Optically
and
acoustically
triggerable
sub-micron
phase-change
contrast
agents
for
enhanced
photoacoustic
and
ultrasound
imaging
Shengtao
Lin
a,
Anant
Shah
b,
Javier
Hernández-Gil
c,
Antonio
Stanziola
a,
Bethany
I.
Harriss
c,
Terry
O.
Matsunaga
d,
Nicholas
Long
c,
Jeffrey
Bamber
b,
Meng-Xing
Tang
a,*
a
DepartmentofBioengineering,ImperialCollegeLondon,London,UK b
JointDepartmentofPhysicsandCRUKCancerImagingCentre,TheInstituteofCancerResearchandTheRoyalMarsdenNHSFoundationTrust, London,England,UK
cDepartmentofChemistry,ImperialCollegeLondon,London,UK d
DepartmentofMedicalImaging,UniversityofArizona,Tucson,AZ,USA
ARTICLE INFO
Articlehistory:
Received14October2016
Receivedinrevisedform10March2017 Accepted8April2017
Keywords:
Phase-changecontrastagent Droplet
Microbubble Ultrasound Photoacoustic
Optical/acousticvaporisation Multispectraloptoacoustictomography (MSOT)
ABSTRACT
Wedemonstrateaversatilephase-changesub-microncontrastagentprovidingthreemodesofcontrast
enhancement:1)photoacousticimagingcontrast,2)ultrasoundcontrastwithopticalactivation,and3)
ultrasoundcontrastwithacousticactivation.Thisagent,whichwename‘Cy-droplet’,hasthefollowing
novelfeatures.Itcomprisesahighlyvolatileperfluorocarbonforeasyversatileactivation,anda
near-infraredopticallyabsorbingdyechosentoabsorblightatawavelengthwithgoodtissuepenetration.Itis
manufacturedviaa‘microbubblecondensation’method.Thephase-transitionofCy-dropletscanbe
opticallytriggeredbypulsed-laserillumination,inducingphotoacousticsignalandformingstablegas
bubblesthatarevisiblewithecho-ultrasoundinsitu.Alternatively,Cy-dropletscanbeconvertedto
microbubblecontrastagentsuponacousticactivationwithclinicalultrasound.Potentiallyallmodesoffer
extravascularcontrastenhancementbecauseofthesub-microninitialsize.Suchversatilityofacoustic
and optical ‘triggerability’ can potentially improve multi-modality imaging, molecularly targeted
imagingandcontrolleddrugrelease.
©2017TheAuthors.PublishedbyElsevierGmbH.ThisisanopenaccessarticleundertheCCBYlicense
(http://creativecommons.org/licenses/by/4.0/).
1.Introduction
Microbubbleultrasoundcontrastagentshavebeenwidelyused asavalueableimagingtoolinclinicalradiologyandcardiology[1]. Atthesametimetherecontinuestobeextensiveresearchefforts focusing on new paradigms for contrast-enhanced ultrasound imaging (CEUS) [2,3], and microbubble-mediated therapy [4,5]. However, these micron-sized microbubbles are limited to the intravascularspace[6].Asameansofexploringtheextravascular space, sub-micron phase-change droplets show widespread interest[7,8].Theycanpotentiallyextravasatethe‘leaky’cancerous vasculatureintointerstitium[9] priortovaporisation,providing extravascularcontrastenhancementuponthephasetransitionof dropletstoechogenicmicrobubbles.Vaporisationcanbetriggered eitheracousticallyor,fordropletscontaininganopticalabsorber,
optically. The optical activation of phase-change droplets can providephotoacousticcontrastenhancement[10].
Existingstudies onsuchdual-modality contrastagents have demonstrated the generation of both optical and ultrasound contrastafteropticalactivation[10–16].Howeverthesestudiesdid not explore the option of acoustic activation. This would add versatilityofvaporisationtriggering,offeringnewpossibilitiesin dual mode imaging, molecular imaging and drug delivery. Furthermore,highboilingpoint(b.p.)perfluorocarbonswereused inthesestudies,i.e.,perfluoropentane(b.p.29C)[10–12,17,18]and perfluorohexane(b.p.56C)[13,14,19].Alowb.p.maybepreferred, tominimiseun-wantedbioeffects[20],especiallywhenactivating indeepertissues.AlthoughDoveetal.[21]engineeredoptically triggered droplets using a low b.p. perfluorocarbon (decafl uor-obutane,DFB,b.p. 2C),theopticalabsorberemployed(i.e.gold spheres) may limit the imaging depth due to the weakly penetratingplasmonicresonancewavelength(i.e.535nm).
In this study, we have employedan easily vaporisable sub-micron, phase-shift droplet made with a highly volatile *Corresponding author. Current address: Department of Bioengineering,
ImperialCollegeLondon,London,SW72AZ,UK.
E-mailaddress:[email protected](M.-X.Tang).
http://dx.doi.org/10.1016/j.pacs.2017.04.001
perfluorocarbonandformulatedviacondensationofpre-formed, lipid-shelled microbubbles. This has previously shown great promise as an extravascular contrast agent for diagnostic and therapeuticultrasound[6,8,22–26],includingpromiseforeventual clinical translation [27,28]. In this paper, we provide the first demonstrationofitspotentialforphotoacousticimaging,andthus asaversatilethree-modeagent.Wedevelopedandcharacteriseda newsub-micronphase-changedroplet(Cy-droplets)by incorpo-rating a near-infrared (NIR) optical absorber, i.e., a Cyanine7.5 bioconjugate,intotheprecursormicrobubblemembranesbefore condensation.Cyanine7.5hasapeakabsorptionatawavelengthof 788nm,offeringrelativelygoodtissuepenetration[29].Herewe demonstratethat theCy-dropletphase transition caneither be triggeredbyapulsedlasertoproducesubstantialphotoacoustic signalenhancementaswellassubsequentultrasoundcontrast,or be triggered acoustically using clinical ultrasound pulses to provideconventionalultrasoundcontrast.
2.Methods
2.1.Cyanine7.5bioconjugationsynthesis
Cyanine7.5 NHS ester (Lumiprobe GmbH, Germany) was
conjugatedto the amine terminus of a commercially available phospholipidwithaPEG2000spacer(DSPE-PEG(2000)-NH2)viaa NHS-mediatedcouplingreactiontoaffordthetargetCyanine7.5 dye-functionalised phospholipid (DSPE–PEG(2000)–Cyanine7.5) after purification by dialysis. DSPE–PEG(2000)–Cyanine7.5 (Fig.1)wascharacterisedbyvariousanalyticalandspectroscopic techniques(seeSupportinginformation,Figs.S1–S2).Inatypical reaction, DSPE–PEG(2000)–NH2 (3.0mg, 1.1
m
mol) and 6m
L of triethylamine were dissolved in 120m
L of dry DMSO. To this solution,120m
LofDMSOcontainingCyanine7.5NHSester(1.7mg, 2.2m
mol)wasaddeddropwise.Theresultingmixturewasallowed toreactatroomtemperatureovernightundercontinuousstirring. Distilled water was then added to the reaction mixture. The solutionwascentrifuged,andthesupernatantwaspassedthrough a0.45m
mfiltertoremoveinsolubletraces.Thesupernatantwas then dialysed using a Spectra-Por1 Float-A-Lyzer1 G2 (Sigma-Aldrich,Milwaukee,Wis)(MWcutoffof3.5–5kDa)againstwater (3500mL). The dialysate, containing the pure product, was lyophilisedandtheresiduedriedinvacuooverP2O5.Alllipidsused inthisstudywerepurchasedfromAvantiPolarLipids,Inc.,USA.2.2.Cy-dropletssynthesis
Thelipid-coated, DFB-filled precursor Cy-microbubbles were manufacturedusingamodifiedformulationdescribedbySheeran etal.[6].Briefly,thelipidmixtureconsistedof 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero -3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]
(16:0 PEG2000 PE) and DSPE-PEG(2000)-Cyanine7.5 9:0.8:0.2 (Fig.2),m:m:m(totallipidconcentrationof0.85mg/mL)dissolved inasolutionofpropyleneglycol,glycerol,andphosphate-buffered saline(PBS)(15/5/8,v/v/v). TheCyanine7.5concentrationinthe lipidsolutionwas13nM.Next,1mLoftheresultinglipidsolution wassealedina2mLglassvialandtheheadspacewasthenpurged with DFB at room temperature. The amount of DFB used to synthesisetheCy-droplets(Fig.2)wasapproximately8.810 10 mLperparticle.TheprecursorCy-microbubbleswereproducedvia mechanical agitation. Finally, the Cy-droplet emulsion was obtainedbycondensingCy-microbubblesusingthemethodofLi etal.[22].
2.3.Controls
Controlsamplesincludedsixgroups:(1)precursor Cy-micro-bubbles, (2) blank-microbubbles, (3) blank-droplets, (4) Cy-solution, (5) blank-droplets in Cy-solution, (6) deionised water (used to dilute all the experiment samples). The lipid-shell compositionsofbothblank-microbubbles(precursor)and blank-dropletswerepreparedinanidenticalfashionexceptforthelipid composition,whichconsistedofDPPC,16:0PEG2000PEinamolar ratio of 9:1. The Cy-solution was prepared using a similar procedure to the precursor Cy-microbubble lipid solution, by substitutingthelipidmixturewithCyanine7.5NHSesterpowder anddispersingintheaforementionedpropyleneglycol,glycerol, andphosphate-bufferedsalinediluentmixture.Theconcentration ofCyanine7.5dyewaskeptthesameacrossallthecontrolsand Cy-dropletemulsion.
2.4.CharacterisationofprecursorCy-microbubblesandCy-droplets
TheprecursorCy-microbubblesandCy-dropletswereobserved usingbothbright-fieldopticalandconfocalmicroscopy.Confocal microscopy(LeicaSP5DMI6000CS,60objective)wasoperated tolocatethefluorescencefromprecursorCy-microbubblesand Cy-droplets.Duetoresolutionlimitationsofthosemicroscopes,only size outliers of Cy-microbubbles and Cy-droplets could be visualised todetermine opticalappearance and the location of fluorescence.Onehundred
m
Laliquotsofdiluted(1:100)stock Cy-microbubbles and Cy-droplet samples were imaged at a plane through the cross section of the samples. The imaging slice thicknesswassetto0.76m
m.Forbright-fieldmicroscopy(Nikon Eclipse 50i, 40objective), 10m
L diluted samples were first introducedintoa haemocytometerandthen sized andcounted accordingtotheprotocoldetailedinSennogaetal.[30].Thesize distribution of Cy-droplets was measured using dynamic light scattering (DLS, MalvernNano ZetaSizer, UK). Before measure-ment,theDLSwas calibratedusingLatexparticleswithamean diameter of 750nm. Following calibration, 10m
L of the stock Cy-droplet emulsion sample was dilutedin 90m
L milliQ water(milliQ,Canada) in order tomeasure the droplet size.The ab-sorptionspectraoftheCy-dropletlipidsolutionswerethen mea-suredusingaUV/VISspectrometer(Lambda25,PerkinElmer,UK).
2.5.Preparationoftissuemimickingphantomforin-vitroexperiments
Two types of tissue mimicking (TM) agar phantoms were prepared for optical vaporisation experiments. A Tubing-TM phantom(Fig.3(1))wasusedforphotoacousticsignalacquisition by embedding a semi-transparent silicone tube (ID=1.5mm OD=1.9mm, Harvard Apparatus, UK) in the centre of the cylindrical agar-intralipid TM phantom (diameter=15mm, length=10cm).Theagar-intralipidgelwasmanufactured follow-ingtheprotocoladaptedfromMadsenetal.[31].Briefly,the agar-intralipid solution was made of 1.5% w/v agar powder (Fisher Scientific,UK)and1%v/vintralipid(20%emulsion,Sigma,UK)in deionised and distilled water. Anotherdispersion-TM phantom (Fig. 3(2)) with the same geometry was used for ultrasound
contrastmeasurementsbeforeandafterlaserillumination.Itwas formulatedbydispersingtheCy-dropletemulsionat0.25%v/vin agar-intralipidgelat36Cbeforegelation.Theimmobilityof Cy-droplets in thedispersion-TMphantom allowedseparate ultra-soundimagingbeforeandafterlaserscan,necessarybecausethe MSOTsystememployedwasincapableofultrasoundimaging.
2.6.Photoacousticandultrasoundimagingexperimentsetup
Forthephotoacousticimagingwithopticalvaporisation,theTM agarphantomswerescannedwiththeMSOTsystem(inVision 256-TF, iThera Medical).Cy-droplets and the six controls described previouslyweredilutedto10% relativetostocksolutionin the distilled/deionised water so as to keep the concentrations of Cyanine7.5dyeidentical.Thetemperatureofthewaterbathwas heldconstantat34Cthroughoutallopticalvaporisation experi-ments.ThedilutedCy-droplet solutionwas introducedintothe tubing-TMphantom,andthecross-sectionswerescannedatthree
Fig.2.SchematicshowingthecompositionofCy-dropletcontrastagent.
positions(20mm apart)alongthelongitudinaldirection (Fig.3 (1)).Ateachposition,photoacousticimagingwasperformedata singlewavelength788nm(peakabsorptionofCyanine7.5)witha fluenceof22.6mJ/cm2usingonepulse(10nspulses,10Hzpulse repetitionfrequency)perimage,resultingin30sacquisitiontime. Foraccurateultrasoundcontrastassessmentbeforeandafterlaser illumination, the problem of imaging a potentially mobile Cy-dropletsolutionhadtobeovercome.Toachivethis,the dispersion-TMphantomwasimagedtwice(Fig.3(2)).ToactivateCy-droplets ladenin the phantom,half of thedispersion-TM phantomwas scannedlongitudinally(alongthephantom’scylindricalaxis)with 5spulsedlaserilluminationateachposition,withapproximately 5cmtotalscanningdistanceand2mmstepsize.Theotherhalfof the phantom was used as control. Cross-sectional ultrasound imagingofbothhalvesofthesamephantomwasperformedwitha VerasonicsV1system (Verasonics,USA) equippedwithan L7-4 (ATL,USA)probe.
For ultrasound imaging with acoustic vaporisation, focused pulses (8MHz,10-cycles,3.39MPa, mechanical index (MI)=1.2, pulse-repetition-frequency=14.3kHz,totaldurationofexposureis 8.8ms)transmittedfromaclinicallineararrayprobeL12-5(ATL, USA) were applied to activatethe Cy-droplets. The ultrasound contrastenhancement was quantifiedusing a custom designed ‘imaging-activation-imaging’ sequence [22,32] on a Verasonics Vantage 256 research platform. Single cycle, low amplitude ultrasound at 4.5MHz (plane-waves of 15-angle spatial com-pounding,106.1kPa,MI=0.05)wasusedateachimagingstepto estimatetheultrasoundsignallevelfromthecontrastagentbefore and after Cy-dropletactivation. Thesame amountof stock Cy-dropletandblank-dropletemulsionwasintroducedintoa2Lwater tank(Fig.3(3))filledwithwaterandequilibratedto37C[33]to achievea finalconcentrationof approximately106droplets/mL. Beforeeachacquisition,thewaterwasmixedtoachievearelatively uniformdistributionofdroplets.Acousticabsorberswereusedto linethewatertanktoreduceultrasoundreflections.
2.7.Photoacousticimagebeamforming
Thephotoacousticimageswerepresentedafterbeamforming the raw radiofrequency (RF) data extracted from the MSOT ultrasound transducer using a customised Matlab (Mathworks, USA)program.Animagewasformedbyapplyingatemporaldelay foreachchannelaccordingtodifferentpositionsalong the270 concaveultrasoundtransducerarrayfollowedbysummingeach image component over all the 256 channels. The geometry parameters were applied according to the MSOT ultrasound transducerdesigndescribedbyDimaetal.[34].
2.8.Dataanalysis
Forthephotoacousticexperiment,boththerawRFdataandthe beamformed photoacoustic images were used to measure the relativephotoacousticsignallevels.Thefirst-pulseresponseofthe photoacousticsignalwaspresentedalongwithsixcontrols.Inthe caseofthebeamformedimages,themaximumpixelmagnitudein theregionofinterest(ROI)wasusedasameasureoftherelative photoacousticimagesignalgeneratedbyCy-dropletvaporisation. For ultrasoundechosignalevaluation,themeanimagepixel magnitudeintheselectedROIwasusedtocomparetheechosignal levelbeforeandaftertheactivationofCy-droplets.Forultrasound imagingwithacousticactivation,anROIwaschosenwithinthe Cy-dropletactivationarea(thefocalzone)andusedforanalysingthe signalbothbeforeandafteractivation.Thedifferenceinmeanpixel magnitude within the ROI before and after the activationwas calculated.
Forstatisticalanalysis,eachexperimentalresultwasproduced byatleastthreeacquisitions.Student'st-testwasusedtocompare thestatisticaldifferencebetweengroupswithp>0.05considered tobenotsignificantlydifferent.
3.Results
3.1.Cy-dropletandprecursorCy-microbubblecharacterisation
ThestockprecursorCy-microbubblesolution(Fig.4b)yieldeda concentrationof5109 microbubbles/mL,andamean bubble diameter of 1.020.40
m
m. DLS (Fig. 4e) revealed an average hydrodynamic diameter of approximately 400nm for both Cyanine-droplets (Fig. 4c) and normal-droplets, withrelatively narrowsizedistributions(polydispersityindex=0.986and0.509, respectively). TheCy-dropletsare metastableintheliquidstate under physiological conditions due to the energy barrier for homogeneousnucleation[35],withavaporizationtemperatureof 75C[36],eventhoughtheyaresuperheated.Fig.5(aandb)show representative confocal microscopic images used to verify the locationoffluorescentlipidonprecursorCy-microbubblesand Cy-droplets. The majority of Cy-droplets were beyond resolution limits.TheCyanine7.5lipidonmicrobubblesappearedasacircular rim while the Cy-droplets that could be seen demonstrated fluorescencethroughouttheirprojectedarea.Theunevencircular projectedappearanceofCy-dropletswaspossiblyduetothelipid monolayeroftheCy-microbubblebeing‘folded’or‘buckled’after condensation[37].Fig.5(candd)showbright-fieldmicrographsof Cy-microbubblesandlargeCy-dropletsrespectively(again,most Cy-dropletswere beyondthe resolution limits). TheabsorptionspectrumoftheCy-dropletlipid solutionhad a peakataround 788nm(half-maximumwaveband710–840nm)(Fig.4d).
3.2.PhotoacousticsignalsandimagingcontrastofCy-dropletsand controls
Therawphotoacousticsignalwasplottedasafunctionoffast time,whichwas theone-waytime-of-flightcalculatedbyusing samplingpointnumberofeachchannel(i.e.2030points)anddata acquisition sampling frequency (40MHz) [38]. Fig. 6 plots the meanoftherawphotoacousticsignalfrom256channelsforthe first-pulseresponseoftheCy-dropletsandsixcontrols,withthe
shadederrorbarfromthreerepeats(acquiredatthreepositions showed in Fig. 3(1)). The signal started to rise at about 25
m
s correspondingtothepositionofthetubing-TMphantom.The Cy-dropletsproducedmorethananorderofmagnitude(maximum meanamplitude14.5a.u.)highersignalamplitudethanthenoise level, whereas none of the six controls produced a detectable photoacousticsignal.TypicalbeamformedphotoacousticimagesofCy-dropletsanda control are demonstrated in Fig. 7. A 10% diluted Cy-droplet suspensionresultedina56.3dBhigherenhancementofthespatial maximum imaging signalthan six controls.Fig.8 presentsthe beamformedphotoacousticimaging signalforthefirsttenlaser pulses.TheveryfirstlaserpulsevaporisedmostoftheCy-droplets inthetube,generatingsubstantialphotoacousticsignal,whileall subsequentpulsesproducedlittlesignal,possiblybecausefew Cy-dropletswereleftand suggestingthattheenhancedsignalwas producedbythevaporisationprocess.Fig.9demonstratesthatthe photoacousticsignalinducedbythevaporisationofCy-droplets (0.25%v/v)immobilisedinthedispersion-TMphantomproduced 8.1dB higher signal magnitude than the control (a ‘blank’ TM phantom).
3.3.PhotoacousticsignalandCy-dropletconcentration
For eventualinvivo use, anunderstanding isneeded of the relationship betweenthe concentration of Cy-droplets and the generatedphotoacoustic signal.ReferringtoFig.10,varyingthe relativeconcentrationofCy-dropletsolutionproduceda substan-tial and significant increase in the first-pulse (vaporisation) photoacoustic signalbetween5% and10% (relativetothestock Cy-dropletsolution)butnosignificantchangefrom10%to25%.
3.4.Ultrasoundechoenhancementviaopticalvaporisation
Fromthedispersion-TMphantomcross-sectionalimaging,the ultrasoundechosignalwas11dBhigherwheretheCy-dropletshad beenexposedtopulsedlaserillumination(Fig.11a)thanwhere theyhadnot(Fig.11b).InFig.11c,thelongitudinalviewofthe Cy-dropletladenphantomdemonstratesadistincttransitioninecho
Fig.5. MicroscopyofoutlierprecursorCy-microbubblesandoutlierCy-droplets(thoselargeenoughtoberesolvable),presentedtoillustratethelocationoffluorescentlipid. (a,b)ConfocalfluorescenceofCy-microbubbleandCy-droplets.(c,d)Bright-fieldmicroscopyofCy-microbubbleandCy-droplets.Thescalebarsare10mm.
strengthat theboundary,indicated bythewhite dashedoverlaid verticalline,betweentheregionthathadbeen(Fig.11c.left)andthat whichhadnotbeen(Fig.11c.right)exposedtothelaser.Anequivalent phantomwithnodropletsorparticlesofanykindembedded(Fig. 11d, e, f) provided confirmation of the lack of echo signal from the backgroundmaterialofthephantom,andthatthisdidnotchangewith exposuretothelaser.Theechosignallayeratthebottomofimages wasduetothereflectionfromanacousticallyabsorbingpadonwhich thephantomwasplaced,usedtoreduceacousticreverberations.
3.5.Ultrasoundimagingwithacousticvaporisationofdroplets
Fig. 12showsrepresentativeultrasoundimagesbeforeandafter acoustic vaporisation of droplets. Increased echo signal after
acousticvaporisationappearedaroundthepre-setfocusingdepth (16mm)of thevaporisingpulsesandgeneratedanaverageof 11.98 and 14.39-fold echo amplitude enhancement for Cy-droplets (Fig.12a,b) and blank-droplet controls (Fig.12c, d) respectively,wherethequantitativecomparisonisprovidedby Fig. 13. There was no significant difference in the results betweenCy-dropletsandcontrolblank-droplets(p>0.05).Afew echoeswereseenbeforeacousticvaporisation,usuallydeepin the watertank (Fig.12a,c), andoutsidethe focaldepth after vaporisation,mostfrequentlyimmediatelybelowthefocalzone (Fig.12b,d).
Data underlying this article is available on request: please contact [email protected]. A general li-cenceappliestoallusersofthedata.
Fig.7.Beamformedphotoacousticimagesof(a)thefirst-pulseresponseof10%dilutedCy-dropletsand(b)thatofarepresentativeimageofallsixcontrols.The‘whitedashed circle’showstheROIfordataanalysis,whichoutlinestheexternalcircumferenceofthetubing-TMphantom.
4.Discussion
4.1.Overallresults
Whenviewedintotality,theresultsdemonstratethatthe Cy-dropletsrepresentanewdual-triggerableanddual-modality sub-micronphase-changecontrastagent,whichcanbeactivatedbya pulsed laser or by diagnostic ultrasound pulses to offer both photoacoustic and ultrasound signal enhancement via optical vaporisation or by ultrasound echo imaging of the resultant gaseousbubbles.Furtherworkisneededtoinvestigateaspectsof theresultsinmoredetailandtodetermineoptical,acousticand signalprocessingparametersforoptimiseduse.
4.2.Comparisonwithotheropticallyactivatedphasechangeand dual-modeagents
Other optically activated phase change agents that produce dual-mode(photoacousticandultrasound)imagingincludethose ofHannahetal.[11],whoincorporatedindocyaninegreen(ICG)
intothealbuminshellsofnanodroplets,andWeietal.[19],who usedananoemulsioninwhichshell-lessdropletswerecoatedwith gold nanospheres. The advantages and disadvantages of each approachhaveyettobefullyuncoveredandstudied.ICGseemsto berequiredinmMamounts(2mMICGwasusedin[11])buthas theadvantagethatitisalreadyapprovedforclinicaluse,andthe shortlifetime(afew
m
s[19])ofthecavitationbubblesgenerated bypulsedilluminationofthenanoemulsionmaynotbeidealfor ultrasoundimaging. Oursis thefirst investigationof whethera combined photoacoustic and ultrasound contrast agent can be generated by condensing precursor fluorescent microbubbles containingalowb.p.perfluorocarbon(i.e.DFB).Inprinciple,any suitabledye(ornanoparticle)couldactastheopticalabsorberin the precursor microbubble shell. Here we used Cyanine7.5 attached by bioconjugation. In combination with DFB, this providedhighlyefficienttriggeringofvaporisationbya commer-cially available photoacoustic imaging system, with only nM amounts of the dye (13nM in this work). The system is also versatile;ouruseofcommerciallyavailableDSPE-PEG(2000)-NH2 forbioconjugationenableseasyattachmentoftargeting ligands (e.g.folateforintracellulardelivery[39])and/orcomplementary imaging units (e.g. Gadolinium(III)-DOTA) to incorporate addi-tionalfunctionalities.Finally,asnotedin[39],theuseofDFBalso enablesefficientuseinthethirdimagingmode,acoustictriggering ofvaporisation,atanFDAapprovedMIconsistentwithclinicaluse. Allotherpublishedmicrobubble ordropletbased dualcontrast agentstudiesthatwecouldfindhaveverydifferentobjectivesand modesofaction,involvingfluorescentdyestoprovideeitherdual ultrasound-fluorescenceimaging(e.g.[40,41])ordualfluorescence and magnetic resonance imaging (e.g. [42]), making direct comparisonwiththepresentworkinappropriate.4.3.Concentrationdependence
ChangingtheCy-dropletrelativeconcentrationfrom5%to10% nearlydoubled thephotoacousticsignal enhancement(Fig.10), probablybecauseagreaternumberofCy-dropletsyieldedmore optical vaporisation events [22] producing a higher density of acoustic sources. Further study is required to determine the linearityandconcentrationrangeofthisdependence.Saturationof thephotoacousticsignalenhancement abovea relative concen-tration of 10% is not at present fully understood,and requires
Fig.9.Photoacousticimagesofthedispersion-TMphantomshowing(a)Cy-droplets(0.25%v/v)and(b)‘blank’dispersion-TMphantomcontrol.Theopticalvaporisationof Cy-dropletsimmobilisedinthephantominduced8.1dBhighersignalenhancementthanthecontrol.Thewhitedashedcirclesindicatethepositionofthedispersion-TM phantomandROIfordataanalysis.
Fig.11.UltrasoundechoenhancementproducedbyopticalactivationofCy-dropletsimmobilisedinthedispersion-TMphantom,andcomparisonwiththecontrolphantom (nodroplets).(a,d)cross-sectionalechoimagesoftheCy-dropletladenphantomandcontrolphantomafterpulsedlaserillumination.(b,e)Cross-sectionalimagesofthe Cy-dropletphantomandcontrolwheretherehadbeennolaserirradiation.(c,f)LongitudinalimagesoftheCy-dropletphantomandcontrol,inwhichthewhitedashedvertical lineindicatestheboundarybetweentheregionsexposedandnotexposedtothelaser.Thebandofechoesatthebottomoftheimageswasduetothereflectionfroman anti-reverberationpad.Whitedashedoverlaidcircleshighlightthephantomcross-sectionalareaandtheROIfordataanalysis.Scalebaris5mm.
furtherstudy,butmaybeassociatedwithsignalsaturationwithin thephotoacousticdetectionsystem.
4.4.Opticalactivationthresholdenergy
Itishelpfultoknowthat,asdemonstratedhere,standardlaser pulsesusedforphotoacousticimagingweresufficienttoactivate theCy-droplets,andthatthiswasachievedwithalaserfluence that was below the safe limit (100mJ/cm2) for human skin exposure[12].Nevertheless,furtherloweringofthethresholdfor vaporisation,while preservinggeneral stability, is preferableto enabledeeptissue opticalactivation. More detailed studiesare neededofthedependenceoftheopticalvaporisationthresholdon laser exposure parameters such as fluence, and on droplet properties.Forexample,thelargerthedropletthelowerthelaser fluenceneededtoachieve phasetransition[12].Inthis study,a polydispersedprecursormicrobubblesizedistributionproduced polydisperseddroplets.Infuturestudiesitwouldbedesirableto selectthesizeofdroplets[28],toinvestigatetheopticalactivation threshold energy as a function of size. It is also important to optimise the amount of Cyanine7.5 on the lipid membrane of precursormicrobubbles,sincethiswillaffecttheopticalactivation threshold.Furthertuningmaybeachievedbyalteringtheboiling pointofthegas,orcombinationsofgases,intheprecursorbubbles [43].Finally,thelipidcompositionoftheshell,particularlylipid acylchainlength,canaffectphase-changeactivationenergy[36], suggestingopportunities,forexample,toreplace 1,2-dipalmitoyl-sn-glycero-3-phosphocholine(DPPC,C16)withalipidpossessinga shorteracylchain,e.g.,1,2-dimyristoyl-sn-glycero -3-phosphocho-line(DMPC,C14).
4.5.Selectionofopticalabsorberandpossibilitiesfor“droplet recognitionimaging”
The peak absorption wavelength of Cyanine7.5 (788nm) is withinthe ‘imaging opticalwindow’ of biological tissue (600– 1300nm)[44]withminimaltissueattenuationthatenablesdeep penetration of light, making the Cy-droplets amenable to enhancingthe sensitivityof whole body small animal vascular andmolecularlytargetedimaging.AlthoughICGalsohas absorp-tion in this window, with the additional advantage that it is approved for clinical use, it has poor photostability, a molar absorption coefficient [29] that is much lower than that of Cyanine7.5 (over 200,000mol 1cm 1L) and its fluorescence
showntobethecase,itbringsabouttwoimportantpossibilities. First,changingtheopticalwavelengthmayallowalimitedformof vaporisationspectroscopyforphotoacousticimagingoftheagent (e.g. starting at wavelengths not expected to cause a phase transition), so that the method may go beyond simple signal enhancementtoagentrecognitionimaging,byanalogytotheway that nonlinear (e.g. pulse-inversion) techniques have taken conventionalmicrobubbleultrasoundimagingbeyondsimpleblood echo signal enhancement. Admittedly, such tissue background-suppressedimagingoftheagentmightbeachieved(intheabsence ofsignificanttissuemotion)alsobyvaporisationcontrast subtrac-tion imaging, but there may be advantages in combining both temporal and wavelength subtraction approaches. Second, the whole cyanine dye family(e.g. from Cyanine3 to Cyanine7.5) is commerciallyavailable,offeringarangeofpeakabsorption wave-lengthsandraisingthetantalisingpossibilityofco-administering variousCy-droplettypesintothebloodstream,eachwithdifferent activationwavelengthsfordifferentialdropletrecognition.Thismay beusefulif,forexample,eachdroplettypewerefunctionalisedto bindtoadifferentmoleculartarget.Thesefeaturesmakecyanines very promising candidates for the optical absorber, particularly duringthisresearchphaseofthework.Althoughcyanineshavenot beenapprovedbyFDAforclinicaluse,otherdyes,ornanoparticles, couldbeeasilysubstitutedandmayoffersimilarpossibilities.
4.6.Synergisticeffectofcombiningultrasoundandopticalenergyfor loweringthevaporisationthreshold
Previousstudieshaveshownthatthesimultaneousdeposition of optical(laser illumination) and acoustic (ultrasound rarefac-tionalpressure)energycanlowerexposurethresholdstoachieve enhanced photoacoustic and/or acoustic signal from various contrast agents [45–47]. Future work willinvolve investigating the vaporisation thresholds of Cy-droplets excited with a nanosecond laser pulse coinciding with various phases of an ultrasoundactivationwave.Inparticular,basedontheobservation in[48],thatvaporisationmicrobubblescanemergefromdroplets throughthefirst rarefactionalphase ofanultrasoundpulse,we hypothesisethatthevaporisationthresholdcanbemostreduced byaligninglaserpulsestoararefactionalphase.Suchsynergism couldbeharnessedtoimprovevaporisationimagingdepth,aswell assensitivityand specificityof targetedmolecularimagingand therapyusingCy-droplets.
4.7.Acousticvaporisationthresholds
Echoesseenbeforeacousticvaporisation(Fig.12a,c),suggest spontaneousvaporisation ofsome droplets.These wereusually spotteddeepinthewatertank,andthereforemaycorrespondto largerdroplets whichwouldmorereadilyundergospontaneous phase change. This would be consistent with the echoes that appearedoutsidethefocaldepthaftervaporisation,whichmost frequentlyappearedimmediatelybelowthefocalzone(Fig. 12b,d), i.e.,theywerepossiblyduetoacousticvaporisationofthelargest dropletswhichwouldhavehadthelowestacousticvaporisation thresholdscomparedwiththemajorityofdroplets.Futurestudies arerequiredtofullycharacterisetheacousticvaporisation.
5.Conclusion
In this study, we have demonstrated the development and characterisationofanopticallyandacousticallytriggerable sub-micronphase-change contrastagent‘Cy-droplets’manufactured with a highly volatile perfluorocarbon via the ‘microbubble condensation’approach.Foropticaldropletactivation,Cy-droplets generated substantial photoacoustic transient signal from the vaporisation light pulse, and gas bubble formation thereafter providesstableenhanced ultrasoundsignal.Foracoustic activa-tion,Cy-dropletscanbevaporisedusingexternalacousticenergy with clinical diagnostic ultrasound pulse parameters, offering ultrasound echoimaging contrast.This versatilityoffers photo-acoustic-ultrasound dual imaging and high selectivity, which would benefit cancer molecular imaging and targeted drug deliveryusingCy-droplets.
Conflictofinterest
Theauthorsdeclarethattherearenoconflictsofinterest.
Acknowledgement
S. Lin was funded byan Imperial College-China Scholarship Council(CSC)scholarship.Thisworkwas partiallysupportedby Cancer Research UK Multi-Disciplinary Project Award (C53470/ A22353),theCancer ResearchUKCancerImagingCentreat the InstituteofCancerResearch,theEngineeringandPhysicalSciences ResearchCouncilStrategicEquipmentGrant(EP/N015266/1),and theNationalInstitutesofHealthGrant#CA185684(T.O.M).The authorsacknowledgeDrEleniBazigouforthehelpwithconfocal microscopy, and Dr Robert Eckersley, Prof. David Cosgrove, Dr Daniel Elson and Dr Ji Qi for the fruitful discussion. Special acknowledgementisgiventotheUltrasoundLaboratoryforImaging andSensing(ULIS)Groupmembers,ImperialCollegeLondon.
AppendixA.Supplementarydata
Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.pacs.2017.04.001.
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ShengtaoLin receivedhis MScdegree inBiomedical EngineeringatImperialCollegeLondonin2014.Heis currentlyaPh.D.studentattheUltrasoundLaboratoryfor ImagingandSensing(ULIS)groupintheDepartmentof Bioengineering, Imperial College London, under the supervisionofDrMengxingTang.Hisresearchinterests include nanodroplet/microbubble contrast agents for photoacousticandultrasoundimaging.
Dr.AnantShahreceivedhisPh.D.degreeinbiophysicsin 2014fromtheInstituteOfCancerResearch,forhiswork onphotoacousticimagingofmolecularmarkersofcancer prognosisandresponseusinggoldnanoparticles.Heis currentlyworkingasa postdoctoralresearcheratthe Instituteof CancerResearch. Hisresearch isoriented towardsassessingthepotentialofphotoacousticimaging forcancertreatmentplanningandtreatmentresponse.
DrJavierHernándezGilisapostdoctoralresearcherin thegroupofProfessorNicholasJ.Long.HereceivedhisB. Sc.inChemistryfromtheUniversityofValenciaandthen obtained a competitivefellowshiptoundertake Ph.D. studies(2008–2012)inBioinorganicChemistryinthe FacultyofPharmacyatthesameUniversity,underthe supervisionofProf.SacramentoFerrer.Duringthistime healsoobtainedaB.Sc.inBiochemistryattheUniversity ofValencia.PriortojoiningImperialCollegeLondonin 2015,hespenttwoyearsinthegroupofProf.JuanMareque atCICbiomaGUNE.Hiscurrentresearchinterestsarefocused onthedesignofnano-andmicroparticlesforMR,PETand Ultrasoundimagingandtheirapplicationsincancer.
AntonioStanziolareceivedhisBScandMScdegreein BiomedicalEngineeringatUniversityofPisaand Chalm-ersUniversityofTechnologyin2014.HeiscurrentlyaPh. D.studentattheUltrasoundLaboratoryforImagingand Sensing(ULIS)groupintheDepartmentof Bioengineer-ing,ImperialCollegeLondon,underthesupervisionofDr Mengxing Tang. His research interestsinclude signal processingforcontrastenhancedultrasoundimagingand beamforming.
Prof.TerryMatsunagais aResearchProfessor inthe Department ofMedical Imaging atThe University of Arizona.HewasVicePresidentforResearchatImaRx Pharmaceutical where he was instrumental in the development of a lipid-coated microbubble that is currentlyapprovedintheUnitedStatesunderthename Definity. While at the University of Arizona, he has focused on the development of novel methods for formulatingphase-changecontrastagentsfromvolatile perfluorocarbongasesalongwithin-vitroandin-vivo studiesfor ultrasound-mediatedimagingandtherapy. HiseducationincludesaPharm.DandPh.D.Fromthe UniversityofCaliforniaSanFrancisco,Aclinicalpharmacy residencyattheUniversityofMichigan,andaNIHpost-doctoralfellowinchemistry attheUniversityofArizona.
Prof.NicholasLongistheSirEdwardFranklandBPChair in Inorganic Chemistry and Head of the Catalysis, Sustainability and Applied Inorganics Research SectionatImperialCollegeLondon.Researchinterests includetransitionmetalandlanthanidechemistryforthe synthesisoffunctionalmolecules,homogeneouscatalysis andinrecentyears,probedesignandnovel methodolo-giesforbiomedicalimaging.Researchhasfocusedonthe designofsingle-anddual-modalityPET,MRIandoptical imaging agents for early detection and diagnosis of disease.He is Co-Director of the KCL-ICL Centre for DoctoralTraininginMedicalImaging.
Dr.JeffBamberhasbeenheadoftheUltrasoundand OpticsPhysicsTeamatTheInstituteofCancerResearch since1986.HeisaReaderinPhysicsAppliedtoMedicine, SeniorTutorandanhonoraryMedicalPhysicistwithin the Royal Marsden and other London hospitals. His degreesare:BScPhysics(UniversityofKentat Canter-bury),MScBiophysicsandBioengineering(Universityof London),andPhDBiophysics(UniversityLondon).Hehas hadsabbaticals at the TokyoInstitute of Technology, Japan,andHewlett-Packard,Andover, USA.Heispast presidentof the International Association for Breast Ultrasound, past vice-president of the International SocietyforSkinImaging,servesontheeditorialboards ofvariousjournalsandasscientificadvisortoanumberofcompanies.
