PENNSTATE

TeamMagneto
FinalReport
06May2015

GregoryLynn
FaranakFoolad
KennethSurano
SamuelVilchez
KatherineArazawa
NoIntellectualPropertyRightsAgreement
NoNonDisclosureAgreement
1

ExecutiveSummary
Humancoronaryarterydisease(CAD),themostcommontypeofheartdisease,isthetop
killerofmenandwomenworldwide,accountingfor25percentofannualdeaths.Earlydetectionof
CADwouldenablebetterdiagnosticanalysisandpossibletreatmentoptionsforpatients.Dr.Jim
AdairandDr.LarrySinowayofPennStateUniversityhavetaskedthisgroupofundergraduatesto
investigatetheuseofnanoparticlestotargetischemiclesions(inadequatebloodsupply)incoronary
arteries.Thenanoparticlesarebasedonmagnetite(Fe3O4),ahighlymagneticmaterial,and
resorbablecalciumphosphosilicate,abiodegradablematerialplatform.Magnetitenanoparticlescan
beimagedwithavarietyofdifferentimagingmethods.Theheavymagnetiteprovidesdarkcontrast
forMRIandXRayCTscanning.Photoacousticscanningisachievedthroughasonicgenerator
causedbyarapidthermalexpansionandcontractionafternearinfrared(NIR)pulselaserexposure.
Thepreliminaryresearchforthisprojectinvolvedacomprehensiveliteraturereviewof
potentialbiomarkers,materialsanalysisforthenanoparticle,andchemicalprototypingofbio
conjugationschemes.BasedonthevaryingcharacteristicsofCADwhichdependhighlyonthe
stageofthedisease,asequentialtripletargetsystemisbeingdesignedtoallowforearly(tyrosine),
middle(antiSRA1),andlate(fibrin)detectionofatheroscleroticplaques.Abioconjugation
methodforeachtargethasalsobeenproposedsothatamultimodalcontrastagentcanbe
conjugatedtothebiologicalmarkers.Testingforproofofconceptinvolvedopticalmeasurements
andimagesofthefluorescent(indocyaninegreen)encapsulatedparticlesinbovinebloodundernear
infraredexcitationbya785nmlaser.Thesetestresultshaveshownsatisfactoryexcitationand
imaginingofthenanoparticleusingacamerawithaNIRcutofffilter(i.e.acamerathancantake
picturesofNIR).A$1000budgethasbeendelineatedforexpensesrelatingdirectlytothisproject
andhasnotbeenexceededfortheproject.
Dr. Adair and Dr. Sinoway will continue take our results and continue work on this project to
develop an improved diagnostic system for CAD. Further work will be expanded in the coming
summer into the nanoparticle fabrication, and our designs will be used as a foundation for new
prototypes. Once full nanoparticle complexes have been synthesized with attached targets, they will
be tested in vitro, in vivo in mice, and in many years, eventually in humans.

Table of Contents
1.0
Introduction.................................................................................................................................5
1.1

Initial Problem Statement........................................................................................................5

1.2

Objectives................................................................................................................................5

2.0

Customer Needs Assessment......................................................................................................7

2.1

Gathering Customer Input.......................................................................................................7

2.2

Weighting of Customer Needs................................................................................................7

3.0

External Search...........................................................................................................................8

3.1

Literature Review....................................................................................................................8

3.1.1 Application of Nanoparticles for Imaging..............................................................................8

3.1.2 Bio-conjugation schemes...................................................................................................11
3.1.3 Potential Bio-markers for Atherosclerosis Lesions...............................................................11
3.2
4.0
4.1
5.0

Existing Products and Patents...............................................................................................14

Engineering Specifications.......................................................................................................16
Establishing Target Specifications........................................................................................16
Concept Generation and Selection............................................................................................18

5.1

Problem Clarification............................................................................................................18

5.2

Concept Generation...............................................................................................................18

5.2.1 Concept A..............................................................................................................................18

5.2.2 Concept B..............................................................................................................................19
5.2.3 Concept C..............................................................................................................................20
5.3

Concept Selection.................................................................................................................20

5.3.1 Concept A..............................................................................................................................22

5.3.2 Concept B..............................................................................................................................22
5.3.3 Concept C..............................................................................................................................22
5.3.4 Pugh Concept Selection........................................................................................................22
6.0

System Level Design................................................................................................................24

7.0

Special Topics...........................................................................................................................26

7.1

Preliminary Economic Analyses - Budget and Vendor Purchase Information.....................26

7.2

Project Management.............................................................................................................27

7.3

Risk Plan and Safety.............................................................................................................28

7.4

Ethics Statement....................................................................................................................29

7.5

Environmental Statement......................................................................................................30

7.6

Regulatory Considerations....................................................................................................30
3

7.7

Communication and Coordination with Sponsor..................................................................30

8.0

Detailed Design........................................................................................................................32

8.1

Target Selection Process...........................................................................................................33

8.2

Target Analysis.........................................................................................................................33

8.3

Production Procedure................................................................................................................34

8.4

Schematics and Drawings.........................................................................................................37

8.5

Test Procedure...........................................................................................................................40

8.6

Economic Analyses - Budget and Vendor Purchase Information.............................................42

9.0

Final Discussion........................................................................................................................43

9.1

Construction Process.............................................................................................................43

9.2

Test Results and Discussion..................................................................................................44

10.0

Conclusions and Recommendations.........................................................................................45

11.0

Self-Assessment (Design Criteria Satisfaction)........................................................................46

11.1

Customer Needs Assessment................................................................................................46

11.2

Global and Societal Needs Assessment................................................................................47

References............................................................................................................................................48
Appendix A: Resumes.........................................................................................................................50
Appendix B: Updated Literature Review............................................................................................56
Appendix C: Updated Gantt chart.......................................................................................................58
Appendix D: Updated test procedures.................................................................................................60

1.0

Introduction

Eachyear,approximately600,000Americansdieofheartdisease.Ofthese600,000victims,
overhalfsufferedfromCoronaryArteryDisease(CAD),whichisthemostcommontypeofheart
disease (Center for Disease Control 2014). CAD is characterized by a buildup or plaque, or
atheroscleroticlesions,overdecadesinthearterieswhichgoesunnoticed.However,buildupof
plaquemayleadtoacuteinflammationandcriticalconditions,suchasplaqueruptureandvessel
occlusion.Currentmethodsforidentifyingtheseatheroscleroticlesionsarelimitedandisunableto
effectivelydiagnosistheonsetofCADforearlyprevention.Thereforeamoreefficientwaytodetect
andlocatetheselesionsbeforetheonsetofcatastrophiceventsmayleadtoimprovedtherapyand
preventionforthesecriticalconditions.

Figure1.Diagramshowingtheprogressionofatherosclerosis(CenterforDiseaseControl
2014)

1.1

InitialProblemStatement

One of the reasons why CAD continues to be such a lethal disease is that medical
professionalslackaviablemethodofearlydetection.TheearlystagesofCADinvolveplaquebuild
upinthecoronaryarteries;unfortunately,itiscurrentlynearlyimpossibleforphysicianstodetect
smallamountsofplaque.Inmanycases,thepresenceofCADdoesnotbecomeapparentuntilits
inflammatoryphase,duringwhichtheplaquerupturesandvesselocclusionoccurs.

1.2

Objectives

Thefirstrequirementistoidentifyaviablebiologicalmarkerforischemiclesionsincardiac
bloodvessels. Thismarkershouldbespecifictoatheroscleroticlesionssothatanydrugdelivery
willnotbedeliveredsystemically,butaccuratelyaffectonlythetargetlesions.Ifthisinformationis
notavailablethroughaliteraturesearch,materialtestingwillbecompletedonischemictissueto
assesspossibletissuemarkers.
5

Thesecondrequirementistodesignachemicalbioconjugationschemetoattachcalcium
phosphosilicatenanoparticlestothematchingchemicalmarkerfoundforischemiclesionsabove.
Thisconjugationschemeshouldbesimple,lowcost,andeasytoreplicate. Theschemeshould
containnoprocessormaterialwhichwillcouldbetoxictobiologicaltissue,orshowadequate
mitigationofpossibletoxicitywhereneededwiththoroughneutralizationandcleaning.
Thethirdrequirementistodesignandcreateaworkingprototypeofbioconjugationscheme
withatheroscleroticlesiontissuetoperformaproofofconcept.Thenanoparticlecomplexesshould
associatewiththelesiontissuewithatleast95%higheraffinitycomparednormalvesseltissue.This
objectiveistertiaryandwillbecompleteduponavailabilityoflabtimeandtissueavailability.

2.0

CustomerNeedsAssessment

2.1

GatheringCustomerInput

On Feb 3rd, the Magneto Team met with Dr. James Adair to discuss expectations and
deliverablegoalsforthesemester.EmailcommunicationwasusedtocontactDr.LarrySinoway,
who graciously provided background information and ideas on the science of atherosclerosis,
althoughnoformalmeetingwilloccurwithDr.SinowayuntilFebruary17th.

2.2

WeightingofCustomerNeeds

Thefollowingweightingisforthecompletedbioconjugatednanoparticleforatherosclerosis
targeting. Toxicityisamainconcernandshouldbeweightedheavily(23%)toensurethatthe
materialsbeingchosendonotcauseadditionalharmtoapatient. Themostimportantmetricisa
uniqueaffinityoftheseparticlesforspecificlesions,sincethetargetmoleculeisonlyasgoodasits
abilitytoassociatespecifically(46%).Totalcost(11%),easeofmanufacture(9%),andclearance
mechanisms(11%)arealsoimportantconsiderations,butconcessionscanbemadetoallowforless
desirablecharacteristics.
Toxicitywasdefinedinthisstudytoensurethattheparticlesonlabmicewithonlyminimal
sideeffects.Oncetheconceptcanbeprovenviable,measurescanbetakentoconfirmtheirsafetyin
humans.Easeofmanufacturingwasdefinedtobethatmanufacturingmusttakelessthan24hours.
Easeofclearance,orcirculationtime,meanstheparticlesmustbeabletostayinthebodyforatleast
72hourstoensureaclearwindowforimaging.Uniqueaffinityassurestheparticlesarebindingto
theirintendedtarget,whichwedefinedasneedingagreaterthan95%affinityforuniquereceptors.
Lastly,costfortheseparticlesmustbeatleastcomparable,preferablycheaper,thantheexpensesfor
currentimagingtechniques,suchasCTscanandMRI.
Table1.AHPPairwiseComparisonCharttoDetermineWeighting

Toxici
Easeof
Easeof Uniqu Cos
ty Manufacturi Clearan
e
t
ng
ce Affinit
y
1.00
0.33
0.50
2.00 0.5
Toxicity
0
3.03
1.00
0.66
6.06 0.6
Easeof
6
Manufacturi
ng
2.00
1.52
1.00
4.00 1.0
Easeof
0
Clearance
0.50
0.17
0.25
1.00 0.2
Unique
5
Affinity
2.00
1.52
1.00
4.00 1.0
Cost
7

Tot
al

Weig
ht

4.33

0.23

11.4
1

0.09

9.52

0.11

2.17

0.46

9.52

0.11

0.99

3.0

ExternalSearch

Anextensiveliteraturereviewwasperformedtoresearchandunderstandessentialaspects
neededforthedesignoftheCADtargetedmagnetiteparticlesfordiagnosticimaging.Themain
topics researched included Application of Nanoparticles for Imaging, Potential Biomarkers for
AtherosclerosisLesions,andBioconjugationschemes.Apatentsearchforsimilarnanoparticlesand
useofnanoparticleswasperformed.

3.1

LiteratureReview
3.1.1 ApplicationofNanoparticlesforImaging
Medicalimaginghasbeenarevolutionarycontributioninmedicine.Earlydiagnosisand
treatmentofseveralchronicdiseaseswasfarfrompossibleafewdecadesago.However,early
detectionofcardiovasculardiseasesremainsachallengeinthemedicalcommunity.Atherosclerosis
isoneoftheleadingcausesofdeathinhighincomecountries(Almeretal.2014),thusearly
diagnosisoftheselesionsisfundamentalincardiovascularmedicine.Macrophages,duetotheir
largeconcentrationinatheroscleroticdiseases,arepromisingtargetsforbioconjugationandmedical
imagingtechniquescanbeusedtodiagnosethesecomplications.Nanotechnologyhasgreatly
contributedtomedicalimagingduetoitslowtoxicity,easybodyclearance,surfaceproperties,and
considerablysmallsizethatallowstoreachsmallervesselsandhigherresolutionforimaging
modalities(Mulderetal.2014).
Imagingtechniquescanbedividedintotwomajorcategories:invasiveandnoninvasive.
However,invasivetechniquessuchasangioplastyorstents,arebeingdisplacedbymajoradvances
innoninvasivetechniquesformedicalimaging.Someofthenoninvasivetechniquesmightinclude
computedtomography(CT)scans,magneticresonanceimaging(MRI),ultrasound,Xrays,
includingnuclearimagingtechniqueslikepositronemissiontomography(PET)andsingleproton
emissioncomputedtomography(SPECT).

Figure2.Currentimagingtechniques(Mulderetal.2014)
Therehasbeenagrowinginterestonusingnanoparticlestotargetspecificregionsandtobe
usedascontrastimagingparticles(Mulderetal.2014).Severaltypesofnanoparticleshavebeen
reportedtobeusedascontrastagentsforearlydetectionofatherosclerosis,someofthembeing
lipidbasedcarriers,carbonbased,viruslike,oreveninorganicparticlessuchasgold,metaloxides,
andsilver(AltinoluandAdair2010;Ankrietal.2014;Malyutinetal.2015).Surfacetreatment
withPEGprovidesgreatcytocompatibilityanditcanincreasebrightnessandsignalintensity
(AltinoluandAdair2010).Nanoparticleshavebeensuccessfullyimplementedindrugdelivery,
earlydetectionofdiseases,andnoninvasiveimaging(Chandramouli,Sanjana,andSwathin.d.).
Also,nanoparticleshavebeenstudiedforlesiontreatment.Aninjectablereconstitutedhighdensity
lipoprotein(rHDL)nanoparticlecarrierthatdeliversstatintoatheroscleroticplaquehas
demonstratedtoreduceinflammationwithin1weekinahighdosetreatment(Duivenvoordenetal.
2014).

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Nanoparticlesexhibitpropertiesthatmakethemidealcarriersformedicalimaging
modalities:Firstofall,ananoparticlesclearanceroute,mechanismandkineticscanbedetermined
bythepropertiesoftheparticle,thusmolecularrecognitioncanbeachievedbyattachingaligandto
theexternalsurfaceoftheparticle.Likewise,aninherentpropertyofmostnanoparticlesisthe
multiplexeffect,thisrepresentsanamplificationofthecontrastsignalin105106timeshigher.A
particlebindstoitstargetandtherelativelylargenumberofmoleculesintheparticlealsocontribute
thesignalmeasuredbytheequipment(Annapragada2015).

Figure3.Exampleofiposomenanoparticle(Annapragada2015)
Nanoparticleshavegreatlyprovenitspotentialinatherosclerotictreatments.Oneofthe
severaltreatmentsisbasedonsuperparamagneticironoxidenanoparticles(SPIONs),whichthanks
totheirinduciblemagnetizationcanbedirectedtoadefinedlocationviaMRimagingandspin
backwardsandforwardathighspeedgeneratingheatandreducingtheharnessoftheplaque
(Chandramouli,Sanjana,andSwathin.d.).
Anextensiveworkonnanoparticleshasbeendonewithinthepastyearsindetectionof
atheroscleroticdiseasesandcancer.Nanoparticleshavebeenusedindrugdelivery,medicalimaging,
genetherapy(Adairetal.2005),asatargetingmechanismtodetectandcounterregulatethe
inflammatoryresponsecausedbymacrophages(Almeretal.2014)inatheroscleroticdiseases.
AlthoughthevastmajorityofnanoparticlesarefocusedinMRimagingduetoitshighresolutionand
itselfbeingthestateofthearttechnologyformedicalimaging,therearealsodifferentnanocarriers
usedinotherimagingmodalities.ArecentcaseofnearIRemittingfluorophoredopedcalcium

11

phosphatenanoparticleswaspresentedasaminimallyinvasive,nonionizingmethodfortissue
diagnosticimaging(AltinoluandAdair2010).
Despitetheextensiveworkdoneonnanoparticles,thereisfewcontributiontomultimodal
particlesthatcanbeusedindifferenttypesofscanningtechniques.Thus,thescientificcommunityis
stillinneedofamultimodalcontrastagentthatcanefficientlydetectatheroscleroticdiseasesandbe
abletotreatthediseaseinatimelymanner.
3.1.2

Bioconjugationschemes

Bioconjugation,ingeneral,istheattachmentofonemoleculetoanotherthroughcovalent
bondstoformamoleculecomplex.Therearemanydifferenttechniquesusedtobioconjugate,with
theapplicationofthefinalmoleculecomplexbeingformedbeingafactorindeterminingwhich
specificschemetouse.Boththeinitialcomponentsandthespecificreagentsusedarevitalin
determiningtheapplication.Becausethepurposeofbioconjugationinthisprojectistoattach
nanoparticlesfortargetinganddiagnosingdisease,aspecificbiomolecule,suchasaminoacids,
proteins,oroligonucleotides,isnecessarytodesignaviablescheme(Hermanson2013).

Figure4.Abasicconceptoftheformationofamoleculecomplexbycovalentlybondingtwo
moleculeswiththeuseofacrosslinkingagent(Hermanson2013)
Allbioconjugationtechniquesrelyonusingreactivefunctionalgroupstocreatethis
crosslinking.Someofthesegroupsincludecarboxylategroups,aminegroups,andhydroxylgroups.
Throughchemicalreactionswithreactivecrosslinkingagentsorsecondaryintermediates,the
moleculecanthenbelinkedwiththeintendedtarget.Knowingthespecificfunctionalgroupsfrom
theinitialmolecule,intermediates,andtargetallowfordesigntoensurethesuccessofthe
conjugation.Whileintermediatescangreatlyincreasethereactivityofsomemolecules,these
reagentscanalsobetoxictothehumanbody.Itisimportantthat,becausetheconjugationfor
targetingmoleculesoccursinvitro,thesereagentsarewashedandpurifiedfromthefinalparticles.
3.1.3

PotentialBiomarkersforAtherosclerosisLesions

Nanoparticleshavebeenshowntobeeffectivecontrastagentsfordiagnosticimaging
applications.Byusingbiomarkerstotargetspecifictissue,theyallowforspecifiedimagingand
diagnostics.Forexample,Barthetal.createdcalciumphosphosilicatenanoparticlesthatencapsulate
indocyaninegreen,afluorescentdye,toimagethenanoparticleswithnearinfraredlight(Barthetal.
2010).Thenanoparticlestargetedtransferrinreceptorsonbreastcancercellsbybioconjugating
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biotinylatedantiCD71tothenanoparticles.Afterthesenanoparticleswereinjectedintomurine
modelsandweregiven96hourstocirculateandadheretothecanceroustissue,thesubjectwas
scannedusinganearinfrared(NIR)light.TheuseoftheantiCD71conjugatednanoparticleswere
determinedtobeeffectivetargetsforthecanceroustissuethanthecontrols.
Usingasimilarapproach,TeamMagnetosobjectiveistodesignandcreateaworking
prototypeofthemagnetitenanoparticlethattargetatheroscleroticlesions.Aviablebiomarkerto
targettheplaquemustbespecifictotheselesionsandmustbeabletobeconjugatedtothesurfaceof
themagnetitenanoparticles.Anoverviewofpotentialbiomarkersfortheselesionsisshownin
figure5(Lewisetal.2011).

13

Figure5.Atablecompilingvariousstudiesinatheroscleroticlesiontargetednanoparticles
(Lewisetal.2011)
Reviewingthetargetingmoietiesreferenceinfigure5,showedpotentialfortheabilityto
targettheplaquewiththemagnetitenanoparticleasacontrastagenttoimproveddiagnosticimaging.
Thesetargetingmoietiesarefurtherreviewedasapotentialviabletargetforourdesignofthebio
conjuagtedmagnetiteparticlebelow.
VascularcelladhesionmoleculeorVCAM1isanendothelialcellsurfacemolecule.MRI
imagingoftheaortawithinjectionofapeptidesequenceconjugatednanoparticletotargetVCAM1
inapolipoprotienEdeficientmice(apoEmice)showedanincreaseincontrastoftheinitial
atherogenesisofthelesion(Nahrendorfetal.2006).Theseparticlesusedphagedisplaypeptide
conjugatednanoparticletargetedforVCAM1.ThoughtheuseofVCAM1mayfalloutsidethe
scopeofthisprojectasweareconsideringimagingmoreadvancedstagesofthedisease.
Anotherpotentialtargetisoxidizedlowdensitylipoprotein(LDL)whichispredominant
factorinatherogenesis.TargetingtheseoxidationspecificepitopeswithMDA2orIK17antibody
fragmentsconjugatedtosuperparamagneticironparticleshavebeenshowntoresultinsuccessful
imagingofplaqueinapoEmice(BrileySaeboetal.2011).TheseresultsshowoxidizedLDLoffers
afeasibleoptionfortargetinglesions.
Macrophageshavearelativelyhighuptakeandareprevalentinplaquebuildupandtherefore,
seemtobethemostcommonlyusedtargetforatherosclerosis(seefigure6)(Weissleder,
Nahrendorf,andPittet2014).Forexample,dextrancoatedparticleshavebeenshowntohaveahigh
bindingaffinityformacrophageswhichcanmakeupabout20%ofthecellswithinplaque
formations.Variousstudieshaveshownuptakeofdextrancoatedparticlesinatherosclerosismodels.
CrosslinkeddextrancoatedironoxidenanoparticleswereshowntouptakebytheplaqueinapoE
micethatweregivenahighcholesteroldietfor10weekstofurtheradvancethestateoftheCAD
(McCarthyetal.2010).Therefore,dextrancoatingsshouldbeconsideredasapotentialbiomarker
forourdesignofmagnetitenanoparticles.
Macrophagescavengerreceptor(SRA1)showpotentialasaviabletargetforCADasitis
overlyexpressedonthesurfaceofmacrophagecellinlesionsbutnotpresentonnormalcellsinthe
artery.MultiplestudieshavetestedtheuseofSRA1targetednanoparticlesandresultedin
successfulimagingresults(Lipinskietal.2009).Inoneinstance,immunomicelleshavebeenusedto
targetandimageplaqueinapoEmiceandprovideda79%SNRincreasepostinjection.
AnothermacrophagetargettoconsiderisscavengerreceptorB(CD36)whichhasalsobeen
demonstratedtobeasuitablebiomarker.Whengadolinium(Gd)nanoparticlecontrastagents
conjugatedwithantiCD36wereimagedusingcardiacmagneticresonance,thepostinjection
imagesshowedincreasedcontrasttonoiseratio(CNR)upto52.5%tothepreinjectionimage
(Lipinskietal.2009).Thisincreasewassignificantlygreaterthantheuntargetednanoparticle
controls.Theseconjugatednanoparticleshavebeendemonstratedtobindtotheatherosclerotic
lesionsandhelpdetectpotentialhighrisklesions.
14


Figure6.Atablegivingcommonlyusedapplicationofnanoparticlestargetedformacrophages
inatherosclerosis(Weissleder,Nahrendorf,andPittet2014)
Furtherresearchandreviewmustbeperformedtodeterminethebestchoiceforthebio
markerbasedontheabilitytobioconjugatethetargettothemagnetitenanoparticles.Thecustomer
needsandengineeringspecificationsmustalsobeconsideredwhenchoosingthetargetandbio
conjugationscheme.

3.2

ExistingProductsandPatents

CurrentdiagnostictoolsforCADinclude:

MRI
CT
Catheterization
Angiography

15

In a clinical setting these methods are not currently used with nanoparticles, therefore current
medicalpracticesandstandardsarenotwithinthescopeofourobjective.

16

AsearchinGooglePatentforpatentsrelevanttoourprojectandthatmaycoincidewithourobjectivewasperformedusingthekeywords
atherosclerosisnanoparticle,coronaryarterydiseasenanoparticle,andischemiananoparticle.Theresultsofthissearcharegivenin
below.
Table2.Patentsearchresults
Patent

ID

Nanoparticlesforimaging
atheroscleroticplaque(Jarrettand
Louie2006)

WO2006012201A1

Nanoparticlebaseddelivery
systemwithoxidized
phospholipidsastargetingligands
fortheprevention,diagnosisand
treatmentofatherosclerosis
(Wang,Li,andFan2014)

US20140287024A1

Nanoparticletargetingto
ischemiaforimagingandtherapy
(Kim,Cao,andMooney2011)

WO2011133685A2

Claim(s)relevanttoproject
1.Amethodofimagingamacrophage,themethodcomprising:contactinga
macrophagewithadetectionagent,whereinthedetectionagentcomprises:adetectable
nanoparticlecore;acoating;andareceptorbindingmoiety,whereinthereceptor
bindingmoietybindstoareceptoronamacrophage;anddetectingsaidagentto
therebyimagesaidmacrophage.
1.Acomposition,comprising:
apluralityofnanoparticlescomprisingoneormoreoxidizedphospholipids
encapsulatedwithin,adheredtoasurfaceof,orintegratedintothestructureofthe
nanoparticles,whereintheoneormoreoxidizedphospholipidstargettoatherosclerotic
lesionsites.
2.Thecompositionofclaim1,whereinthenanoparticlesareselectedfromthegroup
consistingofliposomes,polymerosomes,microspheres,microstructuredlipidcarriers,
nanostructuredlipidcarriers,highdensitylipoproteinsandacombinationthereof.
1.Apharmaceuticalcompositioncomprisinganonliposomalnanoparticlecomprising
anangiogenesispromotingfactorlinkedthereto.
2.Thecompositionofclaim1,whereinsaidfactorisagrowthfactororcytokine.
3.Thecompositionofclaim1,whereinsaidfactorisselectedfromthegroup
consistingofvascularendothelialgrowthfactor(VEGF),basicfibroblastgrowthfactor
(bFGF),plateletderivedgrowthfactor(PDGF),placentalgrowthfactor(PLGF),
Angiopoietin,stromalderivedfactor(SDF),granulocytemacrophagecolony
stimulatingfactor(GMCSF),andgranulocytecolonystimulatingfactor(GCSF).4.
Thecompositionofclaim1,whereinsaidfactorisVEGF.

17

4.0

EngineeringSpecifications

4.1

EstablishingTargetSpecifications

Thetablebelowliststhevariousengineeringmetricsandtheircorrespondingtargetvalues.
Table3.EngineeringmetricsandcorrespondingtargetvaluesforthetargetedmagnetiteNP
EngineeringMetrics
TargetValue
Size
10nm200nm
Affinity
>95%
Circulationtime
72hours
Solubilityremainswithin10%of
Colloidablystableinphysiologicalconditions
originalvalue
Canbetestedonlabmousewithonly
Toxicity
minimalsideeffect
Timetomanufacture
<24hours
Imagecontrast
Increaseinsignal>50%
Theengineeringmetricswereestablishedbasedontherequirementofdrugdeliveryand
imaging.Weusedengineeringmetricsandtargetvaluessimilartothosethatwereusedin
nanoparticleswhichtargetedotherchronicdiseases,suchascancer(Adairetal.2010).Theremust
beanextremelyhighaffinitybetweenthenanoparticleandthetarget(inthiscase,theatherosclerotic
lesions),orelsenoimagingmethodwouldbeabletocapturethelocationoftheplaquebuildup.
Anotherkeymetricisthetoxicity;thenanoparticlesmustbesafetouseonhumans.Allofthese
metricsmustbetakenintoconsiderationduringthedesignprocessinordertomeetourobjectiveof
designingaviabletargetandbioconjugationmethodforearlydetectionofCAD.

4.2RelatingSpecificationstoCustomerNeeds
TheNeedsMetricsMatrix(seefigure7)wascompletedtoascertaintheengineeringmetrics
determinedforourmagnetitenanoparticlealsoencompassesthecustomerneedsdiscussedinsection
2.

18

Figure7.NeedsMetricsMatrix

19

5.0

ConceptGenerationandSelection

5.1

ProblemClarification

Foratheroscleroticdiseases,bioconjugatednanoparticlesarethestateoftheartintermsof
diseasedetectionandtreatment.Bioconjugationhashadahugeimpactonscienceandtechnology.
Bioconjugationisresponsibleforthediscoveryofnewbiomolecules,growthofindustrieswithin
themedical,diagnostics,microelectronics,andmaterialsciencesfields.Moreover,itisduetobio
conjugationtechniquesthatwecandetect,track,image,capture,ortargetmoleculesandtreat
diseases.
Bioconjugationisaveryuseful,yetsimpleway,tocarrytargetednanoparticlediagnostics
andtreatments.Itisbasedontheattachmentofonemoleculetoanotherthroughacovalentorionic
bond.Thus,itisthroughbioconjugationthatweaimtolinkthenanoparticlecarriertoaspecific
moleculeinatheroscleroticdiseasesinordertobeabletodiagnoseandtreatthediseaseinatimely
manner.

5.2

ConceptGeneration

5.2.1

ConceptA

Asproteinsareaprobablepotentialtargetfornanoparticlesindetectionofatherosclerotic
lesions, conjugation using amine functional groups may be a viable scheme. The magnetite
nanoparticlescanbefunctionalizedwithcarboxylategroups,whichareveryreactive.Throughthis
carboxylatefunctionalgroupandtheuseofacarboxylPEGamineoligomer,aconjugationscheme
toattachthemagnetitenanoparticlestotheproteintargetmaybepossible.Amethod,usedbyDr.
Adair,reactsthecarboxylatedmagnetiteparticleswithEDAC,acarbodiimide.EDACisacarboxyl
activatingagentfortheusetoproduceanamidebond.Becausethisintermediateisnotparticularly
stable,theadditionofNhydroxysulfosuccinimide(sulfoNHS)producesamorestableintermediate.
Atthispoint,acarboxylPEGamineisadded,whichreactswiththesulfoNHStoproduceanamide
bond,nowlinkingthePEGtothemagnetiteparticle.BecausethePEGisalsofunctionalizedwitha
carboxylategroup,theprocedureofreactingEDACandsulfoNHStoformahighlyreactiveleaving
groupisrepeated.Theleavinggroupcanreactwithaminegroupsfoundintheprotein,toform
amidebonds,andthusconjugatingthemagnetitePEGcomplextotheprotein.Thisprocessisshown
intheschematicbelow,withtPAastheshownprotein.Ifatheroscleroticlesionsarefoundtocontain
targets with large amounts of free amine groups, this scheme may allow the conjugation of
magnetite,withaPEGchainandmultipleamidebonds.

20

Figure8.Theseriesofreactionsthatleadtotheconjugationofmagnetitenanoparticlestotheprotein
tPA(Adair2015)

5.2.2

ConceptB

TheproposedschemetofollowisfromtheYangGroupBioconjugationMethod,whichuses
ironoxidenanoparticlesand1ethyl3(3dimethylaminopropyl)carbodiimide(EDAC),thisfurther
reactswithSulfoNHStoformtheestersfrompoly(ethyleneglycol)PEGforcrosslinking,lastlyitis
reactedwithtissueplasminogenactivator(tPA)toobtainironoxidenanoparticlesconjugatedwith
PEGandtPA.

Figure9.ProposedbioconjugationschemefromYangGroupBioconjugation(Adair2015)

21

5.2.3

ConceptC

AnothermethodtoattachtPatomagnetitestartswithcitrate,duetoitshighlyreactive
carboxylicgroup.AmaleimidePEGcomplexisusedwithYangsSulfoNHStocreateaPEG
linkergroupbetweenthemagnetiteandtPaprotein.Inthismethod,citratereactswithEDAC
throughacarboxylactivatingprocesstoformanamidebond.SulfoNHSactsasanintermediate
stabilizerduetotheinstabilityoftheEDACcitrateproduct.PEGisaddedthroughaminereactive
crosslinkchemistry,usingtheNHSesterreagentandprimaryamineonamaleimidePEGamine
complex.ThereactionproducesanNHS(hydroxysuccinimide)byproductwhichisextractedoutof
thesolution.Bywayofasulfydrylreaction,aSHgroupreactswithaleimidetoformathioesther
bond.Thismaleimidereactionproducesthefinalbioconjugatedproduct,adheringtPAmoleculesto
magnetitenanoparticles.

Figure10.Sulfhydrylreactionchemistry(Hayworth2015)

Figure11.AnalternativestrategytoconjugatemagnetitetotPaprotein(Adair2015)

22

5.3

ConceptSelection

Inordertodecideuponthethreebestmethodsforbioconjugationofcalciumphosphate
nanoparticles,anextensiveliteraturereviewonpossibletargetstobindtothelesionsitehelpedus
evaluatewhichmoleculesshowinsuchvascularlesionandatwhatstageofthedisease.Therefore,
the three herein presented bioconjugaton methods are targeting abundant cells characteristics of
atherosclerotic diseases that will bind with at least 95% affinity, to target sites such as the
macrophages,Tcells,andmastcells.

23

Figure12.InflammationduetoatherosclerosisinHumanArteries(Hansson2005)

24

5.3.1

ConceptA

Someissueswiththisschemeincludethelackofspecificityaswellasthemultipleuseof
couplingreagents.ThisschemeisdesignedtouseamidelinkagetofirstbondthecarboxylPEG
aminecomplextothemagnetiteparticle,andthenthatentirelinkagetothetargetprotein.Many
proteinscontainthesefreeaminegroups,whichmaymakethebindingtospecifictargetsin
atheroscleroticlesionsdifficult.Ifothertechniquesareabletoconglomeratenanoparticlestothe
lesions,thisschemesefficiencymaygreatlyincrease.Also,theuseofcouplingreagentsmeansthey
mustbepurifiedfromthenanoparticles,astheycanbetoxicinhumans.Thisnotonlyaddsriskof
toxicity,butalsoincreasesthetimeandenergyusedtomanufacturetheseparticles.
5.3.2

ConceptB

Dr.Yangsmethod,however,isnotperfectinordertoperformbioconjugation,theamine
bondscreatedcouldalsoreactwithotherproteinsthatcontainfreeaminegroups(asintroducedin
thescheme1byDr.Adair.Also,sincethismethoddoesnotincludethePEGaddition,thismight
showadecreaseinaffinity.Likewise,thereisadecreaseinsolubilityofthemolecule.Lastly,this
designisaffectedbythelengthofthecrossbridge,whichissignificantlyshorterthanothermethods
duetothelackofPEGaddition.

5.3.3 ConceptC
PEGaddition(alsopresentin1.1)couldincreasetheaffinityofattachedproteinsby
preventingdeformationoftheproteinandreducestericeffectsofthenearbynanoparticle.ThePEG
actsasatether,extendingthetargetmoleculefurtherintothebiologicalmileu.Thistechniquetakes
advantageofasulfhydrylreactionusinganSHgrouplocatedontheproteinofchoice.Sincethe
idealtargetmoleculehasyettobeidentified,difficultiescouldariseintheabsenceofsuchagroup
onthetargetproteinorbysomeinteractionwiththetargetingpathwaytobindwithischemiclesions.
Similartoxicityconcernsof1.1aexistin1.1cduetothetoxicityofcouplingagents.
5.3.4

PughConceptSelection

Toevaluatethequalityofeachbioconjugationmethodweimplementedthedecisionmatrix
method,alsocalledPughmethod.ThefollowingmatrixpresentedinTable4illustratesourselection
criteriaforeachmethodandrelativeimportanceofeachcriterion.Aswecanseeintable4,unique
affinityholdsthehighestweightpercentageduetoitsimportanceforproperdetectionofdisease;
cytotoxicityfollowstherankinordertoavoidmajorsideeffectsinthehumanbody;otherconcerns
suchaseaseofclearance(howlongdoesittakesforthebodytocompletelyeliminatetheparticles),
costs(affordabilityofproduct),andeaseofmanufacturing(repeatability,massproduction)were
considered as well for the different bioconjugation schemes. To summarize, we see that the
preferredmethodwasconceptCduetoitshighscorerelativetotheopposingschemes.

25

Table4.PughConceptScoring

SelectionCriteria

Toxicity
Easeof
Manufacturing
EaseofClearance
UniqueAffinity
Cost

Concepts

b(reference)
Wgtd.
Rating
Score
3
0.69

Weight

Rating

Wgtd.
Score

0.23

0.46

0.09

0.18

4
5
3

0.44
2.3
0.33
3.71

3
3
3

0.11
0.46
0.11
TotalScore
Rank
Continu
e

YesAlt.Dsgn

RelativePerformance

Rating

Muchworsethanreference

Worsethanreference

Sameasreference

Betterthanreference

Muchbetterthanreference

26

c
Rating

Wgtd.
Score

0.92

0.27

0.18

0.33
1.38
0.33
3

4
5
3

0.44
2.3
0.33
4.17

3
No.

1
YesPrim.
Design

6.0

SystemLevelDesign

Figure13.Designofmultimodalnanoparticle(Adair2015)
Ourdesignofmultimodalimagingcontrastnanoparticleconveystheusageoftwo
fundamentalparticlesformedicalimaginganddiagnosis.Image(A)representsthecontrast
nanoparticlesforNIRscans,image(B)representsthecontrastnanoparticlesforMRI/CTscans;at
last,image(C)representsthebioconjugatedmultimodalimagingcontrastnanoparticlewhichwill
befurtherbindtothespecifictargetincoronaryarterydisease(CAD)andatherosclerotic
inflammations.
Ourfirstandthirdpreliminarydesigns(1a,1c)bothmakeuseofanelongatedPEGoligomer
tethertoextendthetargetproteinfromthenanoparticleandincreaseitsaffinityduetoareductionin
stericeffectsanddeformation.Researchintothespecifictargetmoleculewilldecidewhichofthe
overallbioconjugationschemeswewillbeusing.Thisisbecausethespecificchemistryofeach
27

targetmoleculedictatesthebesttypeofbindingchemistryaccordingtotheassociatedfunctional
groups.
Onceanappropriatebioconjugationschemeandtargethavebeenidentified,magnetite
nanoparticleswillbecreated.Magnetitehasparamagneticpropertiesallowingittoprovidegreat
contrastinmagneticresonance(MRI)orcomputertomography(CT)scanning.Nearinfrared(NIR)
contrastagentscanthenalsobeassociatedtothemagnetitecoretoallowforfluorescenceimaging.
Finally,photoacousticimagingcanalsobeachievedbypulsingnanosecondlasersintothebody,and
detectingtheresultingacousticwavesproducedbyarapidwarmingandcoolingofthelaser
absorbingcontrastagents.
Initialresearchhasrevealedthepossibilityofutilizingatritargetsystemthatwouldallow
forspecifictargetingofplaquetissuesaccordingtothestageofdevelopmentwithinthedisease.
EarlydetectioncanbeachievedbyconjugatingTyrosine,whichbindsspecificallytolipidrich
lipoproteinsinprelesionalareasofthearterialwall.Middlestagescouldbedetectedusing
macrophagescavengerreceptorSRA1a(antibody),whichseekareaswithhighmacrophage
concentrationsurroundingplaquesites.Finally,endstagedetectioncouldbeviablethroughtheuse
ofFibrintargetingwheresubtleclottinginducesthedepositionoffibrinogenonthesurfaceof
plaques.Usingthissystem,itwouldbepossibletochartandmonitoratheroscleroticplaque
formationovertimeclinicallyinanovelapproach.
Thisprojectlimitsitsscopetothedesignofthesemultimodalnanoparticles,particularlyin
associatingaspecifictargetmoleculetomagnetite.Furtherresearchanddesigninsystemdevices
usedtocombinethemultimodalimagingforclinicalapproachesarenotinvestigatedinthisproject.

28

7.0

SpecialTopics

7.1

Preliminary Economic Analyses Budget and Vendor Purchase

Information

Basedonthecurrentknowledgeandscopeoftheprojecttheinitialbudgethasbeen
estimatedandcanbeseenintable5.Wehavebeengivena$1,000dollarbudgetforthecompletion
ofthisproject.Thismoneywillbetravel,projectsupplies,thefinalposter,andanyotherrelevant
necessities.Asourprojectisveryresearchoriented,ourcostsatthistimeareverylow.Wehave
accounted$173.85forthecostoftravelexpenses,aswellasourfinalposter.Wehavebudgeteda
totalof$200fortravelexpenses,which$113.85hasbeenaccountedfor.Wehavealsobudgeted
$200foranylabsupplieswemayneed,and$300fortestsubjectsforanyplannedexperiments,
whichcouldincludebloodortissuetakenfrompigs.Thesearejustestimates,andthemoneywillbe
usedasneededasourprojectproceeds.Wecurrentlydonotanticipatepurchasingmaterials;
thereforehavenotbuiltaBillofMaterials.Iftheneedtopurchasematerialsariseswewillcreatea
BillofMaterialsandupdatethereporttoincludeit.
Table5.Currentbudgetforproject
Item
LabSupplies
Test/ExperimentSubjects
Travel
Poster

Cost
$200.00
$300.00
$200.00
$60.00

TotalBudgeted=

29

$760.00

Comments

7.2

ProjectManagement

AGanttchartoutliningtheprojectscheduleinordertoachieveourthreeobjectivestatedinsection1.1.TheGanttchartoutlinesall
theresponsibilities,tasks,progressoftasks,deadlinesandmilestoneswehaveplannedforthedurationofthisproject.

30

Figure14.Ganttchart

31

TeamMagnetoiscomposedofagroupoffiveBiomedicalEngineeringundergraduateseniors:
GregoryLynn,FaraFoolad,SamuelVilchez,KennySuranoandKatherineArazawa.Theresumes
foreachteammemberaregiveninAppendixB.WithastrongbackgroundinBioengineeringand
collectiveexperienceintheChemicalEngineeringandMaterialScienceandEngineeringfields,we
aretechnicallyaccomplishedtoachievethesedeliverables.Inaddition,weareallexperiencedin
projectmanagementwithvariousinvolvementinindustryandacademicresearch.
Beforetheendofthesemesterourobjectiveistocreateaworkingprototypenanoparticlefora
biomarkerforatheroscleroticlesionsbioconjugatedtothesurface.Inordertoaccomplishthisour
maintaskistoidentifyaviabletargetfortheatherosclerosislesionsanddesignabioconjugation
schemetoattachourtargetproteintothemagnetitenanoparticles.

7.3

RiskPlanandSafety

Aswebeginanindepthliteraturesearchforviableischemiclesiontargets,wewilllesson
theriskofusingunreliableinformationbyonlyusingsourcesthathavebeenpeerreviewed,orare
fromagovernmentalagency.Wewillmakesurethatweareperformingworthwhileresearchby
clearlycommunicatingwithoursponsorsearlyon,andfullyunderstandingtheirexpectationsforthe
project.Asourprojectisprimarilyaresearchproject,itisinherentlyopenended.Thereisalarge
riskofchangeinprojectscope;wewillprepareforthisbymeetingwithDr.Adaireachweekto
discusstheprogresssofarandwhatthenextstepsintheprojectshouldbe.Ourprimarydeliverable,
findinganischemiclesiontarget,maybedelayediftherehasnotbeenadequateresearchonthe
topic.Wemustprepareforthisbymakingourselvesawareofhowwecouldtestpossibletargets
ourselvesinordertofindaviableone.
TheRiskPlantablebelowdepictssomeofthemajorrisksthatwefaceinthisproject,aswell
astherisklevel,howwecanminimizetherisk,andourfallbackstrategy.Therisksthatarerated
withahighlevelarethosethatwemustbemostwaryof,andreadytocombatiftheyoccur.

32

Table6.Riskplan
Risk
Changein
projectscope
and/or
objectives

Level

Moderate

Ischemiclesion
targetdoesnot
functionas
predicted

7.4

Savesomeofbudgetfor
unexpectedexpensesdue
tonewprojectobjectives
Addtimetoschedulefor
newtasks
Buildinsafetytime
Reallocateresources
orstaff

Moderate

Performthoroughliterature
reviewearlyontofindoutifan
ischemiclesiontargethasbeen
discoveredbyotherresearchers
earlyinthesemester

Plan
tests/experiments/criteria
todiscovertargets
ourselves
Discussotheralternatives

Low

Understandthesponsors
expectations
Regularlyupdatesponsoron
progressandfeasibilityofgoals

High

Testearlyandoften
Beawareofthedifferences
betweeninvitroandinvivo
testing,aswellasanimaltesting
versushumantesting

High

Sponsornot
satisfied

Communicatewithsponsorona
regularbasistodiscussprogress
andupcomingsteps
Workwithsponsortoestimate
timeandcostpenaltiesofchanges

FallBackStrategy

Constantlytrackprojectprogress
Ensureallteammembersare
participatingequally
Lookforwaystoaccelerate
activities

Scheduledelays

Delaysin
providing
deliverablesto
sponsors

ActionstoMinimize

Discusswaystofixthe
problem
Tryothertargets

EthicsStatement

AmajoraspectofourinitialprojectworkwillincludeintensiveliteraturereviewsonCAD
andcontrastimagingofnanoparticles.Whenstudyingandusingtheworkofotherresearchers,we
willproperlyciteandreferencetheirworksoastoavoidanyformofplagiarism.Ifatargetfor
ischemiaisfoundandfurtherlabresearchisrequiredtodeterminethebioconjugationschemeforthe
nanoparticles,wewilladheretothebiomedicalengineeringresearchobligationsasoutlinedbythe
BiomedicalEngineeringSociety(BMES).Specifically,wewillrespecttherightsofcolleaguesand
33

animalsubjects,andwillfullycomplywithlegal,ethical,institutionalandgovernmentalresearch
guidelines.Throughoutthisproject,ourteamwilluseourknowledgeandabilitiestoimprovethe
safety,health,andwelfareofthepublic.

7.5

EnvironmentalStatement

Wearecommittedtoupholdingthehighestenvironmentalstandardsthroughoutthecourseof
thisprojectsoastominimizethenegativeimpactofourwork,andtocreateasustainablefuturefor
thisresearch.Intheearlystagesofthisproject,whileweperformavastliteraturereview,wewill
save paper by reading and taking notes on computers as opposed to making printouts. Our
environmentalimpactwillalsobeconsideredaswemoveintothelabworkandtestingstagesofthe
project. Magnetite (Fe3O4), which will be used for the biochemical tagging of the plaque, is
naturallyoccurringandbiodegradable.Furthermore,themethodsofdetectionofmagnetite,which
mayincludephotoacousticsandmagneticresonanceimaging,haveveryminimalimpactsonthe
environment.Wearededicatedtoenvironmentalimprovementsthatfosterasustainablefutureand
leadtosocialandeconomicimprovementsinthecommunitywedobusiness.

7.6

RegulatoryConsiderations

Throughoutthecourseofthisproject,wewillneedtokeepinmindthattheendgoalisto
implementanewmethodbywhichphysicianscandetectCADinpatientsduringitsveryearly
stages.Sincehumanpatientswillbeimpacted,oneofourmajorregulatoryconsiderationsis
receivingFoodandDrugAdministration(FDA)approval.InordertopassanFDAreview,wemust
demonstratethattheMagnetitenanoparticlesarenotonlyfunctional,buttheyaresafeforhuman
consumption.Wemustalsoprovethatthattheoverallprocessoftaggingtheischemiclesiontargets,
andthenimagingusingphotoacousticsand/ormagneticresonanceimagingisharmlesstothepatient
(FDA2014).Oncewefeelthatwehaveproofofsafetyandviability,wecansubmitaNewDrug
Application(NDA)totheFDA.

7.7

CommunicationandCoordinationwithSponsor

Table7.ThemeetingscheduleandoverviewofmeetingswithourSponsors
Date&
Time

Typeof
Meeting

Sponsor(s)
Present

Description

2/3/15,
11:00am

Visit

Dr.Adair

PreliminarymeetingwithDr.Adair.
Discussprojectgoalsandobjectives.

2/10/15,
11:00am

Visit

Dr.Adair

PreparationfortriptoHershey.Plan
questionsforDr.Sinoway.

2/17/15,
11:00am

VisittoHershey
MedicalCenter

Dr.Adair&
Dr.Sinoway

TourofHersheyMedicalCenterbyDr.
Sinoway.ReceivefeedbackfromDr.
34

Date&
Time

Typeof
Meeting

Sponsor(s)
Present

Description
Sinowayonhisexpectationsforproject.

2/24/15,
11:00am

Visit

Dr.Adair

WeeklyMeeting.GooverSOW.Discuss
currentprogressandassigntasksfornext
week.

3/3/15,
11:00am

Visit

Dr.Adair

WeeklyMeeting.Discusscurrent
progressandassigntasksfornextweek.

Weekof
3/15/15,
11:00am

ConferenceCall

Dr.Adair&
Dr.Sinoway

WeeklyMeeting.UpdateDr.Sinowayon
currentprogressandreceivefeedback.

3/24/15,
11:00am

Visit

Dr.Adair

WeeklyMeeting.Discusscurrent
progressandassigntasksfornextweek.

3/31/15,
11:00am

Visit

Dr.Adair

WeeklyMeeting.Discusscurrent
progressandassigntasksfornextweek.

4/7/15,
11:00am

Visit

Dr.Adair

WeeklyMeeting.Gooverprototype.

4/14/15,
11:00am

Visit

Dr.Adair

WeeklyMeeting.Gooverposter.

Weekof
4/20/15

ConferenceCall

Dr.Adair&
Dr.Sinoway

Finalmeeting.Discussfindingsandnext
steps.Receivefeedback.

35

8.0

DetailedDesign

Section8.0.1ModificationstoStatementofWorkSections
RevisionstotheProposalSections1through7arelistedas8.0.1.X:
8.0.1.1.Introductionnochange
8.0.1.2.CustomerNeedsnochange
8.0.1.3.ExternalSearch
Anextensiveliteraturereviewhasbeenperformedtocharacterizethepotentialbiomarkers.This
willallowustodiscussthebestoptionsasagroupwithoursponsor.Thereviewsummaryis
giveninAppendixB.
8.0.1.4.EngineeringSpecificationsnochange
8.0.1.5.ConceptGenerationandSelectionnochange
8.0.1.6.SystemLevelDesign
Asummaryofatripleearlymiddlelatestagedetectionsystemusingthreedifferenttargeting
schemes(Tyrosine,SRA1[a],&fibrin)isgiven.
8.0.1.7.SpecialTopicstheGanttcharthasbeenupdatedinAppendixC.

36

8.1

TargetSelectionProcess

Animportantaspectofcoronaryarterydiseasethatbecameapparentasweperformedthe
literaturereviewisthatthediseasecanbesectionedintothreedistinctphases.Thesephasesdifferin
theextentofatheroscleroticplaque,aswellasconcentrationofcertainmolecules.Sincethereare
varyingconcentrationsofdifferingmoleculesforeachstage,itbecameevidentthatasingletarget
wouldnoteffectivelydiagnosecoronaryarterydiseaseduringallstages.Forthisreason,ourteam
hasselectedonetargetforeachstageofCAD.
IntheearlystagesofCAD,atheroscleroticplaqueisinitiatedbythedepositionoflipidrich
lipoproteinsinsusceptiblebutstillprelesionalareasofthearterialwall.Basedonourresearch,we
foundthattyrosinefunctionalizedmicellescouldbeusedasatargettopenetrateandaccumulate
withintheplaqueafterintravenousadministration.Thefactthattyrosinefunctionalizedmicelles
havelipophilicproperties,smallparticlesize,andprolongedcirculationallcontributetoitsviability
asatarget.Thusweselectedtofunctionalizeourmagnetitenanoparticleswithtyrosine.
Duringthemiddlestageofcoronaryarterydisease,thereisgenerallyaconsiderablevolume
ofatheroscleroticplaqueinthearteryandclottingmaybeoccurring.Duringthisstage,thereisa
highconcentrationofmacrophagesinthediseasedartery,becausemacrophagesareassociatedwith
plaques that are vulnerable to rupture. For this reason, we selected a macrophage scavenger
receptor,specificallySRA1(MSR1)forthemiddlestagetarget.Paststudieshaveshownthat
PEGylated,fluorescentparamagneticmicellesthatwereconjugatedwithSRA1antibodiesshowed
uptoa200%signalenhancementofplaqueriddenareas24hourspostinjection.
Thelatestageofcoronaryarterydiseaseischaracterizedbyplaquerupture.Theseruptures
initiate hemostasis and activate thrombin. Unlike in healthy arteries where there is little fibrin
present,diseasedarteriesinlatestageCADgenerallycontainalargeamountof fibrin;thus,we
selectedfibrinforthelatestageCADtarget.

8.2

TargetAnalysis

Detectionofatheroscleroticdiseasescanbeamajorchallengeifthecharacteristicsweaimto
targetarenotwellexpressedinthelesion.Severalconsiderationssuchastargetedmolecule,affinity
ofeachtargettotheinjectednanoparticle,andspecificityofeachnanoparticletoproperlybindtothe
targetatthelesionzone,oughttobecarefullyconsideredtoincreasethelikelihoodofasuccessful
procedure.Herein,wepresenttheanalysisperformedbytheteamtoselectthebesttargetsfor
criticalstagesofdetection(early,middle,andlate)ofatheroscleroticlesions.
Aspreviouslymentioned,earlystagesofatheroscleroticdiseasesarecharacteristicsofhigh
lipidconcentrationinthevessel;therefore,weaimtobindournanoparticlestolipidrichzonesin
thebloodstream.Researchindicatesthattyrosinecanbeasimple,yeteffectiveapproachtoimage
thelipoproteinsoflipidrichzonesintheplaque.Tyrosineisahydrophobic,nonessentialamino
acidwhichcanbefunctionalizedwithmagnetitenanoparticles.Theexposureoftyrosinegroupsat
thesurfacecouldbeusedtodetecthighlipidareas.Previousworkrevealednarrowsizedistribution
37

oftyrosinefunctionalizednanoparticles,significantincreaseinMRIsignalafewhourspost
injection,prolongedcirculation,specificbindingtoatheroscleroticplaqueswithlipidrichareas.
Othercontrastagentshavebeenreportedforearlydetectionsuchasantibodies,proteins,peptides,
smallmolecules,andpeptidomimetics;however,tyrosinehasproventobeapreciseandsimple
approachtodetectthelipoproteinsofthelipiddepositsintheplaque.Thusweexpecttyrosineto
showpromisingresultswhenfunctionalizedtoourparamagneticcalciumphosphatenanoparticles
formultimodalimagingofCADlesions.
Progressionofthediseaseisdeterminedprimarilybychangesinconcentrationsofmolecules
aroundtheplaque,severityofdamage,andevolutionofcellsinthelesion.Consideringthesefactors
iscriticaltobeabletoselectthecorrectmoietiestobindourmagnetitenanoparticleandefficiently
detecttheatheroscleroticlesion.Formiddlestagethereisahighconcentrationofmacrophagesdue
tolocalinflammation.Thereisacorrelationbetweentheamountofmacrophagesandtheriskof
plaquerupture,makingmacrophagesidealtargetstodeterminetheseverityoftheplaqueandto
efficientlytreatthediseasewhilepreventingplaqueruptureandfurtherdamagetothevessel.Anti
inflammatorydrugsarenotsuitableforselectivemacrophageinhibitionduetotheirseriousside
effects.Statinsinmacrophageshaveproventoreducetheuptakeofoxidizedlowdensitylipoprotein
(LDL),leadinguptoa30%reductionofacutecardiacevents,suchasplaquerupture,andacute
coronarysyndromes.However,idealconcentrationstosignificantlyinhibitmacrophageactivityand
proliferationisnotpossibleinhumansduetothelifethreateningadverseeffects.Targetingthe
macrophagesscavengerreceptorsclassA1(SRA1)cangreatlyinhibitmacrophageactivity.Ifwe
downregulatetheactivityofthereceptorthenthereisasignificantdecreaseinmacrophageactivity
whilemaintaininglowadverseeffects.Thesesreceptorshaveproventobeanattractivetargetdueto
theirdirectinhibitionofmacrophages.Ourschemeinvolvedtheuseofantibodiestofunctionalize
thenanoparticles,specificallytheantiMSR1.Thisantibody,onceconjugatedtothemagnetite
nanoparticle,willbindtothereceptorstodownregulateactivityanddetectthemiddlestageofthe
disease.
Lastly,latestageofthediseasepresentsanacuteresponseinthepatientsbloodvessel.Also,
latestageischaracterizedforruptureofthevesselwall,whichleadstomanysuddenchangesinthe
vesselenvironment.Continuousactivationofmacrophages,alongwithmastcells,andTcellsatthe
siteofplaqueaccumulationproducescytokines,proteases,coagulationfactors,andradicalsas
factorsthatdestabilizethelesions.Theseeventsarefollowedbyruptureofplaque,thrombosisand
ischemia.Thereisanactiveinteractionbetweenmacrophages,foamcells,andsmoothmusclecells
whenrelatedtotheprogressionoftheatheroscleroticlesion.Fibrin,asaproteinformedbyreacting
proteasethrombinonfibrinogen,isknownforitsinvolvementintheformationofclotsoverwound
sites.Althoughtheexactroleoffibrinogen/fibrininthedevelopmentofplaquehasnotbeenfully
exposed,numerousinvitrostudiesshowthatfibrindegradationplaysaroleinseveralinvivo
biologicalfunctionsofsmoothmusclecellsandmacrophages.Thisopensthechancetousefibrinas
atargetbeforeandafterplaquerupture.Nevertheless,ourscopeiscenteredtowardstheacute
responseofthebodyandsuddenfibrinhighconcentration.Previousresearchhasshownviability
andhighaffinityoftPAtofibrin,asmentionedearlier.Thisschemepresentsapromisingmethodto
efficientlydetectatherosclerosisatitslatestage(postplaquerupture).

38

8.3

ProductionProcedure

Ourearlystageparticleswillusetyrosinefunctionalizednanoparticlestotargetthehighlipid
concentration regions found in early atherosclerotic lesions. Tyrosine will be conjugated to the
magnetitenanoparticles,thusshouldtargetareasofhighlipidconcentrationinthebloodstream,
whilebeingvisibleusingmultimodalimagingtechniques.Tyrosinecanbecoupledtoanamine
terminatedPEGmoleculethroughtheadditionofEDACfollowedbysulfoNHS.Thiswillbondthe
carboxyl groupof tyrosine to anamine groupin the crosslinker, giving aaminePEGtyrosine
complexshownbelowinFigure15.

Figure15:aminePEGtyrosinecomplex(Detailedschematicsoftheconjugationschemescan
befoundinsection8.4)
ThiscomplexcanthenbereactedagainwiththeYangMethodsEDACandsulfoNHS,as
wellasthemagnetitecitrateparticlestoconjugatethecomplextotheparticle.Thusourmagnetite
nanoparticle will contain a tyrosine molecule, with a PEG tether, to be administered. The
lipophilicity of the tyrosine will attract the particle to high lipid regions in the blood vessels
associatedwithearlystageatheroscleroticdisease.
Forthemiddlestagetargetingofmacrophagescavengerreceptors,anantibodyisnecessary
tobefunctionalizedtoournanoparticles.Theantibodywillbeconjugatedtotheparticle,which
allowtheantibodytobindtothemacrophagescavengerreceptors intheseregions.Antibodies,
specificallyantiMSR1inourdesign,containimmunoglobulindomains,whichcontaintheamino
acidcysteine.Cysteinecontainssulfhydrylfunctionalgroups,thusallowsustousethemethodofan
aminePEGmaleimidelinker.UsingEDACandsulfoNHS,thelinkercanbeconjugatedtothe
magnetitenanoparticle.Thisleavesahighlyreactivemaleimidegrouptoreactwiththesulfhydryl
groupsintheantibody,conjugatingtheantibodytotheparticle.Thisshouldallowustousethis
particle,nowtargetedtowardsMSR1receptors,todetectthemiddlestageofthisdisease.
The late stage particles are designed to target fibrin, as the fibrin is formed from the
activationofthrombinduetoplaqueruptures.Previousstudieshaveshownthatfibrinantibodiesare
difficulttoproduceandcancauseimmunogenicproblemsinvitro.Tissueplasminogenactivator
(tPA)hasbeenshowntobindtofibrinwithhighaffinity,andwithoutthenegativesassociatedwith
fibrinantibodies.Forthistobeviable,thetPAsenzymaticactivitymustfirstbeinhibited.This
neutralizedtPAcanthenbeconjugatedtothemagnetitenanoparticle,andusedtotargetandimage
the fibrin. DpheLproLargchloromethyl ketone (PPACK) is used to inactivate tPA. tPA is
incubatedwithPPACKina7.2pHbuffersolution;afterremovalofunreactedPPACK,tPAactivity
is95%inhibitedbutthehighaffinitybindingtofibrinisretained(Klegerman,2008).Toconjugate
theinactivetPAtothemagnetiteparticles,aPEGmaleimidelinkerisused.Themagnetiteisreacted
with EDACand sulfoNHS, tocreate the reactive leaving group, which when reacted with the
39

crosslinkercomplex,formsanamidebondbetweenthetwo.Thiscomplexisthenreactedwiththe
inactivatedtPAtocompletetheconjugation.SulfhydrylgroupspresentinthetPAreactwiththe
maleimidegroupsontheparticlecomplexestocreatetheconjugation.Themagnetitenanoparticles
arenowtargetedtowardfibrin,andcanbeusedforimagingthefibrindenseregionsassociatedwith
latestageatheroscleroticdisease.
AsallofthesemethodsusetoxicEDACandsulfoNHStocreategoodleavinggroups,the
particles must be thoroughly laundered and purified to ensure safety of use. This can be done
throughmicrocentrifugationtechniques.ThelaunderingwillbedoneaftereachexposuretoEDAC
andsulfoNHS,thuswillbedonetwiceforearlystageparticles(asthesereagentsareusedtwice)
andonceformiddleandlatestageparticles.Thisprocessaddstimeandcosttothemanufacturing
process,butisanecessarystep.
Table8.Experimentalsupplies
MATERIALNAME

COMMENTS

Magnetitenanoparticles
Calciumchloridedehydrate

99+%,ACSReagent

Sodiumhydrogenphosphate

99+%,ACSReagent

Disodiumcitratedehydrate

99+%,ACSReagent

Sodiumsilicatesolution

~14%NaOH,~27%SiO

Bioconjugation
EDAC

FlukaBioChemika99.0%AT

Ethyleneglycol

99.8%anhydrous

Methoxypolyethyleneglycolamine

mPEGamine,MW20kDa

cyclohexane

99.9+%,A.C.SReagent

methanol

99.9+%,OmniSolvspectroscopicgrade

PPACK

usedtoinactivatetPA

40

8.4

SchematicsandDrawings

Figure16:Tyrosine(earlystage)bioconjugationschemeusingmodifiedAdairGroupConcept
A(5.2.1)

41

Figure17:AntiMSR1(middlestage)bioconjugationschemeusingAdairGroupConceptC
(5.2.3).

42

Figure18:tPA(latestage)bioconjugationschemeusingAdairGroupConceptC(5.2.3)for
thetargetingoffibrin.

43

8.5

TestProcedure

TestingfortheeffectivenessofthenanoparticlesasatargetforCADandcontrastagentfor
NIRandMRIcanbeconductedusingapolipoproteinEdeficient(apoE)mice.Variousstudieshave
shownthatapoEmicedevelophighlevelsofcholesterolandahighfatdietwillleadtoprogression
ofatheroscleroticlesionsintheaorticroot(Nakashima,1993).Miceonahighfatdiet(21%fatby
weight)forsixweeksshowedmonocyteadhesionandintermittentoccurrencesoffoamcellsinthe
aorticbranch.Aftereightweeksthemiceshowedintermediatelesionscharacterizedbyfoamcells
entanglingwithsmoothmuscleandpotentialformationofacapontopofthelesion.By15weekson
thehighfatdiet,developmentofplaquewasobservedinthemice.Thesemiceshowcharacteristic
developmentofthedisease.Therefore,wewillbeabletotesttheeffectivenessofthenanoparticleto
detectanddiagnosisvariousstages(early,middle,latedescribedin8.1)ofthediseasebytesting
miceinsequentialtimepointsthatareonthehighfatdiet.
Atotalof9apoEmicewillbegivenahighfatdietandthreemicewillbeinjectedwith
targetednanoparticlesateverytimeinterval(six,eightandfifteenweeks).Aftersixweeksofahigh
fatdiet,onemousewillreceivenanoparticlestargetinglipidrichareasusingtyrosine,onemouse
willreceiveSRA1targetingnanoparticles,andonemousewillreceivefibrintargeting
nanoparticles.Thiswillberepeatedfortheremainingmiceateightweeksand15weeks.Also,asa
control,ninewildtypemicewillbeonthesamehighfatdietandthreemicewilleachreceiveadose
ofoneofthenanoparticlesatthesameweekintervalsastheapoEmice.Afterimagingthemiceat1
HR,12HR,24HR,48HR,and96HRintervalswecanobservethediffusionofthenanoparticles
throughoutthemiceandobserveaccumulationatthelesionsite.Basedontheresultswecan
comparetheeffectivenessofeachnanoparticlebasedontheaffinityandprogressionofthedisease.
TheKodakInVivoFXImagerattheHersheyCenterforAppliedResearch(HCAR)canbeused
observetheaccumulationandaffinityofthenanoparticlesthroughNIRI.Thewellbeingofthemice
willbeobservedoverthelengthofthe15weekprocedurebytheanimallabtechniciansatHCAR.
Thelayoutoftestmethodsisgiveninthetablebelow.

44

Table5.TestProcedure
MouseID
Mousetype

Diet

Controlwildtype

Highfat

InjectionTime
Period
6weeks

Controlwildtype

Highfat

6weeks

SRA1

Controlwildtype

Highfat

6weeks

Fibrin

Controlwildtype

Highfat

8weeks

Tyrosine

Controlwildtype

Highfat

8weeks

SRA1

6
7
8
9
10
11
12
13
14
15
16
17
18

Controlwildtype
Controlwildtype
Controlwildtype
Controlwildtype
ApoE
ApoE
ApoE
ApoE
ApoE
ApoE
ApoE
ApoE
ApoE

Highfat
Highfat
Highfat
Highfat
Highfat
Highfat
Highfat
Highfat
Highfat
Highfat
Highfat
Highfat
Highfat

8weeks
15weeks
15weeks
15weeks
6weeks
6weeks
6weeks
8weeks
8weeks
8weeks
15weeks
15weeks
15weeks

Fibrin
Tyrosine
SRA1
Fibrin
Tyrosine
SRA1
Fibrin
Tyrosine
SRA1
Fibrin
Tyrosine
SRA1
Fibrin

45

Targeted
Nanoparticle
Tyrosine

8.6

EconomicAnalysesBudgetandVendorPurchaseInformation

Magnetitenanoparticleswerealreadysynthesizedasstatedinpreviouswork.Oureconomic
analysisreflectsanapproximatecostofthesynthesisofmagnetitenanoparticles,alongwiththe
costsoftheneededchemicalsforthethreemajorbioconjugationschemesselected;lastly,we
includedtravelexpensesandposterpresentation.TheTable5belowpresentsadetailedlistofthe
itemsalongwiththedateofpurchase,supplier,costoftheitem,andrelevantcomments.
Asofsynthesisandprocedures,thematerialscostmayvarydependingonsponsorresources
andavailabilityofequipment.Thisanalysisconsidersthethreepreviouslystatedtargets,
bioconjugationmethodsforeach,andotherlabequipmentrelevanttotheexperiment.Itisimportant
toemphasizeonthedifferentmaterialsneededthatwilltargetthedesiredmoleculesintheselected
stageofthedisease.Evenifallparticlesusemagnetite,andbioconjugationbasedonEDACand
SulfoNHS,therearekeydifferencesinordertoproperlydetectthetargetedmolecules.Forearly
detectionweintendtousetyrosinetodetecthighlipidzones;middledetectionusestheantiMSR1
antibodytodownregulateSRA1macrophagescavengerreceptorsandminimizetheactivityof
macrophages;lastly,latestagedetectionusestPAplasminogentodetecthighfibrinconcentrationin
damagedzone.
Table6.Purchasesinformation
Date
2/17/2015
4/15/2015
3/25/2015
3/25/2015
3/25/2015
3/25/2015
3/25/2015

Item
TransportationtoHersheyMedical
Center
Poster
EDAC1g
SulfoNHS250mg
mPEGamine100mL
Tyrosine50g
AntiMRS1100uL
Labsupplies
Approx.ofmagnetiteNPs
Calciumchloridedehydrate25g
Sodiumhydrogenphosphate100g
Disodiumcitratedehydrate1Kg
Sodiumsilicatesolution1L
TotalofmagnetiteNPs

46

Supplier

PennState

Cost Comments

$113.85 198miles
$60.00
$57.10
$111.00
$41.90
SigmaAldrich(StLouis, $68.90
MO)
$429.00
$50.00
Sponsordonations
$36.50
$25.60
$40.00
$39.00
$141.10

9.0

Final Discussion

Section 9.0.1 Modifications to Statement of Work and DSR Sections

Revisions to the Proposal and DSR Sections 1 through 8 are listed as 9.0.1.X:
9.0.1.1. Introduction - no change
9.0.1.2. Customer Needs no change
9.0.1.3. External Search no change
9.0.1.4. Engineering Specifications no change
9.0.1.5. Concept Generation and Selection no change
9.0.1.6. System Level Design no change
9.0.1.7. Special Topics
The Gantt chart has been updated and included in appendix C.
9.0.1.8 Detailed Design
The test procedure has been updated and included in appendix D.

9.1

Construction Process

The process of creating the nanoparticles was out of the scope of this project. We were given
nanoparticles created by Dr. Adair and his lab for the imaging done in our proof-of-concept. On the
same note, the bio-conjugation process was not physically carried out as of now because of time and
resource limitation. The chemical overview of the conjugation schemes is found in the Production
Process in Section 8.3.
A bench top optical lab for the proof-of-concept was constructed for basic imaging experiments.
A photo of the finished set-up is shown below in figure 19.

Figure 19. Optical set-up for near-infrared imaging

To construct this, a pegboard with various inserts was used. A laser emitting light at 750 nm was
used as the excitation source. A quartz optical cuvette was used to contain the various liquids tested,
as well as the nanoparticles. The detection device in the figure above is a specially modified camera
that allows for the imaging in the near-infrared region. A photodiode was also used in this same
position. It allowed for power measurements to be made, as well as ensuring optimal position of all
components. The camera/photodiode and cuvette must be in a straight line from the source of the
laser. Each component was approximately 20 cm from each other in our set up.
47

To allow particles to be reused and not directly mix with the blood, a holding tube was created.
An approximately 2 inch piece of 3/16 inner diameter Tygon tube was cut. In one end of the tube,
a 1/4 metal cap was fitted to make a seal. This then allowed for filling the tube to be filled with
nanoparticles, water, or other solutions of interest. The holding tube was then inserted into the
cuvette, also containing a liquid, and kept the liquids from mixing.
The camera used for imaging was modified (previously by Dr. Adair) to allow for the imaging of
the near-infrared region of the spectrum. Various filter were used to help eliminate unwanted sources
of light. The filters used in this experiment were 665 nm, 780 nm, 830 nm, and 850 nm; all 50% cutoff below the given wavelength. A 128 MB MemoryStick memory card was used for the storage of
taken images. The camera used is not particular, but it must be able to detect in the near-infrared.
All materials were used with permission from Dr. Adair, with only the metal cap being bought
externally. Other factors to keep in mind is the minimization of outside light sources, as well as the
use of pipets and gloves to transfer the various liquids. The optical bench top-imaging lab was
created as proof-of-concept for the fluorescence of ICG-encapsulated nanoparticles, and a future
clinical system would be much different in appearance.

9.2

Test Results and Discussion

Our prototype served as proof-of-concept design for imaging of near infrared nanoparticles in
blood to detect atherosclerotic lesions using ICG as a dye. The testing phase was based on
quantitative and qualitative analysis of the collected data. The data was divided in two: measures of
intensity of transmitted light by the laser, and qualitative pictures indicating the amount of brightness
related to each configuration.
The experimental setup allowed us to take accurate measurements of the intensity of the light
transmitted through the cuvette-tube system. We mounted a 3-component set over a fixated table to
minimize movement and diminish scattering of the laser beam. The components in the optical setup
are as follows:
1. This is the first component of the experiment, consisting of a 785 nm laser.
2. This consists of a cuvette and a plastic tube both filled up with different liquids as specified
later.
3. This is the measuring part in which a camera is used for qualitative data and a photodiode is
used for quantitative data (intensity of light passing through the system).
In summary, we were able to demonstrate the little transparency of blood to NIR imaging. Also,
we showed that the addition of ICG can serve as a contrast agent for particle detection in the
bloodstream. Thus, the magnetite nanoparticles can serve as a potential mechanism for multimodal
imaging (NIR imaging and CT/MR imaging). Below in figure 20 and 21 are images indicating our
comparison of blood and blood+ICG on the NIR detection range. Both images were taken using a
filter with a 50% cutoff at 850 nm.

48

Figure 20. Blood with water inside the tube

Figure 21. Blood sample with 20% (v/v) ICG dilution.

As expected, this experiment allowed us to verify the possibility of multimodal imaging
using bio-contrast nanoparticles. We also satisfied our customer needs by efficiently providing a
design to demonstrate the effects of ICG in blood at different wavelengths. Other standards such as
DI water and fetal bovine serum (FBS) were also used as control models. All data is included in the
Appendix.

10.0 Conclusions and Recommendations

This project represents a proof-of-concept for a plausible revolutionary diagnostic strategy for
cardiovascular diseases, including coronary artery disease. Our sponsors, Dr. James Adair and Dr.
Larry Sinoway, proposed an idea of using methodologies for targeting cancer with nanoparticles and
applying them in a novel way with cardiovascular imaging. They theorized that near-infrared
spectroscopy could activate nano-sized magnetite nanoparticles and provide a measurable photoacoustic effect. Targeting these nanoparticles toward atherosclerotic lesions could allow physicians
to quickly map the body from the exterior in a matter of minutes before recommending an MRI for
further testing. The particles would then be perfect for use as a contrast agent, without further
dosage requirements. Our sponsors challenged us specifically with finding a viable target for
atherosclerotic lesions and to develop a bio-conjugation scheme to attach our target to the Adair labs
existing nanoparticles.
Our team approached this project as a three step process in which we would (1) identify a
biological marker for ischemic lesions in cardiac blood vessels, (2) design a chemical bioconjugation scheme to attach our new targets to calcium phosphosilicate nanoparticles, and (3)
create a working prototype of the bio-conjugation scheme with atherosclerotic lesion tissue to
perform as a proof of concept.
49

The first stage of the project was completed sufficiently for our sponsors after an exhaustive
literature review of hundreds of papers concerning atherosclerotic lesions. We found so many
targets that we were able to present targets which were likely to identify different stages of disease in
early, moderate, and late stages. These targets were tyrosine, SR-A1 macrophage scavenger
receptor, and fibrin respectively.
Our work on bio-conjugation schemes was greatly assisted by previous research done by the Yang
Group and Adair Group at Penn State University, and existing methods proved to be adequate for
attaching each of our targets successfully to the nanoparticle scheme. Explicitly detailed schemes
are available in sections 8.3 & 8.4.
Finally, we attempted to create a working prototype with the Adair lab nanoparticles.
Unfortunately, the Adair lab is still working to create the theorized particles and experiencing
difficulty in creating colloidally stable particles using this design. Although success is anticipated in
the near future, we were unable to create a full prototype without this key component. To provide
some leverage to the overall design concept of near-infrared spectroscopy, we performed some short
laser-induced fluorescence experiments by placing indo-cyanic green nanoparticles in different
mediums (water, blood, fetal bovine serum) and took images of the emitted light (a cut-off lens filter
was placed on a camera).
Conclusively, we were able to successfully identify targets as well as bio-conjugation schemes for
each. This was the main goal of our design project and our sponsors have informed us that they are
satisfied with the work produced. Further work will be expanded in the coming summer into the
nanoparticle fabrication, and our designs will be used as a foundation for new prototypes. Once full
nanoparticle complexes have been synthesized with attached targets, they will be tested in vitro, in
vivo in mice, and in many years, eventually in humans.

11.0 Self-Assessment (Design Criteria Satisfaction)

11.1 Customer Needs Assessment
Our team rates our performance to meet customer needs as an 8/10. The customer needs
identified in the section 2.0 in order of importance (based on AHP determined weight) are listed
below.
1. Unique affinity
2. Toxicity
3. Ease of clearance
3. Cost
4. Ease of manufacturing
The scope of the project mainly focused on reviewing and proposing potential biomarkers and
generating conjugation schemes for the proposed targets. Though we were unable to test and confirm
various needs such as ease of clearance and manufacturing, we focused on identifying and choosing
targets and concepts that have been shown to meet these criteria in various studies. All biomarkers
that were reviewed have been repeatedly manufactured in laboratory conditions, shown to be nontoxic and cleared in vivo mice models. These studies also showed proof of significantly higher
accumulation of the targeted nanoparticle in the specified tissue compared to the untargeted
nanoparticle. For example, tyrosine has shown to selectively uptake in the lipid sections of
atherosclerosis (Lewis, 2011). In addition, the chosen bio-conjugation scheme, using maleimide, has
been shown to be an effective method for conjugating proteins, such as tPA, to the surface of our
nanoparticles using a PEG tether (Adair, Unpublished). The cost of the nanoparticles have been
estimated in table 6 above to be approximately $141.10 to manufacture the three types of
50

nanoparticles. This amount has been determined to be feasible as the materials should yield large
amounts of the nanoparticles for testing in vitro and in vivo. Therefore, we were able to accomplish
the goal of the project while focusing on the customer needs and meeting all requirements.

11.2 Global and Societal Needs Assessment

The scope of our project was rather limited compared to the long-term plans for this research,
therefore our team is rating ourselves 5/10 in terms of meeting global and societal needs. We
successfully designed a unique and multifaceted method for detecting atherosclerotic lesions in
patients with CAD. This method involves the use of three biological targets, each unique to a
different stage of CAD. Through this strategy, a larger percentage CAD cases will be able to be
detected since atherosclerosis in a variety of stages can be targeted. Since our method of using
magnetite nanoparticles does not require a large amount of material, it is sustainable and
environmentally friendly.
Our efforts lay the groundwork to successfully diagnose CAD in an affordable and effective
manner. Clearly this ability to diagnose CAD will greatly benefit society since CAD is currently one
of the leading causes of death in men and women. Moving forward, animal testing will be required
to further test the efficacy of this method. Throughout future experiments, we have ensured that the
researchers will follow the ethical standards set forth by the Biomedical Engineering Society.

51

References
Adair,JamesHetal.2005.COLLOIDALLESSONSLEARNEDFORDISPERSIONOF
NANOSIZEPARTICULATESUSPENSIONSwithContributionsfromNSFParticulate
MaterialsCenter,MaterialsResearchInstitute,MaterialsScience&Engineering,The
PennsylvaniaStateUniversity,:93146.
Adair,JamesH.2015.PersonalCommunication.StateCollege,PA.
Adair,JamesH.,MylisaP.Parette,ErhanI.Altinolu,andMarkKester.2010.Nanoparticulate
AlternativesforDrugDelivery.ACSNano.
Almer,Gunteretal.2014.Interleukin10CoatedNanoparticleSystemsComparedforMolecular
ImagingofAtheroscleroticLesions.421122.
Altinolu,ErhanI.,andJamesH.Adair.2010.NearInfraredImagingwithNanoparticles.Wiley
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Ankri,Rinatetal.2014.GoldNanorodsasAbsorptionContrastAgentsfortheNoninvasive
DetectionofArterialVascularDisordersBasedonDiFfUsionReFlEctionMeasurements.
Annapragada,Ananth.2015.AdvancesinNanoparticleImagingTechnologyforVascular
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Barth,BrianM.etal.2010.BioconjugationofCalciumPhosphosilicateCompositeNanoparticles
forSelectiveTargetingofHumanBreastandPancreaticCancersinVivo.ACSNano.
BrileySaebo,KarenC.etal.2011.TargetedIronOxideParticlesforinVivoMagneticResonance
DetectionofAtheroscleroticLesionswithAntibodiesDirectedtoOxidationSpecificEpitopes.
JournaloftheAmericanCollegeofCardiology.
CenterforDiseaseControl.2014.HeartDiseaseFacts.
http://www.cdc.gov/HeartDisease/facts.htm(February10,2015).
Chandramouli,S,SSanjana,andSSwathi.UseofSuperParamagneticIronOxideNanoparticlesin
theTreatmentofAtherosclerosis.IFMBEProceedings46.
Duivenvoorden,Raphaletal.2014.AStatinLoadedReconstitutedHighDensityLipoprotein
NanoparticleInhibitsAtheroscleroticPlaqueInflammation.Naturecommunications.
FDA.2014.Development&ApprovalProcess(Drugs).
http://www.fda.gov/drugs/developmentapprovalprocess/(February8,2015).
Hansson,GranK.2005.Inflammation,Atherosclerosis,andCoronaryArteryDisease.New
EnglandJournalofMedicine(352):168595.
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Hayworth,Douglas.2015.SulfhydrylReactiveCrosslinkerChemistryTle.ThermoScientific.
http://www.piercenet.com/method/sulfhydrylreactivecrosslinkerchemistry(November2,
2015).
Hermanson,GregT.2013.BioconjugateTechniques.3rded.Elsevier.
http://www.sciencedirect.com/science/book/9780123822390.
Jarrett,Benjamin,andAngeliqueLouie.2006.NanoparticlesforImagingAtheroscleroticPlaque.
Kim,Jaeyun,LanCao,andDavidJ.Mooney.2011.NanoparticleTargetingtoIschemiafor
ImagingandTherapy.
Klegerman,M.E.,Zou,Y.,andMcpherson,D.D.2008."FibrinTargetingofEchogenicLiposomes
withInactivatedTissuePlasminogenActivator."JournalOfLiposomeResearch,18(2),95112.
Lewis,DanielR.,KubraKamisoglu,AdamW.York,andPrabhasV.Moghe.2011.PolymerBased
Therapeutics:NanoassembliesandNanoparticlesforManagementofAtherosclerosis.Wiley
InterdisciplinaryReviews:NanomedicineandNanobiotechnology.
Lipinski,MichaelJetal.2009.MacrophageSpecificLipidBasedNanoparticlesImproveCardiac
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Malyutin,AndreyGetal.2015.ViruslikeNanoparticleswithMaghemiteCoresAllowfor
EnhancedMRIContrastAgents.ChemistryofMaterials(27):32735.
McCarthy,JasonR.,EthanKorngold,RalphWeissleder,andFaroucA.Jaffer.2010.ALight
ActivatedTheranosticNanoagentforTargetedMacrophageAblationinInflammatory
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Mulder,WillemJM,FaroucAJaffer,ZahiAFayad,andMatthiasNahrendorf.2014.Imagingand
NanomedicineinInflammatoryAtherosclerosis.ScienceTranslationalMedicine6(239):111.
Nakashima,Yutakaetal.1993."ApoEDeficientMiceDevelopLesionsofAllPhasesof
AtherosclerosisThroughouttheAretialTree."Atherosclerosis,ThrombosisandVascular
Biology.
Nahrendorf,Matthiasetal.2006.NoninvasiveVascularCellAdhesionMolecule1Imaging
IdentifiesInflammatoryActivationofCellsinAtherosclerosis.Circulation.
Wang,Shu,GuigenLi,andZhaoyangFan.2014.NanoparticleBasedDeliverySystemwith
OxidizedPhospholipidsasTargetingLigandsforthePrevention,DiagnosisandTreatmentof
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53

Weissleder,Ralph,MatthiasNahrendorf,andMikaelJPittet.2014.ImagingMacrophageswith
Nanoparticles.Naturematerials.

54

AppendixA:Resumes
Theresumesofalltheteammemberaregivenbelow.

55

56

KENNETH
SURANO

(724) 456-6997
suranoken@gmail.com
111 N. 2nd Street, Greenville, PA 16125
www.linkedin.com/in/kennethsurano

Academicallypreparedwithhandsonexperienceinahighlyregulatedindustry
Fastlearnerwhoismotivatedtodeliverinnovativeresultsquicklyandefficiently
Abletoprovidetechnicalsolutionstoengineeringproblemsandcommunicatethemeffectively
Exceptionalcomputerskills,withanabilitytodevelopnewsolutionsandideas

Education
B.S.inBiomedicalEngineering,ChemicalEngineeringOption
Summary
ThePennsylvaniaStateUniversity,UniversityPark,PA
ExpectedGraduation:May2015
GPA:3.32/4.00
RelevantCoursework:
BiofluidMechanics
SystemsAnalysis

Thermodynamics

BioengineeringResearchandDesign

TransportPhenomena
BiomedicalInstrumentation

Work Experience

NewProductEngineeringIntern
WernerCo.,Greenville,PA
Summer2013,Summer2014

Designedanddraftedcompanydocumentsfortestingproceduresofdifferentstandardstobeusedatfourinternationalanddomesticfacilities

Teamedwithproductengineersandlabtechnicianstotestladdersandclimbingequipmenttopublishedsafetycodesandstandards
PreparedwrittenreportsoftestingresultsanddistributedthemthroughENOVIASmarTeam
Updatedpreviousanddatedbusinesscorrespondenceandreportingfrom1967topresenttohelpintegratetheseintotheuptodatecomputersystem

DeskAttendant
PennStateStrengthandFitness,UniversityPark,PA
January2012Present

Facilitatetheentryandefficiencyofpatronsacrossthreefitnesscenterlocations
Communicateconcerns,issues,andquestionswithpatronsinacustomerserviceenvironment

Computer Skills
MicrosoftOffice
SolidWorks

MATLAB

COMSOLMultiphysics

ENOVIASmarTeam

Minitab

Activities/Honors

PrescottandMaryWillamanScholarship,2014
StruthersFamilyPhiKappaTauResidentScholarship,2013
MostInnovativeDesign,ArcelorMittalSponsoredEngineeringDesignResearchProject,2012
Member,PhiKappaTauFraternity
57

58

204HamiltonHall
UniversityPark,PA16802

G R E G O R Y S.L Y N N

23098SweetbayLane
California,MD20619

//gregory.s.lynn@gmail.com//

NanoMedicineBioengineerpioneeringsolutionstomedicalchallengesthroughtheinvestigationofnanotechnologiesandtraditional
engineeringskills;Determinedtoresearch,design,andprototypeuniquetherapeuticdevicesandtreatmentswithateamofmotivated
innovators.

EMPLOYMENT
EngineeringTechnician,Intern

UnitedStatedNavy,DoD

Summers20092012

SecretSecurityClearancegranted.
ModeledUAVdroneinterferencealgorithms,communicationslaboratorymaintenanceofEMtransceivers,electricalengineering
prototyping.
Installedfiberopticcable,analyzedrobustnessofcybersecurity&physicalsecurity,performeddataqualityscreening.Troubleshot
machinecodeprogramming(Assembly,VisualBasic).
ExtensiveuseofMicrosoftExcel,Word,Access,Outlook,andwikiblogging.
Developmentofsolderingskills.

ResidenceHallAssistant

ThePennsylvaniaStateUniversity

Summer2013Present

Promotesacademicexcellenceandstudentinvolvementthroughweeklyworkshopsandcommunityprogrammingwithateamof12
for600students.
Respondstocrisisscenariosandcoordinatedeffortswithpoliceauthoritiestoenforceuniversitypolicy.

EDUCATION
UniversityPark,PA

ThePennsylvaniaStateUniversity

Fall2011May2015

B.S.E.inBiomedicalEngineeringMay2015//GPA:3.4

CertifiedbytheAccreditationBoardforEngineeringandTechnology(ABET)

MinorinNanotechnology
UndergraduateCoursework
CoreEngineering:DifferentialEquations,CalculusI,II,&III,LinearSystems,Static&FluidMechanics,Thermodynamics
Bioengineering:Cell&MolecularBiology,OrganicChemistry,TissueEngineering,Biomaterials,SurfaceInteractions,
TransportPhenomena,ReactionKinetics
Nanotechnology:FabricationofNanomaterials,SemiconductorDesignFundamentalsMicroelectromechanicalSystems
(MEMS),BiomimeticNanodesign

TECHNICALEXPERIENCE
AcademicLabExperience

BasicPhysiology
Studyofanimalphysiology,cardiacresponse,nervefunction,reactiontodrugexposure,Primarydissectionoffrogs.
PhysiologicalSimulation
ProgrammedsimulationsofcontrolsystemstheoryinMATLAB,e.g.OttoFrankWindkesselmodelforbloodcirculation
circuits.
COMSOLMultiphysicsModelling
COMSOLbaseddesignandsimulationofmultiphysicsanalysisoftissuemechanicsandtransportphenomena(e.g.heat
transferincryofreezingofwarts,lightscatteringinBilibedjaundicetreatment,celltransportandplaqueaccumulationinthe
carotidartery).
BiomedicalMeasurements
Basicelectricaltheory,operational&differentialamplifierdesign,construction,andutilization(e.g.HighPass&LowPass
Filters,Electrocardiogram).

Projects

UnmannedAerialVehicle
ModifiedanRCplanewithaPiccoloautopilot,avionicscontrol,andwebcamtocompeteinthe2009and2010Associationfor
UnmannedAerialSystemsInternational(AUVSI)searchandreconnaissancecollegiatecompetitionsatPatuxentRiverNAS.
59

60

61

AppendixB:UpdatedLiteratureReview
Below is a summary of the biomarkers identified and researched for CAD.

EarlyStageTargets(initialstagesofplaque):
Evansblueanalogrecognizingendotheliumdysfunction
Synthesizedpolymericdrugcarrierwithevansblueanalogasprobingunitforendotheliuminjury.
Specificallyadsorbedagainsttheendotheliuminjuredsiteinextractedporcineaorta
VCAM1
Expressedonendothelialcellsduringinflammation;priortovisibleonsetofdisease
Useofphagedisplaypeptidesonnanoparticleshoweda12foldincreaseintargettobackgroundratios
v3
Integrins,suchas v3,areassociatedwithangiogenesis,andatheroscleroticlesionsarehighlyvascular
comparedtonormalvessels.
Angiogenicbiomarkersareinducedinvasculatureinresponsetocholesterolfeeding,whichresultsin
earlyandcriticalexpansionofthevasavasorumtosupportplaquegrowth.
v3targetednanoparticlesshowedreceptorbindingspecificitythroughinvivoexperimentation.
Tyrosine(lipidrichareatargeting)
Thedevelopmentofatheroscleroticplaqueisinitiatedbythedepositionoflipidrichlipoproteinsin
susceptiblebutstillprelesionalareasofthearterialwall.
Bindingthelipidrichareasinatheroscleroticplaquecouldhelptodiscriminatetheunstableplaquefrom
stableplaque,asalargelipidcoreincombinationwithathinfibrouscapstronglycorrelateswithplaque
instability.
Lipophilicproperties,smallparticlesize,andprolongedcirculationallowtyrosinefunctionalizedmicelles
topenetrateandaccumulatewithintheplaqueafterintravenousadministration.
Targetingthemicelleswithanaminoacidisadifferentmechanismthantargetingusingmorecomplex
moleculessuchasantibodies,proteins,orpeptides.
MiddleStageTargets(plaqueformation,clot):

SRA1(MacrophageScavengerReceptor)
Macrophagesareassociatedwithplaquesvulnerabletorupture
InvivoMRIshowedtargetedimmunomicellesshowed79%increaseinsignalatatheroscleroticaortas
comparedtoonly34%foruntargeted
Pegylated,fluorescent,paramagneticmicellesdevelopedandconjugatedwithmacrophagereceptor
specificantibodies
24hpostinjectionupto200%signalenhancement
Macrophages(CD68,MAC3)
MacrophageshaveSIGNR1dextranreceptors.
Dextrancrosslinkedmagnetitenanoparticleshavebeenshowntohave
CD36(MacrophagescavengerreceptorB)
Maybeapotentialbiomarkerforidentificationoflesionspronetorupture
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 Gdlipidbasednanoparticleswereusedtotargethighrisklesions;MRIimagingoftheaortainapoE
miceshowedasignificantcontrasttonoiseratioincreasewiththetargetedNPversusthecontrol
untargetedNP
OxidizedLDL(OxLDL)
Predominantfactorinatherogenesis
relatedwithinitiationandprogressionofatheroscleroticplaques
associatedwithdestabilizationofplaque
Inducesmacrophageapoptosiswhichmightaccelerateplaquerupture(duetoaformationofanecrotic
coreofoxidizedlipidsandmacrophages)
MDA2orIK17targetednanoparticleshavebeenobservedtobetakenupbylipidrichfoam
cell/macrophages
Phosphatidylserine(PS)atsurfaceofmacrophageburdensandapoptosis
Apoptosisandmacrophageburdencorrelatewithplaquevulnerability.Thesecellsarecharacterizedby
theexposureofphosphatidylserine(PS)attheirsurface.
smallmicellarfluorescentannexinA5functionalizednanoparticleforMRIT1weightingcontrastagent
Contrastenhancementpersistedinmiceupto7daysafterinjection
ProvedtobebetterthanMacrophageScavengerReceptorfortargetingatsimilardoses
Substratepermeablepolymernanocarriers(PNC)loadedwithcatalase(adetoxifyingenzyme)
Thetargetistheendothelialcells,toprotectagainstvascularoxidativestress
Vascularoxidativestressisimplicatedinacutelunginjury(ALI),ischemiareperfusion,stroke,
myocardialinfarction,inflammation,andothers
LateStageTargets(pastplaquerupture):
Fibrin
Rupturesinitiatehemostasisandactivatethrombin.Subtleclottingcanbeindicatedbythedepositionof
fibrinogeninside/onsurfaceofplaques.
Clotbindingpeptidecysteineargineineglutamicacidlysinealanine(CREKA)
CollagenIVtargetingpeptideKLWVLPK
Exploitsvascularbreachesbytargetingtheunderlyingbasementmembrane
Utilized60nmnanoshellstodeliverpaclitaxelPLAconjugatesunderslowreleaseconditions(2weeks).

63

Appendix C: Updated Gantt chart

This Gantt chart was last updated on March 31, 2015.

64

This Gantt chart was last updated on May 5, 2015.

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Appendix D: Updated test procedures

As it was unfeasible, due to time and costs, to follow the test procedure in section 8.0, we decided
to perform preliminary tests on the nanoparticles to show proof of concept. The test procedures are as
follows.
Bovine blood, serum and water will be used to measure and record images of the intensity of light
passed through a 1 cm cuvette containing the blood, serum and water. To show the excitation of the
indocyanine green (encapsulated in the nanoparticles) can be visible through the blood, serum and water
we inserted a tube containing the nanoparticles in the cuvette and measured the response using a camera
with various cut off filters. We compared these results to the cuvette with an inner tube filled with water
and free indocyanine green to compare the differences. The water tube was used as a control for the
effects of the tube on the recordings. The free indocyanine green was used to support the proof of concept
the indocyanine green will degrade and is not effective for imaging if it is not encapsulated. The table
below summarizes the procedure performed. Two pictures were take per filter (e.g. two pictures were
taken for the water cuvette with water tube using 665 nm cut-off filter).
The setup was mounted into a board to ensure the laser, cuvette and camera remained unmoved and a
level was used to determine the board laid flat on the table (see figure 22 below); all pictures were taken
in dark conditions.
Table 7. Procedure for measuring the results of the 785 nm laser light through the cuvette and tube. The
table summarizes what medium the cuvette and tube contain and the filters used to image the cuvette and
tube.
# of
Cuvett
Cut off filter
Inner tube
pictures
e
(wavelength of cut-off)
(per filter)
Water
665 nm, 780 nm, 830nm, 850nm
2
Water
Free indocyanine green
665 nm, 780 nm, 830nm, 850nm
2
Indocyanine green encapsulated NP 665 nm, 780 nm, 830nm, 850nm
2
Water
665 nm, 780 nm, 830nm, 850nm
2
Bovine
Free indocyanine green
665 nm, 780 nm, 830nm, 850nm
2
serum
Indocyanine green encapsulated NP 665 nm, 780 nm, 830nm, 850nm
2
Water
665 nm, 780 nm, 830nm, 850nm
2
Bovine
Free indocyanine green
665 nm, 780 nm, 830nm, 850nm
2
blood
Indocyanine green encapsulated NP 665 nm, 780 nm, 830nm, 850nm
2

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Figure 22. Schematic of experimental setup to visualize the indocyanine green encapsulated particulates
under excitation of near infrared (785 nm).

67