY EXPRESSED IN THE LUNG CANCER TYPE IN OUR IN VIVO TREATMENTS, WE USED AGAIN THIS ORTHOTOPIC MODEL SO IT’S HARD TO SEE THE TINY TUMORS ON THE SCREEN HOWEVER CAN LOOK AT THE BOLD CHANGE IN TUMOR VOLUME HERE AND SEE THE VEHICLE VERSUS RNA, SIS PLATIN AND COMBINATION YOU CAN SEE THERE’S A DIFFERENCE IN SURVIVABILITY AND IN FOLD CHANGE OF TUMOR VOLUME WE ALSO CAN CHECK AND SEE WHETHER OR NOT WE HAVE MODIFIED THE PATHWAYS WE THINK WE’RE MODIFYING AND WE SEE FROM THIS SERIES OF STAINING AGAINST PERK AND A RANGE OF PROTEINS THAT WE HAVE DOWN REGULATED THE PATHWAY AND REINSTITUTED THE P53 PATHWAYS AND WE HAVE ENABLED

CELL DETH THROUGH CASPASE THREE SO I’M COMING TO THE END OF MY TALK I WILL GIVE YOU A VERY SHORT EXAMPLE MENT THIS IS A MICRO SECOND OF EXTENSION OF OUR NANO PARTICLE PROJECT AND I’M BRINGING IT UP BECAUSE ONE OF THE EXCITING THINGS ABOUT WORKING AT M. I.T AND AT THE COKE INSTITUTE IS BEING ABLE TO CORK ACROSS DISCIPLINES A LOT OF THE WORK I DESCROIBED PREVIOUSLY HAS BENEFIT FRIDAY THE COLLABORATIONS OF MICHAEL YAFFE, A SYSTEM BIOLOGIST AND A RANGE OF CLINICAL RESEARCHERS AT DANA-FARBER AND MASS GENERAL HOSPITAL I ALSO HAVE ANOTHER COLLABORATOR, ANGIE BELCHERAND SHE ARRANGES INORGANIC SYSTEMS USING BIOLOGICAL METHODS SHE HAS BEEN LOOKING AT WAYS TO DEVELOP NANO PARTICLES THAT ABSORB NEAR IR AND IT WOULD ALLOW US TO GET DEEPER PENETRATION OF THE TUMORS AND THIS WORK MY POST DOC LEE AND ANGIE’S POST DOC MR. DANG HAS WORKED TOGETHER IN WHICH THE CORE CONTAINS ONE OF ANGIE’S DOWN CONVERSION NANO PARTICLES AND THE OUTER LAYERS CONTAIN AN LBL FILM WHICH COULD CONTAIN SiRNA OR CHEMO THERAPY AGENT SO WE ACTUALLY LOOKED AT A LAYER BY LAYER TO COMPARE WITH ANGIE’S DOWN CONVERSION NANO PARTICLE AND THIS IS JUST SHOWING THOSE CHARACTERISTICS AND IMAGES OF THOSE PARTICLES AND WHAT WE FOUND IS THAT BY USING ANGIE’S SYSTEM, AND COMPARISON WITH THE OTHERS, WE CAN COMPLETELY ESCAPE A RANGE OF AUTOFLUORESCENCE THAT WE EXPERIENCE AND SINGLE WALL NANO TUBES AND ACTUALLY SEE WHAT’S GOING ON INSIDE OF THESE ANIMALS WITHOUT THAT AUTOFLUORESCENCE USING THE NANO PARTICLE AND QUANTUM DOT IN FACT THE DOWN CONVERSION, NANO PARTICLE, WE CAN SEE FINE STRUCTURE OF BLOOD VESSELS, MUSK LO SKELETAL STRUCTURE AND THE BEATING OF THE HEART IS MUCH MORE VISIBLE SO WE’VE BEEN ABLE TO APPLY THIS TO THE IMAGING OF OVARIAN CANCER TUMORS BY USING THAT HYALLUREONIC AX SID BINDING CAPABILITY AND HERE WE’VE SHOWN IN ORTHOTOPIC OVARIAN CANCER MODEL WE GET A HUGE AMOUNT OF CONCENTRATION OF THESE NANO PARTICLES IN THE HEDGIONS WHERE WE HAVE THE TUMOR AND HERE YOU CAN SEE JUST IMAGES THAT ARE SECTIONED IN WHICH WE COMPARE THE NANO PARTICLE ACCUMULATED IN TUMOR VERSUS HEALTHY TISSUE AND YOU CAN GET DOWN TO SUBMILLIMETER SCALE RESOLUTION WITH THESE SYSTEMS AND ACTUALLY SEE AND DIFFERENTIATE THE TISSUE BETWEEN TUMOR AND PANCREAS, GUT, LIVER, AND OF COURSE WE GET A NICE VIEW OF SOLID TUMOR TISSUES SO RIGHT NOW WE’RE DEVELOPING THIS TECHNOLOGY TO SEE IF WE CAN GENERATE THESE SYSTEMS IN WHICH YOU MONITOR TUMOR SUPPRESSION AT THE SAME TIME WE’RE DELIVERING THERAPY SO IN CONCLUSION, I TALKED ABOUT THE FACT THAT WE CAN USE THIS VERY MODULAR WATER BASED APPROACH TO INCORPORATE A RANGE OF DIFFERENT MOLECULES AND THAT THEY CAN BE USED FOR VERY SIGNIFICANT WOUND HEALING APPLICATIONS INCLUDING THE CRANIAL DEFECT EXAMPLE I DIDN’T TALK ABOUT BUT WE’VE ALSO HAD COLLABORATIONS WITH MEMBERS OF THE COKE INSTITUTE INCLUDING DERILLEGALS IRVIN ON INCORPORATIONS ON LAYER BY LAYER FORMS OF VACCINES AND I TALKED ABOUT HOW WE CAN USE THESE KINDS OF LAYER BY LAYER FILMS, NANO FILMS ON THE NANO SCALE AND GENERATE VERY EFFECTIVE TARGETING NANOG’S PARTICLES I THINK THIS IS A VERY MODULAR APPROACH AND THIS IS WHY WE’RE EXCITED ABOUT IT IT CAN BE APPLIED TO A BROAD RANGE OF SURFACES AND CAN ALSO BE ADAPTED FOR USE WITH A RANGE OF TOOLS FOR HEALTH APPLICATIONS SO WHAT WE’RE SHOW SUGGEST THAT ELECTROSTATIC IS A CRITICAL TOOL THAT CAN BE USED FOR CONTROL RELEASE AND THAT THAT APPROACH CAN INTRODUCE FUNCTIONALITY THAT CAN LEAD TO PROMISING BIOMEDICAL APPLICATIONS I’D LIKE TO THANK THE CONTRIBUTORS TO THE WORK I HIGHLIGHTED MY STUDENTS AND POST DOCS IN IF BOLD, AS WELL AS COLLABORATORS AND A LOT OF OUR BONE WORK MICHAEL JAFFEE, BIOLOGIST AT THE KOCH INSTITUTE AND WORKING WITH MIKE BIRRER, AND I WOULD LIKE TO ACKNOWLEDGE THE NIH F FUNDING AN NUMBER OF EARLY DISCOVERIES AND

FOR CONTINUING TO SUPPORT OUR WORK AND THE OVARIAN CANCER RESEARCH PROGRAM AND RESEARCH FOUNDATION AND WELCOME ANY QUESTIONS THAT YOU MAY HAVE THANK YOU [ APPLAUSE ] >> THANK YOU VERY MUCH FOR A FASCINATING TALK IF YOU HAVE QUESTIONS COME TO THE MICROPHONE SO THOSE WATCHING CAN HERE AND PLEASE START HERE >> THANK YOU FOR A WONDERFUL PRESENTATION, WHAT I WAS INTERESTED IN UNDERSTANDING IS HOW CONSISTENT IS THE LOADING OF YOUR EXPERIMENTAL YOUR ACTUAL THERAPEUTIC AGENT TO THE FILMS OF THE NANO PARTICLES AND WHAT LEVEL OF VARIATION DO YOU TOLERATE? AS WE SEE THIS MOVE FORWARD TO THE CLINICAL, WHAT DO YOU THINK THE FDA WILL SAY ABOUT THIS? >> EXCELLENT THEY’RE EXTREMELY CONSISTENT ONE OF THE NICE THINGS ABOUT THIS POLYELECTROLYTE APPROACH IS THAT CHARGE IS VERY PREDICTABLE SOD IF YOU ASSEMBLE THESE SYSTEMS UNDER THE SAME CONDITIONS, YOU END UP WITH THE SAME AMOUNT ABSORBED IF WE LOOK AT OUR–YOU SAW SEVERAL CURVES IN WHICH WE’RE BUILDING CURVES AND YOU SEE THESE CURVES, THE LINEARITY IS BEAUTIFUL AND IT’S ALWAYS THERE SO WE CAN GO BACK TO THE SAME CONDITIONS AND GENERATE THE SAME FILMS THIS HAS BEEN SHOWN BY THE FACT THAT FOR EXAMPLE, IN THE BONE WORK, WE PASSED ON THIS FORMULATION FROM ACROSS THREE STUDENTS AND A POST DOC, DIFFERENT HANDS, SAME LOADING AND I THINK THIS WILL HELP US WITH THE FDA, THAT WE CAN ACTUALLY USE IT AS A VERY REPRODUCIBLE EFFECT YOU HAVE TO DO SOMETHING QUITE INTENTIONAL TO DISRUPT THE SORT OF ELECTROSTATIC ABSORPTION BEHAVIOR OF THESE SYSTEM SYSTEM >> THAT’S CALLED A BIOLOGICAL REPLICATE WHEN IT’S MORE THAN ONE POST DOC DOING THE SAME EXPERIMENT WHAT HAPPENS TO THESE NANO PARTICLES YOU PUT INTO THE BODY, WHERE DO THEY GO? HOW DO THEY EVER DISAPPEAR? >> VERY GOOD QUESTION THEY HAVE A VERY LONG HALF LIFE THEY WILL ACCUMULATE IN THE LIVER AND KIDNEY AND THIS IS WHY WE’VE BEEN TRYING TO MEASURE SIGNS OF DISTRESS, MEASURE BODY WEIGHT AND WE ALSO MEASURE CYTOKINES TO SEE IF THERE’S ANY INFLAMMATORY RESPONSE OR ANY OTHER KIND OF RESPONSE WE’VE ALSO TAKEN OUT THE LIVER AND RESECTED IT TO SEE IF THERE’S ANY LONG-TERM ACCUMULATION OF THE LIVER WE FIND THAT THEY PASS OUT OF THE LIVER SO IF YOU WAIT ROUGHLY 24 HOURS, TO 36 HOURS LATER, YOU YOU ARE NOT SEEING THESE NANO PARTICLES IN THE LIVER ANYMORE SO OVER A PERIOD OF TIME THEY’RE ACTUALLY SOMEHOW BREAKING DOWN AND GETTING OUT >> THEY ARE >> THEY’RE GETTING–THAT IS A GOOD POINT EACH OF THESE FOR THE NANO PARTICLE APPLICATION CONTAINS DEGRADABLE COMPONENT SO WE’RE USING A PHOSPHOLIPID, LIPID STUDIES OF MULTIPLE ENDOCRINE AND THEN LAYERING IT WITH THE HYALLUREONIC ACID CONTAINING SYSTEMS SO WE TRY TO MAINTAIN A SYSTEM THAT’S GOING TO BE ABSORBED OR BROKEN DOWN? >> OVERHERE? >> CONGRATULATION FOR PUSHING THE DIFFICULT FRONTIER OF NANO MEDICINE SO IN ONE OF THE STUDIES IT SHOWED [INDISCERNIBLE] SO AS YOU’RE PUTTING THE LAYER YOU HAVE TO BE CAREFUL WHICH MIGHT BE THE ACCEPTABLE VERSES THE OTHER [INDISCERNIBLE] SO IN TERMS OF MULTILAYERS PARTICLES, I WAS LOOKING AT IT YOU HAVE THE LAYER OF HYALLURE OWNIC ACID IS THERE ANY [INDISCERNIBLE] TO PREVENT PHAGOCYTIC REGULATION OF THE PARTICLES >> WE ACTUALLY CONSIDERED [INDISCERNIBLE] VERSES THE HYALLUREONIC LAYER AND THERE ARE A FEW PAPERS IN WHICH WE CAP OFF THE FINAL LAYER WITH A PEG BRUSH WE ACTUALLY FIND THAT THE HALF LIVES ARE VERY SIMILAR OR BETTER FOR THE HYALLUREONIC SYSTEM AND THEIR REASON FOR THIS AND IN FACT THERE ARE OTHER RESEARCH LABS WHO HAVE USED HYALLUREONIC ACID AS WELL, THEY’RE USING IT MORE FREQUENTLY AND SEAN CHIN IS ONE LAB THAT LOOKED AT THIS TO ONE EXTENT BUT THERE ARE SEVERAL REASONS, ONE IS THE ACID ITSELF HAS A GREAT RELATIONSHIP WITH WATER AND THAT REASON THAT WATER IS HELPING US WITH THESTERRIC PREPOLINGS BUT PROVIDES A BIT OF A CLOAKING LAYER BEHAVIOR, IT PREVENTS OPERATING GLOBALLY MYSELFATION SO PROTEINS TEND TO ABSORB AS READILYOT SURFACES BUT THE PRESENCE OF THE LAYER BY LAYER FILM HAS INFLUENCE SO WHEN THIS TAKES PLACE, THE POLYELECTROLYTE IS ON A POSITIVELY LINED SURFACE, IT’S NOT ANOTHER FLAT SURFACE BUT IT’S A POLYMER CHAIN AND WE GET THESE LOOP LOOPY STRUCTURES THAT ABSORB SO THE POLYMER CHAIN IS DIFFERENT WE THINK WE HAVE THIS BRUSH LIKE STRUCTURE FORMED FROM THESE LOOPS, WHICH IS ACTUALLY PROVIDING AS MUCH DENSER CLOUD

OF WATER AND ASSOCIATED IONS THEN YOU WOULD GET IF YOU HAD AN EXTENDED LYNNIAN CHAIN EXTENDING FROM THE SURFACE OF THE POLYMER, OF THE NANO PARTICLE >> OVER HERE? >> HI, VERY INTERESTING TALK, POLYSACCHARIDES YOU KNOW IS VERY COMMON COMPONENT, THERE ARE 23 DIFFERENT [INDISCERNIBLE] THERE ARE PNEUMOCOCCAL FOUR AND THEY ARE MOSTLY NEGATIVELY CHARGED THAT WE KNOW, HAVE YOU THOUGHT OF USING ANY OF THEM, THE VACCINE AS YOUR CODING YOUR LAYER? >> AH, VERY INTERESTING >> SO LIKE DELIVERING THE VACCINE AS WELL AS OTHER PURPOSE TOO >> EXCELLENT IN FACT I HAD A VERY INTERESTING CONVERSATION TODAY WITH JOE BARSTON, HE WAS WORKING ON GLYCOLYTIC MATERIAL, AND HE CAN CREATE GLYCO PEPTIDES THAT ARE HIGHLY IMMUNO GENIC, THIS IS INTERESTING TO US BECAUSE WE COULD COMBINE THOSE INTERACTIONS WITH OUR LAYER BY LAYER SYSTEM, INITIATE AN IMMUNE RESPONSE AND RELEASE AN ANTIGEN AND HAVE CONTROL OF THE RATE OF THAT PRESENTATION OF ANTIGEN THAT’S INTERESTING TO US, WE HAVE A COLLABORATOR DERILLEGALS URBAN WHO IS USING LAYER BY LAYER APPROACH FOR CANCER THE AND WE HAVE OUR FIRST STUDENT WHO WILL WORK ON THIS SO THESE KINDS OF IDEAS ARE VERY HELPFUL AT THIS STAGE I DO THINK IT’S INTERESTING, WHAT WE LEARNED FROM THE MICRONEEDLE WORK, I DIDN’T TALK AS MUCH ABOUT IS THAT WE CAN COAT A NUCLEOTIDES CLAYIC ACID ON THESE OR COAT A PROTEIN ON TO THESE LAYER BY LAYER FILMS AND GET A SLOW RELEASE AND FOR VARIOUS REASONS GETTING THE SLOWER RELEASE OF THE ANTIGEN GIVES US A MORE SUSTAINED MEMORY T-CELL EFFECT AND THAT MAY BE SOMETHING THAT WE CAN USE FOR IMMUNOTHERAPY AGAINST CANCER WE CAN GET THE SUSTAINED RELEASE >> THE [INDISCERNIBLE] CAN COME IN TROVALL FORMS, ONE IS [INDISCERNIBLE]: AND THE OTHER IS CHEMICAL CONJUGATE FOR [INDISCERNIBLE]: I HAVE ANOTHER QUESTION THAT IS ONE OF THE MOST COMMON NEGATIVELY CHARGED [INDISCERNIBLE] HAVE YOU EVER THOUGHT OF USING [INDISCERNIBLE]? >> COULD YOU SAY IT ONE MORE TIME >> POLYNUCLEIC ACIDS? >> YES >> YEAH REGULARLY NEGATIVELY CHARGED? >> WE HAVE, SO CO INSURE MARKET DENTALLY, WE HAVE A PROJECT IN WHICH WE’RE GENERATING VERY LARGE HAIR PIN SHAPED RNA ESSENTIALLY, IT’S A KIND OF MULTIPERIODIC RNA STRUCTURE AND THAT WORK WAS REALLY DIRECTED TOWARD A DIFFERENT PART OR APPROACH TO SiRNA THERAPY BECAUSE THEY BREAK DOWN IN THE PRESENCE OF DICER AND HAIR PINS THAT CAN BE USED FOR DELIVERY BUT WE’RE DISCOVERING THAT THOSE STRECTURES ARE ACTIVATING THE NCAPPA BETA THEMSELVES AND WE’RE GETTING AN INFLAMMATORY RESPONSE THAT’S SPECIFIC OR AT LEAST HIGHLY AMPLIFIED IN CANCER CELLS SO WHAT YOU’RE SAYING IS I THINK WE’RE BEGINNING TO OBSERVE THESE MOLECULES >> VERY CLEVER >> SO RECOGNIZING THAT WE ARE A BIT AFTER THE HOUR AND I KNOW PEOPLE HAVE THINGS THEY NEED TO DO MAYBE OTHER WHO IS HAVE QUESTIONS CAN POSE THEM TO OUR SPEAKER AS WE ALL MIGRATE TOWARDINGS THE LIBRARY FOR A RECEPTION WITH SOME COFFEE AND COOKERS BUT THREES JOIN ME IN THANKING DR >> [ APPLAUSE ] HAMMOND AGAIN

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