>>WELCOME EVERYONE TO OUR FINAL NIH BME SIG SEMINAR. THANK YOU EVERYONE, BOTH IN THE AUDIENCE AND OUR SPEAKERS THROUGHOUT THE YEAR FOR MAKE THANKSGIVING A HUGE SUCCESS. AND SO FOR OUR CAP STONE SEMINAR, I AM HONORED TO INTRODUCE DR. LOLA ENIOLA-ADEFESO, WHO IS THE UNIVERSITY DIVERSITY AND SOCIAL TRANSFORMATION PROFESSOR OF CHEMICAL ENGINEERING, BIOMEDICAL ENGINEERING AND MACROMOLECULAR SCIENCE AND ENGINEERING AT THE UNIVERSITY OF MICHIGAN AND IS ALSO THE CONFOUNDER AND CHIEF SCIENTIFIC OFFICER OF ASALEXA BIO. SHE RECEIVEDDARY UNDERGRADUATE DEGREE AT UMBC AND Ph.D. AT UPENN. AND SHE HAS A LAB AT MICHIGAN WHERE SHE STUDIES ACTIONS BETWEEN BLOOD LEUCOCYTES AND ENDOTHELIAL CELLS AND LINING AND HOW THEY CHANGE DURING THE INFLAMMATION RESPONSE. SHE IS A FELLOW OF THE AMERICAN INSTITUTE FOR MEDICAL AND BIOLOGICAL ENGINEERS AND BIOMEDICAL ENGINEERING ASSOCIATE. SHE IS ALSO VERY PRODUCTIVE AT CHAMPIONING WOMEN IN UNDERREPRESENTED MINORITIES STUDENTS AT THE UNIVERSITY OF MICHIGAN IN AN EARLY EDUCATION PROGRAM SUCH AS THE NEXT PROP PROGRAM. SO WE'RE VERY HAPPY THAT LOLA COULD JOIN US. >>THANK YOU SO MUCH FOR THAT INTRODUCTION. I'M EXCITED TO BE TELLING YOU ALL OR AT LEAST IN OPPORTUNITY TO TELL YOU ALL ABOUT THE RESEARCH THAT'S BEEN GOING ON IN MY LAB AT MICHIGAN. WE'RE PRIMARILY LOCATED IN CHEMICAL ENGINEERING AND WE HAVE INTEREST IN UNDERSTANDING HOW WE MIGHT DESIGN VASCULAR TARGETED DELIVERY SYSTEMS. ESSENTIALLY THE IDEA OF THIS IS TAKE POLYMERIC PARTICLES AND PACKAGED DRUGS WITHIN THEM AND THIS IS THEADAD LAYER HERE IS THAT WE LOOK TO DECORATE THE SURFACE OF THIS DRUG CARRIERS WITH PEPTIDES, LIGAND ANTIBODIES THAT MIGHT HELP US RECOGNIZE DISEASE MARKERS OR EPITOPES THAT ARE EXPRESSED ON THE VASCULAR WALL. A COUPLE THINGS TO POINT OUT. ESPENTIALLY WHAT WE'RE DOING IS MIMICKING WHITE CELL COUNTS, MONOCYTES USE THIS APPROACH ALL THE TIME IN MAKING A DECISION OF HOW THEY'RE GOING TO A TISSUE SPACE WHEN THEY GET A SIGNAL EITHER WE HAVE AN INFECTION, INJURY OR ANYTHING ELSE IN BETWEEN. AND SO, WITH THAT UNDERSTANDING, THE QUESTION WE ASK IN THE WORLD OF VASCULAR TARGETED DELIVERY SYSTEM IS A, CAN WE RECOGNIZE DISEASE EXPRESSED PROTEIN PATTERNS THAT ARE ON THE BLOOSES VESSEL WALL WITHIN THE SYSTEM AND ARE WE ABLE TO BUILD ON THOSE COUNTER PATTERNS ON A DRUG CARRIER WHICH ALLOWS US THEN TO DESIGN THE SYSTEM TO BE INJECTABLE, RIGHT? AND OF COURSE WE'RE INTERESTED IN INJECTABLE SYSTEM BECAUSE THE BLOOD VESSEL IS 1 OF THOSE ORGANS THAT HAVE IMPOSSIBLE TO BUILD MATE INTERACTIONS THROUGHOUT THE ENTIRE BODY. SO REALLY ANYWHERE THAT YOU WANT TO GET INSIDE A HUMAN, CAN YOU ACCESS THAT LOCATION USING THE BLOOSES VESSELS AS YOUR HIGHWAY. THE PROBLEM OF COURSE IS ONCE WE INJECT THESE TYPES OF SYSTEM SYSTEM INSIDE THE CIRCULATORY SYSTEM, WE LOSE THE ABILITY TO CONTROL THEM, SO THIS, IS WHERE THAT TARGETING COMES IN TO PLAY. SO THE SYSTEM, THIS IDEA HAS BEEN PROPOSED AND EVALUATED FOR A WIDE VARIETY OF HUMAN AILMENT, A LOT OF THE WORK YOU WILL SEE WILL BE IN CANCER, THERE'S A FEW MORE WORK THAT'S COMING ONLINE NOW IN TERMS OF CARDIOVASCULAR DISEASE SYSTEM AND THAT'S BEEN THE SPACE THAT MY LAB HAS BEEN INVOLVED IN. WE'VE ASKED A QUESTION FROM THE BEGINNING, FROM WHEN WE OPEN OUR DOORS IN TERMS OF HOW DO WE DESIGN VASCULAR TARGETED CARRIER SYSTEM THAT MIGHT HAVE APPLICATION IN CARDIOVASCULAR DISEASE. WE WEREN'T INTERESTED IN ATHEROSCLEROSIS AS A DISEASE MODEL. AND WE ASK THE CLIT RATTURE TO SEE WHAT DO WE KNOW THE INFORMATION MG GAP IN THE SPACE OF DESIGNING TARGETED DELIVERY VEHICLES. WE KNOW A LOT ABOUT THE KINDS OF POLYMERIC MATERIALS THAT WE CAN USE, WE TEND TO GRAVITATE TOWARDS THE SYNTHETIC DEGRADABLE POLYMERS BECAUSE WE HAVE A LOT OF CONTROL IN HOW WE SYNTHESIZE THEM AND HOW THEY BREAK DOWN INTO THE TYPES OF DRUGS THAT WILL BE PACKAGED IN THERE BUT THERE ARE OF COURSE MANY OPTIONS IN TERMS OF HYDROJELL BASED SYSTEM, LIPID BASED SYSTEM, LIP O STUDIES OF MULTIPLE ENDOCRINE MISELLS AND WITH THE LAST 2 YEARS WE'VE SEEN THE WIDE EXPRESSION OF THE LIPID NANO PARTICLES AS ANOTHER WAY OF DELIVERING THERAPEUTICS INTO THE HUMAN BODY. WE SPENT A LOT OF TIME ON THE ENGINEERING SIDE LOOKING AT THE SURFACE OF THIS PARTICLES ESPECIALLY WHEN WE THINK ABOUT PUTTING THEM IN SYSTEMIC CIRCULATION IN THE BLOOD STREAM. ONE OF THE THINGS WE KNOW ABOUT BLOOD IS IT HAS BIOMOLECULES IN PLAZ MIDS NA, PLACENTA MAZARA PROTEINS THAT TEND TO IMMEDIATELY COVER THE SURFACE OF OUR DRUG CARRIERS AND WE NEED TO INSURE IN SOME CASES THAT WE MITIGATE THAT BECAUSE THAT OFTEN IS THE WAY THAT THE BODY GETS THE SIGNAL THAT HAVE YOU A FOREIGNENTITY WITHIN THE BLOOD AND THAT COULD TRIGGER THE PROCESS OF CLEARING THOSE PARTICLES IMMEDIATELY FROM CIRCULATE. THE PROBLEM THERE OF COURSE IS, IF YOU PUT SOMETHING IN THAT'S MEANT TO GO AND DELIVER A PACKAGE SOMEWHERE, IF YOU CLEAR IT FROM THE BLOOD SPHREEM BEFORE IT GETS THERE, THEN YOU HAVE A SCENARIO WHERE YOU'RE NOT ABLE TO GET THE THERAPY TO THE INTENDED PLACE. YOU MIGHT THINK OF IT AS THAT UPS GUY TRYING TO DELIVER AND A BUNCH OF DOGS IN THE NEIGHBORHOOD PREVENTED THEM FROM GETTING CLOSE TO THAT MAIL BOX TO DELIVER THE PACKAGE. SO WE SPENT A LOLT OF TIME ON THE SURFACE CODING THAT SPACE. BUT 1 OF THE THINGS WE NOTICED IN OUR DISCUSSION OR IN OUR LITERAL SEARCH AT THE BEGINNING WAS THAT THERE WAS NOT MUCH INFORMATION THAT WE COULD FIND ABOUT TRANSPORTS. WHAT HAPPENS TO THIS DRUG CARRIERS ONCE WE INJECT THEM INTO THE BLOOD STREAM. AND AT FIRST WE WERE SURPRISED BUT THEN WE THINK ABOUT IT SOME MORE, IT MAY BE IT'S NOT SURPRISING BECAUSE AS YOU SEE HERE, THIS IS OFTEN HOW WE DEPICT BLOOD VESSEL WHEN IS WE LOOK AT OUR TEXTBOOKS WHRKS WE LOOK AT OUR PUBLICATIONS, HOLLOW TUBE, SIMPLE, FEW THINGS FLOATING AROUND AND REALLY THAT CAN BIAS THE KINDS OF WAYS WE THINK ABOUT DESIGNING THINGS THAT GO INTO THE BLOOD STREAM AND HONESTLY WE WEREN'T THINKING ABOUT TRANSLATING BLOOD UNTIL WE ENCOUNTERED THIS IMAGE WHICH IS OF COURSE AN SEM OF A BLOOD VESES THEY'LL IS SPLIT OPEN AND YOU SEE IMMEDIATELY THAT BLOOD EXISTS IN BLOOD VESSEL AND THAT IT IS NOT THE SIMPLE FLUID WE MIGHT ENVISION IT TO BE BUT INSTEAD IT'S A COMPLEX ENVIRONMENT THAT WE NEED TO BETTER UNDERSTAND TO BE ABLE TO INFORM OUR DESIGN. SO THAT REALLY WAS THE FIRST THING THAT LED US TO THIS IDEA OF UNDERSTANDING WHAT BLOOD IS AND WHAT RESIDES WITHIN BLOOD AND HOW CAN WE USE THAT UNDERSTANDING TO BETTER TUNE THE DESIGN OF OUR TARGETED DELIVERY SYSTEM. SO HOW DO WE UNDERSTAND THIS, BLOOD IS A TISSUE, AN ORGAN 1 MIGHT EVEN SAY. IT'S A COLLECTION OF CELLS THAT INTERACT WITH EACH OTHER TO CARRY OUT VARIOUS FUNCTION. THE ONLY DIFFERENCE IS THAT THIS TISSUE OR ORGAN IS DYNAMIC AND FLUID IN ITS NATURAL STATE. OKAY, RED CELLS ARE THE MOST PREVALENT, HIGHLY DENORMABLE, THEY ARE DISSHAPED, OUTNUMBER WHITE CELLS 1 TO 700, I BELIEVE AND WHAT THAT MEAN SYSTEM FOR EVERY 1 WHITE CELL, THERE IS 700 RED CELLS AND OF COURSE WE HAVE THE PLATELETS, THEY'RE NOT AS SCARS AS THE WHITE CELLINGS BUT THEY'RE ALSO STILL OUTNUMBERED, I BELIEVE 1-17 WITH THE RED BLOOD CELLS. --I HIGHLIGHT THIS INFORMATION BECAUSE OF COURSE IN THE GRAND SCHEME OF THINGS, THE RED CELL'S JOB IS TO DELIVER GAS EXCHANGE FUNCTIONS. THE WHITE CELL AND PLATELET HOWEVER, THEIR JOB IS REQUIRES THEM TO BE CLOSE TO THE BLOOD VESSEL WALL. THE IMMUNE CELLS, WHITE BLOOD CELLS NEED TO FIND THOSE PROTEINS, BY O MARKERS THAT ARE EXPRESSED THAT ARE SIGNALING DISEASE OR DAMAGE AND THE PLATELETS JOB IS TO QUICKLY LOCALIZE AND PLUG ANY HOLE THAT COMES THROUGH AND BRICKS THE BARRIER OF THE BLOOD VESSEL AGAIN TO PREVENT US FROM BLOODING TO DEATH. SO THESE 2 CELL TYPE VS TO BE AT THE VESES WILL WALL BUT WHEN YOU LOOK AT THAT IMAGE FROM THE PRIOR SLIDE, IT'S VERY CLEAR THAT IF WE ARE GOING TO DO THIS IN A 1 TO 1 RACE, LIKE YOU KNOW EVERYBODY LINES UP AND I'M TALKING ABOUT RACE BECAUSE WE HAD A--IT'S TRACK RESORD, YOU LINE EVERYBODY UP AND THEN YOU SAY GO, THE WHITE CELLS AND PLATELETS ARE NOT GOING TO WIN THAT RACE BECAUSE WE'RE GROSSLY OUTNUMBERED BUT NATURE INVOLVED BLOOZ IN A VERY, VERY INTELLIGENT WAY. IT TURNS OUT THE SIZE AND SHAPE ARE NOT ACCIDENTAL. BECAUSE THE BLOOD CELLS ARE HIGHLY DISK LIKE, HAD WHEN THEY ARE FLOWING AT HIGH SHARE, YOU SEE THEM MOVE TO THE CENTER OF THE BLOOD VESSEL SO YOU END UP WITH THIS PLUG IN YOUR BROOD VESES THEY'LL IS MOSTLY RED CELLS AND AT THE EXTREME NEAR THE VESSEL WALL IS WHERE YOU HAVE THE WHITE CELLS AND PLATELETS CONCENTRATED AND YOU SEE THAT IN THIS VIDEO THAT BLUR IN THE MIDDLE ARE THE RED CELLS AND YOU CAN NICELY VISUALIZE THE WHITE CELLS CLOSE TO THE WALL BECAUSE THAT AREA IS DEVOID OF RED CELLS. WE CALL IT THE RED CELL FREE OR CELL FREE LAYER. SO THAT, WOS OUT GREAT BECAUSE THE WHITE CELLS ARE NOT AS DEFORMABLE AND THEY'RE NOT EXPERIENCING THAT LIFT TO THE CORE. WHY IS THIS IMPORTANT FOR VASCULAR TARGETED SYSTEM? WELL, WE ARE DESIGNING THIS PARTICLES TO BE ABLE TO ALSO PROBE AND FIND THOSE BLOOD VESSEL WALL PROTEINS SO THEY MUST BE WAIBL TO LOCALIZE TO THAT WALL AREA FOR FUNCTIONALITY. SO WITH THAT IMAGE OF BLOOD THAT I SHOWED EARLY ON, AND THIS UNDERSTANDING OF DISTRIBUTION OF BLOOD CELLS WITHIN BLOOD, THE IMMEDIATE QUESTION THAT POPPED INTO OUR HEAD AT THE TIME WAS HOW PARTICLES OF DIFFERENT SIZES THAT ARE POLYMERIC BASED THAT WE WANT TO USE FOR DRUG DELIVERY, HOW DID IT GET TO THE BLOOD VESSEL WALL IN THIS SCENARIO. SO WE HAVE 1 OF 2 CHOICES AT THE TIME OF IN TERMS OF ANSWERING THE QUESTION. WE CAN GO IN A MOUSE MODEL, ANIMAL MODEL AND TREAT THEM WITH OUR TARGETED PARTICLE SYSTEM AND SEE WHERE THEY LAND. IT'S NOT AN EFFECTIVE WAY TO DO RESEARCH BECAUSE AGAIN, WE ARE NOT NECESSARILY LOOKING FOR ANYTHING SPECIFIC BUT TO UNDERSTAND DISTRIBUTION. FOR WHAT WE'RE LOOKING FOR IT WAS A COMPLEX ASSAY TO BE GOING IN ANIMAL MODEL. OR WE CAN GO INTO FLUIDIC SYSTEM FOR US AGAIN AS ENGINEERS THIS WAS A SWEET SPOT AND WE CAN TELLING YOULY WORK WITH HUMAN TISSUE BECAUSE BLOOD IS 1 OF THOSE TISSUES THAT WE'RE ABLE TO GIVE UP WITHOUT DETRIMENT TO THAT HUMAN. SO THAT'S THE APREACH WE TOOK, COUPLE WAYS OR ASSAYS WORK, FLUIDIC CHANNELS, MOST THE TIME THEY'RE PARALLEL PLATES, STRAIGHT CHANNELS AND WE'VE DONE THE ASPECT RATIO SO THAT THE DISTRIBUTION IN THE UP AND DOWN DIRECTION IN TERMS OF CELLS AND PARTICLES IS IMPORTANT. WE CUT THE BASE LINE OF THE SURFACE WITH THE CHANNEL WITH THE ENDOTHELIAL CELLS AND THESE LINE THE LUMEN OF BLOOD VESSELS IN MOST ORGANS WERE USING HUMAN UMBILLICCAL CORD DERIVED EPITHELIAL CELLS IN THIS ASSAY AND THEN WE FLOAT HUMAN BLOOD THROUGH THAT SYSTEM. WE WERE GETTING DONATIONS FROM PATIENTS, FRESH, THESE ARE HEALTHY DONORS AND WE STARTED ASSAYS, WE JUST FOCUSING ON RED CELLS IN PLASMA BECAUSE IN THAT IMAGE, WE RECKON THAT IF THERE'S ANY CELL IN BLOOD THAT'S GOING TO PRIMARILY HAVE AN IMPACT ON HOW OUR PARTICLES DISTRIBUTIONED IT HAS TO BE THE RED CELLS AND WE PUT PLASMA IN THERE SO THAT WE HAVE A NICE REPRESENTATION OF WHAT THE BLOOD IS DOING WHEN IT'S FLOWING. AND SO THIS IS WHAT OUR ASSAY WOULD LOOK LIKE, THESE ARE THE RED CELLS GOING BY, WE SEE THE PARTICLES IN THE BACKGROUND. IF I INCREASE THE FLOW SPEED, YOU SEE THE THAT THE RED CELLS MOVED AWAY FROM THE SURFACE AND THAT IS REPRESENTING THE PACKING OF THE CENTER OF FLOW THEY WAS TELLING INVIVO. WE'RE ABLE TO REPLICATE THAT IN OUR FLUIDIC SYSTEM SO WE'RE COMFORTABLE THAT WE CAN LEARN SOME THINGS IN THIS MODEL IN TERMS OF HOW PARTICLES WILL NAVIGATE THE BLOOD STREM. SOY PARTICLES ARE MADE OF POLYSTYRENE, IT'S A--WE DO NOT HAVE DRUGS LOADED IN THIS BECAUSE AGAIN WE WERE ANSWERING THE QUESTION OF TRANSPORT AT THIS TIME AND WE CAN TARGET THE SURFACE OF THE PARTICLES WITH TARGETING LIGAND. WE WERE USING SIDE LIEWRIS A WHICH IS A CARBOHYDRATE THAT IS NATURALLY EXPRESSED BY WHITE BLOOD CELLS IN HUMAN BLOOD AND WE KNEW THAT THAT CARBOHYDRATE HAS E-SELECTINOT ENDOTHELIAL CELLS WHEN IT'S EXPERIENCING INFLAMMATION SO REALLY IT'S A GOOD MODEL FOR STASKULAR STAGGERLING. SO PARTICLES ARE IN, WE LOOK FOR DIFFERENT FLOW PATTERNS, WE LOOK FOR FIXED CONCENTRATION OF PARTICLES IN BLOOD, WHAT ARE THE CHANCES THOSE BARLS WILL LOCALIZE AND BIND IN HUMAN BLOOD FLOW? OKAY, DATA POINT. IT DIDN'T TAKE US LONG IN OUR ASSAYS TO SEE A PATTERN EMERGE. WHEN PARTICLES, SPHEREICAL PARTICLES WERE 2-MICRON AND LARGER WE SEE A SIGNIFICANT LEVEL OF BINDING, IN FLOW COMPARED TO WHEN THEY WERE SMALLER IN THIS CASE, 200 NANO METERS SIZE WHICH IS THE PERFECT SIZE FOR DRUG DELIVERY, THEY ARE SMALL ENOUGH THEY CAN CIRCULATE THE SYSTEM WITHOUT US BEING CONCERNED ABOUT IMPEDING BLOOD FLOW AND WHEN BOUND TO THE VASCULAR WALL, WE CAN THINK ABOUT THEM GOING THROUGH THE BLOOD VESSEL WALL TO BE ABLE TO GET CLOSER TO THE TISSUE SPACE WHERE IN MANY CASES IS WHERE WE WANT TO DELIVER THE THERAPEUTIC. SO THIS IS A SMALL CHANNEL 25-MICRON HEIGHT. WE ARE SEEING ISSUES WHERE THE SIZE THAT WE REALLY DESIGN FOR VASCULAR TARGETING DOES NOT SEEM TO BE GETTING TO THE WALL AND FINALLY THIS, IS IMPORTANT BECAUSE ALL OF THESE PARTICLES WERE FED IN THE SAME BLOOD STREAM CONCENTRATION. AND OF COURSE, IF WE GO INTO A LARGER CHANNEL AND WE EVEN MIX UP THE BLOOD IN RECIRCULATING FLOW WHICH IS RELEVANT FOR CARDIOVASCULAR DISEASE BECAUSE WHEN YOU HAVE THE BRANCHING AREAS AND YOU HAVE BULGING, YOU HAVE THIS EDDIE THAT TENDS TO FORM AND THAT'S REEL KEEPSAKES VAPT FOR ATHEROSCLEROSIS BECAUSE YOU TEND TO SEE PLAQUE DEVELOP IN THOSE AREAS OF FLOW SEPARATION. WHEN WE LOOK AT PARTICLE DISTRIBUTION IN THAT KIND OF FLOW MODEL, WE SEE AN EVEN BIGGER GAP BETWEEN THE MICRON SIZE PARTICLES AND THE SMALLER NANO SIZE PARTICLES THAT WE'RE INTERESTED IN, OKAY, AND PUT IN MORE NANO PARTICLES IN, DID NOT HELP. IN FACT, IT SEEMS THAT THE MORE NANO PARTICLES WE PUT IN, WE JUST WENT TO FILL IN THE CORE OF THE RED CELL FLOW AND WE KNOW THIS BECAUSE WE'VE DONE ASSAYS WHERE WE PUT OUR CHANNELS ON A CONFOCAL MICROSCOPE AND WE SCAN FROM BOTTOM OF THE CHANNEL WHERE THE TARGETING IS HAPPENING, ALL THE WAY TO THE OTHER END OF THE CHANNEL AND ONLY WHEN WE GET INTO THE RED CELL CLOUD DO WE SEE THE NANO SIZE PARTICLES LIGHT UP WHEREAS THE MICROPARTICLES WE FIND THEM VERY NICELY AT THE WALL. OKAY, SO HERE IS THE SUMMARY HERE WHEN--WHEN PARTICLES ARE SMALLER THAN 1 MICRON, WE'RE SEEN THAT THEY'RE NOT ABLE TO COME TO THE VASCULAR WALL AS MUCH AS WE NEED THEM TO. NOW, I DO ACKNOWLEDGE THAT THE CHANNEL HEIGHT HERE, 25-MICRON IS MAYBE THE SIZE OF ARTERIOLES AND VENOUSLIULES AND FOR PEOPLE WHO ARE INTERESTED IN TUMOR DISTRIBUTION, MAYBE IT'S NOT FULLY REPRESENTING THE ENVIRONMENT THERE BECAUSE THERE WE TEND TO THINK ABOUT THE CAPILLARYS THAT ARE GOING TO BE AROUND 7-9-MICRON IN SIZE. BUT IT IS USEFUL TO HIGHLIGHT THAT THE VASCULATURE IS A CONNECTION OF NETWORKS OF AND ROADWAYS AND HIGHWAYS AND YOU MIGHT THINK OF IT IN THAT SPACE THAT I CAN'T GET TO MY RURAL NEIGHBORHOOD WITHOUT HAVING TO GO THROUGH MAJOR HIGHWAYS, RIGHT? THOSE ARE WHAT FEEDS DOWN EVENTUALLY INTO THE TUMOR SPACE AND YOU SEE HERE IN THIS IMAGE AS THE VESSELS BRANCH, THEY'RE DOING SO IN AN ASSIMETRICAL WAY, NUMBER 2 THAT WE TEND TO SIPHON BLOOD OUT OF THE EDGE OF THE PARENT VESSEL BECAUSE THE NATURAL TENDENCY, THE BODY WANTS TO DEDENSIFY THE RED CELL POPULATION AS WE GO INTO SMALLER AND SMALLER CHANNEL BECAUSE AGAIN IN THE CAP ILLEGALSARY WE WANT TO MAKE SURE BLOOD IS STILL FLOWING. SO THE POINTING THAT IF THE NANO PARTICLES ARE NOT GETTING TO THE WALL, IT'S QUITE POSSIBLE THAT PART OF THE ISSUES WE SEE WITH LOCALIZATION INTO TUMOR SPACE AND LINKED TO THIS INABILITY TO GET DOWN TO THE WALL. OKAY? SO FOR US IN THE AREA OF DRUG DELIVERY, ESPECIALLY FOR INJECTABLE SYSTEM, THIS IS A BIG DEAL BECAUSE THERE ARE NUMEROUS BENEFITS FOR US TO BE ABLE TO USE NANO SIZE CARRIER IN THIS WAY. AND 1 OF THE QUESTIONS WE WERE GETTING EARLY ON IS WHETHER THE FACT THAT WE WERE DOING THIS EXPERIMENT INVITIO WAS LOCALIZED TO THE MICRO PARTICLES BECAUSE OF COURSE IN THE LAB WE HAVE THE EFFECT OF GRAVITY IN A WAY THAT YOU DON'T SEE WHEN YOU'RE IN A BLOOD VESSEL INSIDE A LIVING ORGANISM. BUT RATHER THAN DWELL ON THAT QUESTION OF BIAS, WE ASK A QUESTION THAT MIGHT HELP US, WHETHER DENSITY OR THE WEIGHT OF THE PARTICLE HAD ANYTHING TO DO WITH HOW THEY WERE BEING DISTRIBUTED TO THE WALL IN A FLOW SYSTEM. IMPORTANTLY WE FOUND OUT THAT THAT TIME THAT POLYSTYRENE WAS NEUTRALLY BUOYANT IN BLOOD. IT MEANS IF YOU SIT BLOOD DOWN AND SPRINKLE POLYSPHERES YOU THERE IS NO SETTLING YOU WILL SEE BECAUSE IT SITS NICELY WHEREVER IT'S MIXED IN. SO THE REQUESTY IS IF WE FOUND HEAVIER PARTICLES ARE WE ABLE TO BENEFIT THEIR DISTRIBUTION TO THE WALL AS FLOOD IS FLOWING AND AS LUCK WOULD HAVE IT, WE WERE ABLE TO FIND SILICA PARTICLES IN THE NANO SIZE RANGE, 500 NANO MEETS AND SILICA HAS TWICE THE DENSITY OF BLOOD AND TITANIA HAS ABOUT 4 TIMES THE DENSITY OF BLOOD, SO THE QUESTION IS, IF WE TREAT THEM THE DAME WAY WE DID WITH POLYSTYRENE, TARGET THE SURFACE DO WE SEE IMPROVEMENT OF BINDING OF SMALL PARTICLES IN THE BLOOD FLOW? AND THE GOOD NEWS IS YES, WE SEE HERE THAT SILICA HAD TWICE THE DENSITY OF BLOOD HAD MORE THAN TWICE THE RESPONSE WHEN IT COMES TO LOCALIZING AND ADHERING TO THE BLOOD VESSEL WALL COMPARED TO POLYSTYRENE, SO THAT'S EXCITING. EVEN MORE SO IF I FLIP THE CHANNEL SO THAT I'M FORCING PARTICLES TO GO AGAINST GRAVITY IN THEIR BINDING, WE'RE SHOW THANKSGIVING DISTRIBUTION PATTERN REMAINS THE SAME. SILICA IS STILL BENEFITING FROM ITS DENSITY, SUGGESTING TO US THAT TRULY INTERACTION OF THOSE PARTICLES WITH RED CELLS AND THE COLLECTION THAT HAPPENS AND BE FILTERING THAT HAPPENS IS REALLY WHAT DETERMINES WHO'S GETTING TO THE WALL AND WHO'S NOT AND THERE SILICA WAS GAINING MOMENTUM BY WAY OF DENSITY AND GETTING MORE TO THE WALL. NOW YOU WOULD HAVE ALL NOTICED HERE THE TITANIA THAT HAD THE 4 TIMES THE DENSITY OF BLOOD DID NOT GIVE US THAT BENEFIT, IN THE DIRECTION OF GRAVITY AND ONCE WE TURN THE CHANNEL UPSIDE DOWN AND WAS FORCING IT TO GO AGAINST GRAVITY, YOU SEE ANY BENEFIT YOU WERE GETTING WAS ELIMINATED. SO HERE 1 OF 2 THINGS COULD BE HAPPENING. IT COULD THAT DISCIPLINARY TIANYLIA IS TOO HEAVY, ANY INTERACTION WITH THE CELL WAS NOT MOVING INTEREST ANY PRECIABLE DIRECTION OR IT COULD BE THE MATERIAL INTERACTION IS CAUSING SOME SORT OF INTERACTION WITH THE RED CELL AND SO THEN THEY'RE NOT ABLE TO COME DOWN TO THE WALL. BECAUSE INHERENTLY WHEN WE DID THIS ASSAY WE DID THE DENSITY AND MATERIAL CHARACTERISTIC THERE. SO TO GET MORE INFORMATION ABOUT THIS, WE DECIDED TO GO INTO A BIOCOMPACTIBLE MATERIAL, SAY LPGA AND WE WOULD FIND A WAY OF PLAN EPIGENETICCULATING THAT PARTICLE SO THAT WE CAN CHANGE THE DENSITY BACK AND FORTH. AS LUCK WILL HAVE IT PLGA AS A POLYMER HAS ABOUT 1.4 TIMES THE DENSITY OF BLOOD, IT'S SLIGHTLY MORE DENSE, RIGHT? NOW, WHY IS THIS SOMETHING THAT WOULD EXCITE US? WELL, WE SAW THAT SILICA HAVING JUST 2 TIMES THE DONEESITY OF BLOOD, WE WERE SEEING SIGNIFICANT SIGNALING IN HOW THOSE NANO PARTICLES DISTRIBUTED SO WE STARTED OUR ASSAY BY MAKING GAPA PARTICLES AND TARGETING THE SURFACE AND SEEING WHAT WE COULD READ IN TERMS OF FLOW. AND ALL WORKED OUT GREAT. YOU SEE HERE IN BUFFER FLOW WHEN WE COMPARE ADHESION OF POLYSTYRENE TO FLOW TO RECATALOGGIVE SURFACE IT WAS SIGNIFICANTLY HIGHER THAN WHAT WE WERE GETTING FOR POLYSTYRENE OF THE SAME SIZE. SO THEN WE WENT INTO HUMAN BLOOD ASSAY AND WE WERE SURPRISED WE DID NOT SEE ANY BINDING WHENEVER THE PARTICLES WE PUT IN BLOOD WAS PLGA. SO WE WERE SURPRISED AT THIS BECAUSE OF COURSE PLG IS THE MATERIAL WE HAD ALL BEEN USING AT LEAST DECADE OR 2 PRIOR TO THAT AND I REMEMBER BEING A GRADUATE STUDENT WORKING WITH BEST POLYMER AS A DRUG DELIVERY MATERIAL. SO BECAUSE OF ALL THE WORK WE'VE DONE IN THE PAST WITH POLYSTYRENE, RED CELLS AND HUMAN BLOOD, WE SUSPECTED THAT THIS ISSUE MUST BE PLASMA. AND THIS IS PLAUSIBLE BECAUSE OUR ASSAYS WERE ONLY USING PARTICLES THAT ARE TARGETED, WERE NOT CODING THEM WITH ANY KIND OF NONFOULING MATERIAL. SO THAT MEANS OUR PARTICLES ONCE WE PUT THEM IN HUMAN BLOOD WILL GET PLASMA PROTEINS ABSORBENT TO THE SURFACE AND NOW WE'RE SEEING THAT NEGATIVE IMPACT OF THAT WHEN NAPARTICLE IS PLGA AND WE DID A LOT OF ASSAYS BACK AND FORTH THAT I'M NOT GOING TO GO INTO, YOU WILL FIND THEM IN THIS PUBLICATION LISTED HERE, WE ARE ABLE TO NARROW IT DOWN TO IMMUNO GLOBUE LYNNS, IGA AND IGM. PLGA AS A MATERIAL SEEMS TO HAVE A HIGH AFFINITY FOR THIS AND SO WHEN YOU GO INTO A BLOOD OF A HUMAN THAT HAS HIGH CONCENTRATION OF THAT, IT FOUND THE SURFACE QUICK WHICH ACTIVATES OR AT LEAST CHANGES THE CONFIRMATION OF THE TARGETING LIGAND IN A WAY THAT THEY'RE NOT ABLE TO BIND THE BINDING PARTNERRENER AT THE BLOOSES VESSEL WALL. SO THE GOOD NEWS IS THAT WE FIX THAT PROBLEM BY MAKING THOSE NONFOULING SO WE REDUCE THAT INTERACTION AND TARGETING CAN STILL WORK. HOWEVER, THIS WAS FOR ME, I REMEMBER SAYING TO MY GROUP, THIS IS STRANGE THAT WE'RE JUST SEEING THIS IN 2015 WHEN PLGA AS A POLYMER HAS BEEN AROUND FOR A LONG TIME AND WE'VE BEEN PROPOSING A USE OF THIS IN THIS CONTEXT. SO WE--I CHALLENGED THEM TO LOOK IN THE LITERATURE SEE IF WE MISSED ANYTHING AND THEY CAME BACK, IT TURNS OUT THAT PLGA WAS EITHER DISCUSSING THE CONTEXT OF DRUG LOADING, DRUG DEGRADATION RATE, CELL CULTURE OR ANIMAL MODELS INJECTED INTO A MOUSE AND ACTIVITIES WERE CONFIRMED THERE SO THAT LED US TO THE HYPOTHESIS OF WHETHER SOMEHOW THE EXPERIENCE OF PLGA PARTICLES IN MOUSE BLOOD WAS DIFFERENT FROM THE EXPERIENCE IT HAS IN HUMAN BLOOD. AND TO ANSWER THE QUESTION, WE BROUGHT THE MOUSE BLOOD INTO OUR SYSTEM AND WE REPEATED SOME OF THESE ASSAYS COMPARING PARTICLES IN BLOOD FLOW, THAT HAD--THAT HAD PLASMA, OF THE MOUSE OR IN BLOOD, RED BLOOD CELL THAT HAS BUFFER SO WE REMOVE PLASMA. AND YOU CAN SEE HERE THAT THE PARTICLES BOUND FINE WHEN THE BLOOD WAS MOUSE, WHETHER IT WAS MOUSE PLASMA OR WE SOAK IT IN PLASMA AND RUN IT AND BUFFER IT, X, Y, Z. SO WE'RE SEEING DIFFERENCES IN HOW THE PLGA PARTICLES WAS BEHAVING IN A MOUSE VERSUS HUMAN. NOW SINCE WE ALREADY DID A LOT OF WORK TO SHOW THAT THIS IS AN IMMUNO GLOBUE LYNN EFFECT, AND THAT WE KNOW--WE KNOW THAT THE PLASMA IN A MOUSE DOESN'T HAVE HIGH IMMUNO GLOBUE LINCOLN TENT, WE WENT TO AN ANIMAL MODEL THAT SHOULD BE COMPARABLE TO HUMAN AND WE WANTED SEE SEE WHETHER WE WOULD SEE THE BINDING BEHAVIORAL IN BINDING OR LACK OF BINDING AND SURELY WHEN WE REDID THE ASSAY WITH PIG BLOOD THAT HAS THE HIGH COMPONENTENT WE SEE THAT THE PLGA WAS ELIMINATED SO FOR FIRST TIME WE'RE REALLY GETTING A JOLT THAT BLOOD IS AN ORGAN OR TISSUE, WHATEVER YOU WANT TO SAY IT, AND THAT TISSUE IS NOT NECESSARILY THE SAME TISSUE IN THE ANIMAL MODELS THAT THAT WE WANT TO USE AND WE NEED TO UNDERSTAND WHAT THOSE DIFFERENCE ARE AND SO WE DUG MORE AND WE FOUND OUT THAT RED CELL GEOMETRY, VARIES EVER SO SLIGHTLY BETWEEN HUMANS AND THE ANIMAL MODELS THAT WE LIKE TO USE, MOST NOTABLY THE MOUSE. THE RED BLOOD CELL DIAMETER IS SMALLER AND THE VOLUME IS SMALLER. SO IT'S A SHORTER ROUNDER CELL, WHAT MEANS THAT THE RED CELLS IN A MOUSE ARE PROBABLY LESS DEFORMABLE COMPARED TO HOW DEFORMABLE THOSE OF HUMANS ARE AND WE HYPOTHESIS THAT THOSE CHANGES WILL CHANGE HOW THOSE CELLS ARE DISTRIBUTED WHEN THE BLOOD IS FLOWING WHICH MIGHT EFFECT HOW PRODUCTS ARE LOCALIZED AND SURE ENOUGH WE FOUND A GOOD CORRELATION BETWEEN THE RACIAL PARTICLE DIAMETER TO THAT OF RED CELL DISCIPLINARY AMTERAND THE BINDING AND THAT FOR EACH TYPE OF BLOOD THERE'S A MAXIMAL OPTIMAL RATIO THAT FAVORS HIGH BINDING. WHAT THIS SIMPLY MEANS IS THAT FOR RED BLOOD CELL DIAMETER LIKE HUMANS THAT ARE LARGE, LARGER PARTICLES DO BETTER IN TERMS OF LOCALIZING AND BINDING AND THAT'S WHAT WE FOUND IN THE PRIOR DATA. AND IN A MOUSE WHEN IT'S SMALLER THEN WE SEE NO BIG DIFFERENCE BETWEEN A NANO CARRIER AND A MICROCARRIER. SO AGAIN, SOME OF THE DATA WE WERE GETTING WITH HUMAN BLOOD AND NANO PARTICLES COULD BE EXPLAINED WHY WE HAD NOT SEEN MUCH OF THAT IN A MOUSE SYSTEM. THE OTHER PIECE AS YOU LOOK AT LEUCOSITE COMPOSITION, YOU SEE HERE IN UH ATHLETICS MANS YOU MOSTLY HAVE NEUTROPHILS. AND THOSE AREINIT CELLS AND THEY ARE FRONT LINE IN TERMS OF INFECTION CLEARANCE AND IMPORTANTLY IN THE CONTEXT OF THIS ASSAY, THEY ARE LARGER WHITE CELLS THAT TEND TO BE BETWEEN 10 AND 12-MICRONS. SOME WOULD ARGUE 15-MICRON IN DIAMETER WHEREAS THE MOUSE BLOOD AND MOSTLY LYMPHICIDES THAT ARE SMALLER, 78-9 MICROIN DIAMETER. FROM A BIOMECHANICAL STANDPOINT WHEN A DRUG CARRIER IS AT THE BLOOD VESSEL WALL, IT IS GOING TO ENCOUNTER THE WHITE BLOOD CELL BECAUSE THEY ARE ALSO LOCALIZED THERE AND THE KINDS OF COLLISION THEY GET BETWEEN TWOAZ 2 MATTERS AND IT WILL BE LINKED TO THE SIZE OF THE PARTICLES AND YOU SEE HERE A VIDEO OF THE PARTICLE BOUND AND YOU SLEEP APNEA AND OBESITYY THAT IT COLLIDES WITH THE WHITE CELL OR THE WHITE CELL COLLIDE WITH IT AND RIPPED IT UP. SO YOU CAN IMAGINE THAT WHEN THE WHITE CELLS IN NUTRIFILL, THAT COLLISION HAS THAT POTENTIAL OF RIPPING THE PARTICLE OFF LIKE YOU AWE IN THAT ASSAY, THAT'S HUMAN BLOOD, THAT WILL BE A NUTRIFILL. WHEREAS IF MOST OF WHAT YOU'VE SEEN ARE LYMPHOCYTE MAY BE MORE DRIVEN THERE. NOW THIS ISSUE HERE WE CAN FIX BY SIMPLY ADDING MORE TARGETING LIGAND ADHESION LIGAND DENSITY TO BE ABLE TO W STAND THOSE KINDS OF COLLISION. BUT, YOU KNOW REALLY WE LEARNED A LOT THEY WANT TO QUICKLY SUMMARIZE HERE BEFORE I PIVOT TO SORT OF HOW WE'RE TAKING THE INFORMATION WE'VE LEARNED AND USING THAT TO LEARN MORE ABOUT BLOOD AND HOW THAT HAS INFORMED SOME OF THE WAYS WE'RE THINKING ABOUT TARGETING PARTICLES IN BLOOD. SO SIZE MATTERS IN HOW THE PARTICLE GETS TO THE VESSEL WALL, FOR HUMAN BLOOD THAT FAVORS THE SIZE PARTICLES BETWEEN 2 AND 3-MICRONS SEEMS TO BE A SWEET SPOT AND FOR NANO CARRIERS WE CAN MAYBE ALTER DENSITY, WE CAN ACHIEVE THIS INTERACTION AND HOPEFULLY I WILL GET TO THE END SOME OF THE WAYS WE ARE TRYING TO GET NAVIGATE THE NANO PARTICLE ISSUES AND NOW WE'RE RECOGNIZING THAT BLOOD IS A DIFFERENT TISSUE THAT WE NEED TO PAY ATTENTION TO AND I WILL COME BACK TO THAT IN A MINUTE. BUT THIS WAS AN INTERESTING OBSERVATION BUZZ. THAT NUTRIFILL WAS ALSO TRYING TO BIND BECAUSE WHAT YOU CAN'T SEE BEHIND THE CLOUD OF PLASMA IS A LAYER SO IT HAS ENCOUNTERED OUR PARTICLE AND BOTH PARTICLES AND NUTRIFILLS LEFT THE WALL AND WERE NO LONGER BINDING, AT LEAST NOT IN THIS FIELD OF VIEW. SO IT WAS THE FIRST TIME WE WERE JOLTED TO THIS IDEA THAT PARTICLES COULD BE ALTERING THE BEHAVIOR OF BLOOD CELLS AND THAT'S AN IMPORTANT QUESTION TO SIT WITH BECAUSE IF I'M DESIGNING DELIVERY VEHICLES THAT'S SUPPOSED TO DELIVER DRUG, IN MANY CASES WE WOULDN'T WANT TO INTERFERE WITH THE NATURAL IMMUNE RESPONSE THAT WE HOPE CAN PARTNER WITH US IN DISEASE SCENARIO. YOU CAN THINK OF CANCER FOR EXAMPLE, THAT THIS WOULD NOT BE A DESIRABLE EVENT. SO THAT LED US TO RETOOL OUR THINKING AND ASKED THE QUESTION, HOW DO THE PRESENCE OF PARTICLES, VASCULAR TARGETED ALTER THE BEHAVIOR OF WHITE CELLS, RIGHT? AGAIN, WHITE BLOOD CELLS IN THIS CASE, THESE ARE MOSTLY NUTRIFILLS IN HUMAN BLOOD, WHERE YOU HAVE INFLAMMATION OR SOME SORT OF INJURY OR DAMAGE OR INFECTION, THEY GET THOSE CUES FROM THE BLOOD VESSEL WALL, THEY BIND AND EVENTUALLY GO INTO THE TISSUE SPACE. SO YOU WANT THIS HAPPENING, THIS IS WHAT THAT LOOKS LIKE IN OUR SYSTEM WITH THE HEALTHY HUMAN BLOOD. IF WE PUT TARGETED PARTICLES, IT'S THE SAME ASSAY, IT'S WHOLE BLOOD AND YOU CAN BARELY SEE ANY IMMUNE, WHITE CELLS, NUTRIFILLS BOUND IN THIS SARKS SAY, INSTEAD I'LL OUR TARGETED PARTICLES HAVE OUTCOMPETED THE WHITE CELL. SO THAT WAS FASCINATING FOR US TO SEE. THAT NEUROECTODERMAL NATIONAL PHENOMENA DO THIS OVER LOWER CONCENTRATION BUT OVER HIGH CONCENTRATION CAN YOU SEE THAT EFFECT WITH SMALLER PARTICLES AS WELL AND THAT REALLY, THE PARTICLES HAVE TO BE BOUND AND COVERING THE SURFACE FOR THIS KIND OF OUTCOMPETING TO HAPPEN. BUT BEFORE WE KIND OF COMMUNICATED THIS OBSERVATION, WE WANTED TO MAKE SURE IT'S NOT AN AFTER FACT OF OUR INVITIO SYSTEM. SO WE ASKED WHETHER WE CAN SEE THIS KIND OF PARTICLE BASED INTERFERENCE OF IMMUNE CELLS ACTION INVIVO, IN A MOUSE MODEL. AND SO WE LEVERAGE VITAL MICROSCOPY TOOLS THAT WE HAVE AT MICHIGAN WHERE WE CAN TAKE A MOUSE, THIS IS A MESSENTERIEN TARY THAT'S TRANSPARENT, WE DROP LOCAL AGANISTOT VESSEL WE WANT TO IMAGE SO THAT INDUCES LOCAL INFLAMMATION YOU MIGHT CALL THAT INJURY, AND SEE YOU HERE WITHIN 3 MINUTES NUTRIFILLS ABOUND AND WE KNOW THIS ON NUTRIFILLS BECAUSE WE CHARGE THEM AS WELL AND OVER TIME IT'S NICELY COVERED, BOUND AND THAT'S AGAIN A POSITIVE REACTION TO INFLAMMATION. IF WE NOW PUT OUR TARGETED PARTICLES IN, YOU SEE IN THE SHORT-TERM, NUTRIFILLS ARE THERE TRYING TO BIND BUT OVERTIME JUST LIKE IN OUR INVITRO SYSTEM WITH HUMAN BLOOD, WE OUTCOMPETE THOSE CELLS TO BIND AND YOU KNOW, AGAIN, HIGH NUTRIFILL BOUND RESPONSE TO INFLAMMATION, REDUCED IMMEDIATELY WITHIN MINUTES ONCE WE PUT TARGETED PARTICLES. NOW, WE WERE NOT EXPECTING THIS, CONTROL PARTICLES THAT WERE NOT TARGETED IGG COATED DID NOT BIND WHICH IS GOOD BUT AT THE END OF 10 MINUTES WE NOTICE YET AGAIN THAT THE NUTRIFILLS WERE ALSO STILL NOT BOUND. SO YOU HAVE AN EMPTY VESSEL THAT'S INFLAMED AND YOU DON'T SEE PARTICLES BECAUSE THEY'RE NOT TARGETED BUT YOU'RE NOT ALSO SEEING NUTRIFILLS BOUND AND THAT WAS NOT SOMETHING WE WERE EASILY OBSERVED INVITRO WITH OUR HUMAN SYSTEM AND SO THAT LED US IMMEDIATELY TO THINK PHAGOCYTOSIS IN OUR INVITRO SYSTEM, WE WERE USING ANTICO AGULATED BLOOD AND CITRIC WAS OUR ANTICO AGULATE AND THAT MEANS WE REALLY DIALED DOWN THE IMMUNITY IN THOSE CELLS IN BLOOD TO EAT ANYTHING. AND THERE JOB AS NUTRIFILLS IS TO EAT STUFF AND YOU SEE HERE IN THE ZOOM OF THIS ASSAY, THERE WERE PARTICLES SITTING RIGHT ON TOP OF A NUTRIFILL SO THAT YOU KNOW THAT THAT CELL IS ABOUT TO EAT THAT PARTICLE. OKAY. SO FAB O SIGNIFYITOSEIS MAY BE PLAYING A ROLE IN THIS UNEXPECTED RESULT. WE CAN CONFIRM THIS BY SIMPLY TAKING OUR PARTICLES, AND PUTTING A NONFOULING ACT POLYMER. IN THIS CASE, WE KNOW THAT IF WE GRAFT POLYETHYLENE GLYCOL CHAINS ON TO A DRUG CARRIER SURFACE THAT CREATES A HIGH LAYER WHICH PREVENTS A LOT OF PROTEINS WHICH WILL BE MOSTLY HYDROPHOBIC FROM GETTING AND STICKING TO THE SURFACE OF THAT CARRIER. IN DOING SO, WE KNOW THAT WE SLOW DOWN FAB O SIGNIFYITOSEIS DRASTICALLY AND IF THAT WORKS OUT WELL, THEN WE SHOULD SEE THAT THE USE OF THIS PARTICLE SHOULD MEAN THE SAME LEVEL OF BINDING AS IF PARTICLES WERE NOT INJECTED AT ALL. BECAUSE THEY'RE NOT TARGETED AND SURE ENOUGH, WE SEE NICELY THAT WHEN WE USE NONTARGETTED PEGGULATED PARTICLES WE GET THE SAME LEVEL OF NUTRIFILL BOUND TO THE VASCULAR WALL AS IF THERE WERE NO PARTICLES AND ANY USE OF PARTICLES TARGETED OR NOT WAS GETTING IN THE WAY OF THOSE CELLS. SO FAB O SIGNIFYITOSEIS WAS IMPORTANT HERE. WE WENT AND CONFIRMED THIS IN OUR HUMAN ASSAY. WE CHANGED THE ANTICO AGULATE FROM ACD TO HEP A RIN AND WE LET THE CELLS EAT THE PARTICLES ISSUES, ABOUT 2 HOURS BEFORE WE SEND THEM IN THE ADHESION ASSAY AND YOU SEE HERE, WHEN PHAGOCYTOSIS WAS NOT TURNED ON, BLOCKING OF NUTRIFILL ADHESION WAS MOSTLY ADHESION LINKED BUT WHEN WE LET THEM EAT FOR A COUPLE OF HOURS, YOU SEE THERE WAS NO DISCERNIBLE DIFFERENCE WHETHER THEY WERE TARGETED OR NOT. SO WE CONFIRMED THIS PHAGOCYTOSIS PIECE THERE AND IF WE WERE TO SUMMARIZE WHAT WE HAVE HERE, IS WHEN YOU INJECT PARTICLES INTO THE BLOOD, IF THEY ENCOUNTER NUTRIFILLS IN BLOOD WHICH THEY WILL BECAUSE THEY LIVE THERE, THAT IF THOSE PARTICLES CAN BE EATEN BY NUTRIFILLS, STILL EAT THEM AND THAT PROCESS CAN DRIVE THE CELLS AWAY FROM THE BLOOD AND WE KNOW THEY'RE LEAVING THE BLOOD BECAUSE WHEN WE DO NUTRIFILL COUNT IN THE BLOOD AFTER THE ASSAY, WE SEE A LOT MORE IN BLOOD WITH INFLAMMATION THAT MAKES SENSE, THEY RAMP UP AND IF WE NOW LOOK AT THE BLOOD OF PARTICLE TREATED INFLAMED WE SEE A SIGNIFICANT REDUCTION IN THOSE CELLS IN BLOOD. SO THEY'VE BEEN DRIVING AWAY FROM BLOOD AND WE FOUND THEM IN THE LIVER. THIS IS PARTICLES, WE KNOW THAT TARGETED DRUG CARRIERS OFTEN DEPOSIT AND DELIVER OVER TIME BASED ON THEIR SIZE SO WE'RE NOT SURPRISED THERE, BUT WE'RE SURPRISED TO SEE A LOT OF NUTRIFILLS ACCOMPANYING THEM IN THE LIVER AND WE KNOW THAT IS A LOT MORE BECAUSE IF WE LOOK AT THE COUNT OF NUTRIFILLS IN THE LIVER AFTER THIS ASSAY, SEEEE A LOT MORE IN THE LIVER WHEN THE PARTICLE TREATMENT HAS BEEN GIVEN COMPARED TO JUST INFORMATION. SO THIS IS A LOT OF INTERESTING PIECE OF DATA THAT WE WOULD THEN FOLLOW UP IN TERMS OF THINKING ABOUT DISEASE TARGETING AND TREATMENT. BUT WE WANTED TO TAKE A PAUSE AND GO WHAT IS THIS THING WE'RE SEEING WITH NUTRIFILLS AND PHAGOCYTOSIS, THE QUESTION MAY NOT SEEM--MAY SEEM WEIRD TODAY BUT WHEN WE WERE MAKING THIS OBSERVATION WE DIDN'T KNOW MUCH ABOUT NEUTROPHILS AND PHAGOCYTOSIS, IF YOU SAID TO ANY OF US PHAGOCYTOSIS WE WOULD IMMEDIATELY TALK ABOUT MACROPHAGES, AND THE ONLY DIFFERENCE IS THAT MACROPAGES LIVE IN TISSUE SPACE AND NUTRIFILLS ARE THE 1S THAT YOU WILL FIND MAINLY IN HUMAN BLOOD CIRCULATING SO ANY KIND OF PARTICLE THERAPEUTICS THAT GO IN BLOOD WILL NEED TO ENCOUNTER NUTRIFILLS, YET THERE WERE VERY LITTLE ABOUT HOW THIS CELLS FAG O SIGNIFYITOSED IN THAT TON TEXT AND THIS WAS SURPRISING BECAUSE THIS IS A NUTRIFILL IN OUR ASSAY THAT YOU CAN SEE QUICKLY AND EFFECTIVELY THAT WAS WITHIN 2 OR 3 MINUTES OF A PARTICLE WITH NO PROBLEM. OKAY, SO WHAT DOES THIS MEAN FOR TARGETED, INJECTABLE DELIVERY SYSTEM, WE NEED TO UNDERSTAND HOW THESE CELLS BEHAVE. SO WE SET UP ASSAYS TO TRY AND PROBE AND UNDERSTAND THE CELLS ISSUES WE WERE USING POLYSTYRENE CAR BOXALATED AS A HIGHLY FAG O SIGNIFYITOSIBLE PARTICLE AND THE PEGGULATED VERSION AS THE CONTROL THAT SHOULD NOT BE FAG O SIGNIFYITOSED AND WE KNEW THAT WORKED BECAUSE I SHOWED YOU THE DATA WITH THE MOUSE INVIVO WHERE IT HELPED US TEASE OUT THE MECHANISM OF THE BLOOKING THERE. SO WE PUT THAT IN OUR ASSAY AND MUCH TO OUR SURPRISE WE WERE FINDING THE OPPOSITE WAS TRUE WHENEVER WE FED HUMAN NEUTROPHILS IN HUMAN BLOOD, PEGGULATED PARTICLES WE WERE SEEN INCREASE UPDIAMOND CIRCLE ACHIEVER AND WE DIDN'T BELIEVE THE DATA SO ANY HUMAN IN THE HALLWAY IN OUR LAB, WE GOT THEIR BLOOD AND WE WERE SEEING CONSISTENTLY, THERE WAS A PREFERENCE FOR PEGGULATED PARTICLES THEN AGAIN VERY STRANGE. WE WANTED TO MAKE SURE WE WERE NOT LOSING OUR MINDS SO WE REPEATED THE ASSAYS PUBLISHED IN LITERATURE THAT TENDS TO BE MONOSITTIC CELL LINES, CHP 1, BONE MARROW DERIVED MACROPHAGES OR INVIVO IN A MOUSE MODEL, WE ALWAYS SAW THAT PEGGULATION DID REDUCE EATING OF THOSE PARMS AND WE DID TRANSLATE TO MORE CIRCULATION INVIVO, SO THIS WAS A NUTRIFILL THING BUT THEN WHEN WE ZOOM IN MORE, WE WERE SEEING THAT IT PERHAPS IS A HUMAN BLOOD THING SO WE REPEATED THIS ASSAY BY SWAPPING OUT THE MEDIA AND PUTTING HUMAN PLASMA, WE SEE THAT THE EFFECTIVENESS OF PEG WENT AWAY WHEN THAT PHAGOCYTOSIS WAS DONE IN HUMAN PLASMA. SO THEN IF YOU LOOK AT THE PROTEIN PROFILE ON YOUR PARTICLE THAT WRA PROTEIN DID IT GRAB, YOU SLEEP APNEA AND OBESITY A LOT OF PROTECTION WHEN THERE'S NO PRODECLARATION, AND THERE'S A REDUCTION WHEN HAVE YOU PEG, BUT THERE'S STILL PROEN TOOS THAT REMEANS AND THIS IS NOT NEW. MANY PEOPLE HAD PUBLISHED ON IT'S VERY DIFFICULT TO GET A HUNDRED% WIPE OUT ON PROTEINS ON PARTICLE SURFACE WITH PEGGULATION AND THE SYSTEM MADE THOSE QUITE ATTRACTIVE FOR EATING AND WE THINK THAT FACTORS THAT COMPLEMENT THE SYSTEM IS THE CULPRIT HERE. OKAY, SO, NUTRIFILLS EAT DIFFERENT LEAN BLOOD FROM WHAT WE KNOW, PEGGULATION DID NOT SEEM TO PROTECT THEM FROM FROM EATING AND THAT LED US TO ASK QUESTIONS IN TERMS OF WHAT OTHER THINGS ARE DIFFERENT FOR MACROPHAGES THAT MAY NOT BE TRANSFERRING FOR NUTRIFILLS AND WE KNOW THERE'S THIS LANDMARK WORK FROM [INDISCERNIBLE] GROUP THAT SHOWED THAT MACROPHAGES DID NOT EAT ELONGATED RODS. THEY WERE USING A LOT OF BIG, BIG RODSES ARE 20-MICRON IN LENGTH BUT WE WANT TO ASK IN THE CONTEXT OF DRUG DELIVERY WHEN WE LOOK AT NANO RODS AND MICRORODS DOES THIS TRANSFER TO NUTRIFILL AND SO WE PLAYED AND WERE ABLE TO FABRICATE ROTS OF DIFFERENT VOLUME AND ELONGATION WHERE WE CAN FIX THE ASPECT RATIO, RASE MODELIO OF LUNG, SIDE, TO SHORT SIDE AND WE NEED THEM TO NUTRIFILLS IN HUMAN BLOOD AND IMMEDIATELY WE SAW THAT NUTRIFILLS WERE NOT BEHAVING IN THE WAY WE EXPECTED. AGAIN, HERE, MACROPHAGES DID NOT EAT ROD IN PRIOR PUBLICATION IN OUR SYSTEM, IT DOESN'T MATTER WHETHER WE'RE NANO RODS OR MICRON RODS, THEY EAT THEM AND IT'S NOT JUST THE MENTION BEING MEASURED HERE BECAUSE THIS ROD HERE IN THE BOTTOM HAS 2-MICRON MAJORAXIS AND THEN YOU COMPARE THAT TO A 2-MICRON SPHERE, YOU SEE THAT THERE WAS A SIGNIFICANT PREFERENCE FOR THE ELONGATED PARTICLES RATHER THAN THESE PARTICLES. WE WENT BACK AND MADE SURE THAT WHAT WE WERE SEEING WERE REAL AND AGAIN THE PRIOR LITERATURE FERS ACCURATE. MACROPHAGE AND MONOSITTIC CELL LINES DO NOT CARE FOR EATING RODS AND THAT THIS WAS A IN NUTRIFILL SPECIFICALLY AND A LOT OF PEOPLE ASK, WHY IS THIS? PART OF IT? IT COULD BE THE JOB OF NUTRIFILLS EVOLUTION BASE WHERE HUMANS ARE BIGGEST PROBLEM WAS BACTERIA AND YEAST INFECTION AND THAT THIS CELLS WERE TO TO MICE TO HELP US CLEAR THOSE INFECTIONS AND THE GEOMETRY OF THOSE CELLS BACTERIA AND YEAST WILL BE ELONGATED SO THEN THEY HAD PRACTICE OF EVOLUTIONARY PRACTICE OF DOING SO. THIS IS THE POINT WHERE WE'RE SAYING, OKAY, NOW WE'VE IDENTIFIED AN OPPORTUNITY TO SINGLE HOW THE NUTRIFILLS IN BLOOD, IF WE WANTED TO DELIVER SOMETHING TO NUTRIFILL, WE NOW KNOW HOW TO DO IT AND AVOID OTHER CELLS, MONOCYTES WHICH ARE IMPORTANT IN REFLENNISHING OUR MACROPHAGES OF DENDRITIC CELLS AND THINGS LIKE THAT SO WE STARTED THIS COMPANY FOCUSED ON TARGETING NUTRIFILLS AND NUTRIPHILIC INJURY AND THESE ARE ESSENTIALLY DECEASES, MOST OF THEM ACUTE INFLAMMATORY RESPONSE THAT CAN CAUSE A SIGNIFICANT DAMAGE TO OUR TISSUE, GOOD EXAMPLE OF THIS IS ACUTE LUNG INJURY, PART OF WHAT WE WERE SEEING WITH COVID EARLY ON AND THE NEED FOR VENTILATORS AND FOR TREATMENT WAS LINKED TO THE DAMAGE THAT THE IMMUNE CELLS, NUTRIFILLS WERE DOING TO THE LUNGS IN THEIR ATTEMPT TO GO HELP. SO THE QUESTION WE ASKED THEN IS CAN WE USE THIS KNOWLEDGE OF, ABILITY OF OUR PARTICLES TO ENGAGE NUTRIFILLS IN BLOOD STREAM AND DIVERT THEM FROM AN INNAMED TISSUE TO AS A TREATMENT MECHANISM AND SO WE USED THESE MOUSE MODEL WHERE WE CAN INJURY THE LUNG OF A MOUSE IN THIS CASE, WE'RE USING LPS, A BATTERY OF FRAG MENLT AND THEN WE TREAT THE CELL WITH THE ANIMAL PARTICLES THROUGH THEIR SYSTEMIC CIRCULATION. THIS IS WHAT THE LUNG LOOKS LIKE ON A HEALTHY MOUSE. IT'S MOSTLY MACROPHAGES YOU SHOULD NOT HAVE NUTRIFILLS IN THERE. WHEN THERE'S INFLAMMATION IN THE LUNG, THE AIR WAY IS SWAPPED WITH NUTRIFILLS AND THAT PROCESS AGAIN IT COULD BE DAMAGING AND GET IN THE WAY OF BREATHING. BUT WHEN WE INJECT AND TREAT OUR MOUSE WITH THE PARTICLES YOU SEE THAT WE BEAT BACK THE COUNT OF NUTRIFILLS IN LUNGS, RIGHT? IT'S NOT BACK TO BASE LINE BUT IT'S A SIGNIFICANT REDUCTION, TELLING US THAT THESE KINDS OF BLOOD STREAM MITIGATION APPROACH COULD WORK AND IT'S EXCITING BECAUSE IT COULD WORK ON THAT CONDITION OF ACUTE DISEASE. HOWEVER, THIS WORK WAS DONE WITH POLYSTYRENE, WE NEEDED TO SHOW THAT WHEN WE USE DEGRADABLE SYSTEM THAT WE CAN SEE THE SAME EFFECT. AND SO WE MADE PARTICLES FROM FROM THE ACID WHICH IS BASICALLY A POLYMER THAT'S MADE UP OF SALLY SILIC ACID WHICH IS ESSENTIALLY THE ACTIVE GROUP OF ASSPRIN O THE WHOLE PARTICLE IS AN IN ANTIFLAMMATORY 18th AND WE DID THAT AT THE TIME BECAUSE THERE WAS CONCERN WITH PUTTING A LOT OF LACTIC ACID FROM PLGA INTO THE LIVER SO WE WANTED TO USE A PARTICLE SYSTEM THAT WE KNOW THAT WHEN IT DEGRADES IT HAS AN ANTIINFLAMMATORY PROPERTY. SO WE REPEATED OUR ASSAYS AND MUCH TO OUR SURPRISE, WE WERE SEEING BETTER ACTIVITY IN TERMS OF KNOCK DOWN OF NUTRIFILLS FROM THE LUNG AIR WAY THAN WHAT WE WERE SEEING WITH OUR MODEL PARTICLES AND WE TEASE THAT OUT SOME MORE AND STARTED TO NOTICE THAT OUR PARTICLES ARE THERAPEUTIC PARTICLES WAS REDUCING CELL COUNT, REDUCING INFLAMMATORY CYTOKINES IN THE AIR WAY OF ANIMALS, IN A WAY THAT A SYSTEMIC TREATMENT OF ASPIRIN WAS NOT DOING IN A WAY THAT SYSTEM TEMMIC TREATMENT OF OTHER PARTICLES ARE NOT DOING SO THERE'S ADDITIONAL BENEFIT WE'RE SEEING FROM THIS PERMERAND WE WANT TO--POLYMER AND WE WANT TO MAKE SURE WHAT WE WERE SEE SUGGEST REAL SO AGAIN WE COME BACK TO HUMAN BLOOD AND WE WERE ALLOWING THE PARTICLES OF DIFFERENT MATERIALS, PLGA, POLYA WHICH IS OUR THERAPEUTIC PARTICLES AND WE WERE LOOKING AT HOW CELLS UPTAKE THEM AND YOU SEE THERE'S NO DIFFERENCE IN HOW MUCH THE PARTICLES WERE EATEN BUT, IF WE LOOK AT EXPRESSION OF INFLAMMATORY PROTEINS ON THE SURFACE OF THE ANIMAL, OF THESE CELLS WE DO SEE, THAT WHEN THERE IS UPTICK BY THE THERAPEUTIC PARTICLE, THERE IS SOME ANTIINFLAMMATORY ESSENTIALLY DRIVING THE NUTRIFILLS TO ANTIINFLAMMATORY PHENOTYPE WHICH COULD EXPLAIN THE ADDED BENEFIT WE'RE SEEING IN THE SYSTEM. OKAY, DOES THIS WORK IN THE LIFE INFECTION MODEL? THE ANSWER IS YES, WE SWITCHED TO PSEUDOMOANUS INFECTION IN THE LUNGS AND SAW THE SAME TREATMENT AND SAME THERAPEUTIC BENEFIT. IN FACT WE SAW ONLY THE THERAPEUTIC PARTICLES HAVING A POSITIVE EEIVELGT ON INFLAMMATORY EFFECT, WE SEE ALL MARKERS OF INFLAMMATION GO DOWN IN THAT SPACE, WE SEE THAT THERE'S LESS LEAKINESS OF THE BLOOD VESSEL WHEN WE TREAT WITH OUR PARTICLES IN, IN A CASE WHERE THE ANIMALS WILL END UP IN DEATH, WE WERE ABLE TO RESCUE SOME OF THOSE ANIMALS AS TREATMENT. SO WE'RE EXCITED ABOUT THAT. ALL RIGHT, SO, WE STILL HAVEN'T FORGOATEN THIS ISSUE OF NANO PARTICLES AND WE WANTED TO DO A FEW THINGS TO TRY AND RECTIFY IT, PLGA HAD PLASMA ISSUE AND WE WERE SED TRACKED ON THAT, WE STARTED IT OUT SO NOW WE WERE COMING BACK AND WE WANTED TO LOAD GOLD NANO PARTICLES IN TO SEE WHETHER WE CAN CHANGE THE DENSITY OF THOSE PARTICLES TO BE BIGGER SO WE CAN SEE THE EFFECT OF DENNITY IN A FIXED MATERIAL TYPE. SO WE CAME UP WITH AN ASSAY WHERE WE WOULD TAKE PLGA DISSOLVED IN ORGANIC SOLVENT, EMULINGSIFY IT IN A WATER SOLVE AND THEN THE WE WILL HAVE DROPLETS OF PLGA SOLUTION IN THE OIL FACE. RATHER THAN LOAD NANO PARTICLES ALREADY PREFORMED WHICH WOULD BE TOO HEAVY WE DECIDED WE WOULD USE A GOLD PRECURSOR AND WE WOULD THEN SYNTHESIS IT WITHIN THE OIL DROPLET AND SOLIDIFY THE OLD DROPLET TO GET NANO PARTICLE LOADED PLGA. THE PROCESS WORKED WELL EXCEPT THAT WE NOTICE THAD THE GEOMETRY OFFER PARTICLES WERE OFF. IF THIS WAS AN OIL AND WATER SCENARIO, THAT SHOULD BE SPHEREICAL PARTICLES. SO WE STARTED TO DIG MORE AND MORE EXPW LANDING ON THIS 1 WERE NOT PLGA AND THESE WERE PARTICLES AND THE BIOSALT WAS THE SODIUM CORRELATE THAT WE WERE USING TO STIPULATE THE DROP AND USING IT IN THE OLFACTORY SYSTEM, SO OF COURSE WE DID ASSAYS TO CONFIRM THIS AND AFTER THE INITIAL SHOCK AND DISAPPOINTMENT OF OUR ORIGINAL ASSAY NOT WORKING WE CHALLENGED THE STUDENT TO ASK IS THERE A POTENTIAL TO USE THIS IN ANY BENEFICIAL WAY AND IT TURNS OW THE ANSWER IS YES, BIOSALTS ARE NATURALLY OCCURRING. THEY HAVE POSITIVE FUNCTIONS ORIGINATING FROM THE LIVER AND WHEN THEY ARE MISSING IN INDIVIDUALS WHO CANNOT SYNTHESIZE THEM IT'S A BIG DEAL AND THEY HAVE TO TAKE REPLACEMENT THERAPY THEY TOOK ORAL DOSES OF COLIC ACID. IN THE OTHER SITE, SOME BILE SALTS ARE MODFIGHTED AND GO INTO OUR DISCIPLINARY JECTIVE TRACT AND LIES FAT, SO WE FIGURED OUT IF WE TOOK THE REDUCED SOLUTION AND INJECT IT INTO LOCAL FAT TISSUE WE CAN ACTUALLY REDUCE FAT. SO IT WAS BEING USED AS A FAT REDUCTION THERAPY. SO WE NOW SEE SITUATIONS WHERE BILE SALTS ARE THERAPEUTIC BAH THEY'RE NOT FUNCTIONING WELL BECAUSE OF THE MODE IN WHICH THEY'RE BEING DELIVERED. IF WE SAY OUR PARTICLES CAN CONTROL RELEASE OUT, BIOSALT, NOW WE POTENTIALLY HAVE A TARGETED MECHANISM FOR DELIVERING THIS DRUGS IN THIS CONDITIONS. SO THE FIRST THING WE ASK IS, ARE PARTICLES ERODABLE, THE ANSWER IS YES, RELEASE BACK OUT ACTIVE SALTS AND WE CAN CONTROL THE SIZE OF THE PARTICLE SO WE CAN USE THEM FOR ORAL THERAPY, INJECTABLE THERAPY AND THERAPY OR LOCAL TISSUE DELIVERY LIKE WE'RE DOING WITH THE FAT LYSIS PROJECT AND THAT'S ESSENTIALLY HOW WE TESTED THE EFFICACY, THE DEOXY CORAALATE, IT ENDS UP WITH CHIBELLA, WE SHOW WE CAN MAKE PARTICLES FROM THAT, WHEN WE UNDERSTAND THE PROCESS BY WHICH WE TEMPLATE THIS AND WE SHOW WE CAN SEE MILD TOLERATION OF THE MILD PARTICLE COMPARED TO THE SOLUTION VERSION AND THAT WHEN WE DO TREAT THE MICE, WE SEE ACTIVITIES IN FACT, LISINE WHERE THIS IS A NATURAL FAT HEALTHY FAT CELL ANDS THIS IS WHAT HAPPENS WITH THE CELLS BEING LIESED AND GONE WHEN WE TREAT WITH PARTICLES. SO THAT'S EXCITING AND WE FIGURED OUT HOW TO MAKE ALL KINDS OF OTHER TYPES OF BILE SALT RELATED PARTICLES THAT ALLOWS US FOR TARGETING MULTIPEEL DISEASE. SO THAT'S EBTS SIGHTING. SO I WILL TAKE THE LAST 2 MINUTES HERE TO TELL YOU HOW WE'RE STILL OBSESSED WITH THIS NANO PARTICLE PROBLEM AND HOW WE--1 OF THE WAYS THAT WE THOUGHT ABOUT NAVIGATING THAT. ESSENTIALLY WE HAD HAD USED IT IN THE LAB COME IN AND WE'RE HAVING DISCUSSION ABOUT THIS PROJECT OF NANO PARTICLE IN THESE STUDENTS SAID WHY NOT JUST USE MICROPARTICLES AND THEN YOU GO YEAH, YOU'RE RIGHT, THE CELLS THAT DO WELL ARE GETTING TO THE BLOOD VESSEL IN BLOOD ARE MICRON IN SIZE AND THAT OF COURSE IS WHAT WE'RE SEEING WITH THE PARTICLES. THE PROBLEM OF COURSE IS WITH POLYMER SYSTEM, MOST OF THEM WILL MAKE PEOPLE NERVOUS FOR USING AS INJECTABLE SYSTEM. BUT WHAT IF WE MADE THEM DEFORMABLE LIKE THE CELLS. RIGHT? SO THAT LED TO PROPROJECT WHERE WE MAKE A CONTROL OF THEIR IMAGE, THEY DRY OUT, COLLAPSE, EXPAND AND THEN WE SHOWED THAT THOSE DEFORMABLE PARTICLES WORK NICELY. THE MICROPARTICLE BIND NICELY IN MOUSE MODEL, AND THEY OUTPERFORM ANY NANO PARTICLES WE WERE USING, HOWEVER, THERE IS STILL THE BENEFIT OF NANO PARTICLES CROSSING THE BLOOD BARRIER INTO THE TISSUE SPACE SO HOW DO WE FIX THIS KNOWING THAT OUR MICROPARTICLES CAN'T DO THAT, SO WHAT WE'VE DONE NOW IS IN A RECENT PAPER, DESCRIBED THIS IDEA OF USING THE DEFORMABLE MICROPARTICLES TO GET NANO PARTICLES TO THE BLESS VESSEL WALL. WE LOAD THEM FOR EXAMPLE HERE WITH 50 NANO METER POLYSTYRENE AND WE SHOWED THAT THOSE PARTICLES CAN BIND NICELY IN FLOW WITH THEIR NANO PARTICLE CARGO. THE QUESTION IS WHETHER THIS NANO PARTICLE CARRYING MICROPARTICLES CAN PERFORM THE SAME LEVEL OR BETTER THAN FREE NANO PARTICLES THEMSELVES AND THE ANSWER IS YES, EVEN WHEN THE NANO PARTICLES ARE 3 TIMINGS MORE THAN THE HYDROGELS ARE CARRYING WE WERE ABLE TO DELIVER MORE NANO PARTICLES TO THE VESSEL WALL INVITROW AS WELL AS INVIVO. THIS IS THE ASSAY HERE IN A MOUSE VESSEL AND WE'RE SHOWING VERY NICELY THAT WE GOT MORE NANO PARTICLES TO THE WALL THAN MICROPARTICLES. OF COURSE, THE QUESTION THAT YOU MAY HAVE IS WELL, NANO PARTICLES HAVE LONG CIRCULATION AND SO OVER TIME THOSE FREE NANO PARTICLES MAY OUTCOME PETE THE HYDROGEL SYSTEM BECAUSE THEY'RE MICRON IN SIZE. SO WE DID THE ASSAY WHERE WE FOLLOW THIS PARTICLE FOR AN HOUR AND WE SEE HERE THAT THE MICROPARTICLES THAT'S BOUND WITHIN 5 MINUTES IS A HYDROGEL MICROPARTICLES THEY STAY BOUND FOR UP TO AN HOUR. WE THINK UP TO 4 HOURS. WHEREAS, WHEN WE PUT NANO PARTICLES, THEY OVER THAT 1 HOUR PHASE DID NOT OUTCOME PETE THE MICROPARTICLES. SO FINAL CONCLUSION, WE LEARNED A LOT IN TERMS OF HOW PARTICLES NAVIGATE BLOOD. WE THINK THAT 1 WAY THAT WE MIGHT NAVIGATE GETTING NANO PARTICLES IN THE WALL MIGHT BE TO USE SOMETHING LARGER TO GET THEM, IN THIS CASE, THE VULNERABLE PARTICLES AND THAT IF YOU CAN DESIGN THOSE MICROPARTICLES TO GET DOWN IMMEDIATELY AND BIND, THEY WILL STAY BOUND AND IN THAT CASE, LOCALIZATION OUTCOMPETES LONGER CIRCULATION. SO WITH THAT I WOULD LIKE TO ACKNOWLEDGE MY LAB AND GENEROUS FUND FREE RADICALS GENERATED THE NIH, THE HYDROGEL NANO PARTICLE PLASMA PROTEIN DATA WAS DONE BY NIH FUNDING AND NIH'S FUNDING OUR WORK TO LOOK AT THE THERAPEUTIC PARTICLES IN ACUTE RESPIRATORY DISTRESS. SO WITH THAT I HOPE WE HAVE SOME TIME TO GET QUESTIONS GOING. THANK YOU. >> THANK YOU SO MUCH FOR THE FANTASTIC TALK, --I THOUGHT IT WAS A GREAT EXAMPLE OF THE INTERSECTION BETWEEN BASIC BIOLOGY AND ENGINEERING WHICH I KNOW QUITE A FEW FOLKS HERE ARE INTERESTED IN. WE DO HAVE A FEW QUESTIONS FROM A VARIETY OF DIFFERENT FACETS OF YOUR TALK. ONE OF WHICH KIND OF ON THE BIG BROADER VIEW POINT IN TERMS OF GLOBAL HEALTH IS HOW DO DISEASE STATES SUCH AS SICKLE CELL EFFECT PARTICLE TRANSPORT AND ARE THOSE CERTAIN CONSIDERATIONS THAT NEED TO BE IN PLACE FOR, YOU KNOW THERAPEUTICS BEYOND THE--EUROPEAN WESTERN MARKETS. THAT IS A FANTASTIC QUESTION AND MY LAB DOES HAVE A COUPLE OF PUBLICATION IN THAT SPACE. >> SICKLE CELL OF COURSE COMES IN IF YOU KNOW THE DISEASE, YOU KNOW THAT ONCE--1 OF THE THINGS THAT HAPPENS IS THAT THE RED CELLS IN THOSE PATIENTS ARE A LITTLE BIT RIGID. THE HBS CELLS ARE RIGID. AND IT TURNS OUT THAT WHEN WE THINK SICKLE, WE THINK OF THE SICKLE SHAPE BUT THOSE SICKLE CELL ARE TRANSIENT SO THAT A PATIENT THAT HAS SICKLE CELL DISEASE WILL HAVE NORMAL LOOKING RED CELLS THAT ARE RIGID. AND WE'VE SHOWN IN OUR WORK THAT IF YOU REGIDDIFY THE RED CELLS IN A HEALTHY BLOOD, YOU ACTUALLY CHANGE THE DISTRIBUTION OF WHITE CELLS SO THAT IN A SICKLE CELL PATIENT, WE THINK THAT THAT RIGIDITY ACTUALLY REDUCES THE ABILITY OF THOSE WHITE CELLS TO GET TO THE WALL AND BIND IN THE WAY YOU SAW IN OUR ASSAY, WHICH HAS POTENTIAL LINK TO THE PRESENTATION OF THAT DISEASE. THEY TEND TO BE PRONE TO INFECTION, THIS COULD BE WHY AND INDEED IF WE SHOW THAT WE PUT DRUG CARRIERS IN A DRUG THAT HAS RED CELLS, WE ACTUALLY REDUCE, WE REDUCE THE LOCALIZATION AND ABILITY OF THOSE DRUG CARRIERS TO REACH THE BROOD VESSEL WALL BUT IT'S LINKED TO THE PARTICLES SO WE HAVE THE PAPER OUT THERE, PUBLISHED, I ENCOURAGE THE AUDIENCE WHO ASKED THE QUESTION TO LOOK THOSE OBVIOUSLY INTERESTING THINGS THERE. >> FANTASTIC. SO MOVING A BIT TO KIND OF SOME OF THE THERAPEUTIC WORK YOU HAD SHOWN. WITH THE CELL MEDIATE DAMAGE WITH THE SEVERAL APPLICATIONS YOU LISTED HAVE YOU LOOKED AT ALL INTO CENTRAL NERVOUS SYSTEM APPLICATION FOR THINGS LIKE STROKE WHERE OF COURSE POST INFARCT INFLAMMATION IS MORE DAMAGING THAN THE INITIAL INSULT ITSELF. >> YEAH, NO, AGAIN A FANTASTIC QUESTION. THIS ALL DISEASES IN THE PORTFOLIO THAT WE'RE HAVING THAT, WE'RE LOOKINGA THE TO SEE WHETHER WE CAN HAVE BENEFIT HERE AND CNS DISEASE ARE EXACTLY IN THE WHEEL HOUSE. WE HAVEN'T ACTUALLY GONE IN AND GOTTEN DATA ON THAT BECAUSE THE CHALLENGE THERE IS THE RIGHT ANIMAL MODEL. BUT IN OTHER PLACES WHERE WE HAVE THE ANIMAL MODELS WE'RE SEEING EVIDENCE THAT IF THERE IS SIGNIFICANT INJURY LINKED TO THIS NUTRIFILL CELLS THAT WE CAN HAVE THERAPEUTIC IMPACT IN THERE, SO I DON'T--I SUSPECT THAT THAT WILL BE THE SAME IF WE CAN GET THE RIGHT ANIMAL MODEL FOR THAT. >> YEAH, WE WILL SNEAK 1 MORE QUESTION IN BEFORE WE RUN OUT OF TIME WHICH IS MORE ON THE BIOLOGICS SIDE WITH THE NUTRIFILL PHAGOCYTOSIS OF THE THESE, DO YOU SEE THE SUBSETS THAT ARE MORE DRIVERS OF THIS? OR IS IT KIND OF A GENERIC PHENOTYPE? >> THAT IS A GREAT, GREAT QUESTION AND THAT'S EXACTLY SOME OF THE EXPERIMENTS AND RESULTS THAT WE'RE TRYING TO GET NOW. THIS CONCEPT OF NUTRIFILL SUBSET IS ACTUALLY A RELATIVELY NEW CONCEPT AND THEY DO INDEED EXIST. AND WHAT WE'RE TRYING TO DO NOW IS TO GO IN AND SEE IF WE IDENTIFY NUTRIFILLS IN THE BLOOD THAT DID NOT UPTAKE PARTICLES, THEY PHENOTYPICALLY DIFFERENT FROM THE 1S THAT DO UPTAKE PARTICLES? WHAT DOES THAT MEAN? AND EVEN IN OUR INVIVO ASSAYS, WHEN WE DO TREAT AND WE GET THE BLOOD BACK, WE CAN SEE WHICH BLOOD CELLS HAVE PARTICLES EMBEDDED WITHIN THEM AND WHEN 1S DON'T. AGAIN, CAN WE BEGIN TO GET A SENSE OF WHETHER THERE ARE DIFFERENT SUBSET, THE NEW NUTRIFILLS THAT ARE BACK IN BLOOD OR THEY EXIST IN NUT RIFILLS THAT REALLY JUST HAVE A DIFFERENT PROPENSITY FOR EATING CELLS. SO THE FASCINATING--I'M SO EXCITED ABOUT NUTRIFILLS IN WAYS THAT AND HOW MUCH WE STILL HAVE TO DISCOVER IS SUPER EXCITING. >> FANTASTIC. WELL WE ARE RIGHT AT THE 2:00 MARK, SO WE HAVE REACHED THE END OF OUR TIME HERE. HOPEFULLY WE CAN HAVE IN COME IN PERSON AT SOME TIME TO COME AND INTERACT WITH SOME OF THE DISOF THES HERE BUT WE GREATLY APPRECIATE YOUR SEMINAR AND THINK SO MUCH TO EVERYBODY THAT WAS ABLE TO JOIN US FOR OUR LAST SESSION, OUR LAST TALK FOR OUR VERY FIRST YEAR OF THE BIOMEDICAL ENGINEERING SCIENTIFIC INTEREST GROUP SERIES HERE AT THE NIH SO THANK TO YOU EVERYBODY WHO CAME OUT AND THANK YOU SO MUCH DOCTOR FOR YOUR PRESENTATION TODAY. THANK YOU ALL.