ContentslistsavailableatScienceDirect
Colloids
and
Surfaces
B:
Biointerfaces
j ou rn a l h om epa ge :w w w . e l s e v i e r . c o m / l o c a t e / c o l s u r f b
Light-triggered
release
from
dye-loaded
fluorescent
lipid
nanocarriers
in
vitro
and
in
vivo
Redouane
Bouchaala
a,b,
Nicolas
Anton
c,
Halina
Anton
a,
Thierry
Vandamme
c,
Julien
Vermot
d,
Djabi
Smail
b,
Yves
Mély
a,
Andrey
S.
Klymchenko
a,∗aCNRSUMR7213,LaboratoiredeBiophotoniqueetPharmacologie,UniversityofStrasbourg,74routeduRhin,67401IllkirchCedex,France bLaboratoryofPhotonicSystemsandNonlinearOptics,Instituteofopticsandfinemechanics,UniversityofSetif1,19000Algeria
cCNRSUMR7199,LaboratoiredeConceptionetApplicationdeMoléculesBioactives,UniversityofStrasbourg,74routeduRhin,67401IllkirchCedex,France dIGBMC(InstitutdeGénétiqueetdeBiologieMoléculaireetCellulaire),InsermU964,CNRSUMR7104,UniversitédeStrasbourg,1rueLaurentFries,67404
ILLKIRCH,France
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received2November2016 Receivedinrevisedform11May2017 Accepted12May2017
Availableonline15May2017 Keywords:
Photo-release Controlledrelease Lipidnanoemulsions Lipophilicdyes
Fluorescencecorrelationspectroscopy Fluorescencemicroscopy
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b
s
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Lightisanattractivetriggerforreleaseofactivemoleculesfromnanocarriersinbiologicalsystems.Here, wedescribeaphenomenonoflight-inducedreleaseofafluorescentdyefromlipidnano-dropletsunder visiblelightconditions.Usingauto-emulsificationprocesswepreparednanoemulsiondropletsof32nm sizeencapsulatingthehydrophobicanalogueofNileRed,NR668.Whilethesenano-dropletscannot spontaneouslyenterthecellsonthetimescaleofhours,afterilluminationfor30sunderthemicroscope atthewavelengthofNR668absorption(535nm),thedyeshowedfastaccumulationinsidethecells.The samephenomenonwasobservedinzebrafish,wherenano-dropletsinitiallystainingthebloodcirculation werereleasedintoendothelialcellsandtissuesafterillumination.Fluorescencecorrelationspectroscopy revealedthatlaserilluminationatrelativelylowpower(60mW/cm2)couldtriggerthereleaseofthedye
intorecipientmedia,suchas10%serumorblanklipidnanocarriers.Thephoto-releasecanbeinhibited bydeoxygenationwithsodiumsulfite,suggestingthatatleastinpartthereleasecouldberelatedtoa photochemicalprocessinvolvingoxygen,thoughaphoto-thermaleffectcouldalsotakeplace.Finally,we showedthatilluminationofNR668canprovokethereleaseintothecellsofanotherhighlyhydrophobic dyeco-encapsulatedintothelipidnanocarriers.Theseresultssuggestdye-loadedlipidnano-dropletsas aprospectiveplatformforpreparationoflight-triggerednanocarriersofactivemolecules.
©2017ElsevierB.V.Allrightsreserved.
1. Introduction
Triggeredrelease of active molecules fromnanocarriers has beena subject of intensive researchin thelast decades[1–6]. Varioustriggers,suchaspH,reductivepotential,enzymes, tem-perature,ultrasound,magneticfieldandlighthavebeenutilized. Lightisparticularlyattractiveinthisrespect,asitcanbefocused atadesiredpositionofaspecimenwithhighspatiotemporal reso-lution.Themostcommonapproachesforlightcontrolledrelease utilize molecules that undergo light-controlled isomerization, photo-polymerization and photo-cleavage [7,8]. Thus, azoben-zenecis-transisomerizationcan triggerrelease fromliposomes
[9]polymer vesicles and micelles [10,11]and inorganicporous nanomaterials[12,13].Photocleavagereactionsino-nitrobenzyl
∗ Correspondingauthor.
E-mailaddress:andrey.klymchenko@unistra.fr(A.S.Klymchenko).
[14–16],coumarin[17],spiropyran[18,19],malachitegreen[20], cynnamicacid[21,22]derivatives,etccanproducephoto-driven hydrophobicityandchargechanges,polymer/lipidfragmentation andde-crosslinking.However,majorityofexamplesofthe light-triggeredrelease fromnanocarriersreportedtodaterequireUV lightortwo-photonexcitation[17,23].Theotherapproachutilizes surfaceplasmonand photo-thermaleffects, totriggerthecargo release. In this case, mostcommonly goldnanoparticles, nano-rodsornano-shells,areusedincombinationwithpolymer,lipid nanocarrierorthermo-sensitivecovalentbonds[24–28].The plas-monabsorptioninthesenanostructureswassuccessfullytuned, sothat theycouldbeactivatedwithalmostanydesired excita-tionwavelengthfromUVuptonear-infrared(NIR)region[29,30]. However, photothermal effects using gold nanostrcutures gen-erally requirerelativelystrongilluminationpowers [31], which canbeharmfulforthebiologicalsample.Upconverting nanopar-ticles,which convertNIRlight into visibleand furtherproduce photochemicaleffectshouldbealsomentioned,butsofarall exam-http://dx.doi.org/10.1016/j.colsurfb.2017.05.035
Fig.1.Concept:lipidnanocarrierandtheeffectoflightontheencapsulatedNileRedderivativeNR668.
plesofphoto-releasewithupconversionnanoparticlesarelimited toinorganicsystems[32,33].Inordertodeveloporganic photo-responsivesystems one shoulduse dyes encapsulatedinside a nanocarrier.Inthiscase,dyesoperateasphotosensitizers gener-atinghighlyreactivesingletoxygenand/orasconvertersoflight energyintoheat.Asphotosensitizers,organicdyes,duetotheir capacitytodisruptbiological membranes,wereusedfor photo-chemical deliveryintothecells of molecules and nanoparticles
[34–37].Theuseoforganicdyesastriggersofmolecularrelease fromananocarrierweremainlyrealizedinlipidvesicles,where local photothermal or photochemical effects were proposedas drivingmechanismsofphoto-release[38–42].Thephotothermal effectswerealsoreportedforNIRdyesloadedintonanocarriersin cancertherapyapplications[43–45].
Lipidnanoemulsions are animportant nanocarrierplatform, astheycanbereadilypreparedfromFDAapprovedagents and theirhydrophobiccorecanencapsulatedrugsandcontrastagents
[46–50]. Previously, the group of Texier suggested these lipid nanocarriersasimagingagentsbyloadingtheircorewithapolar cyanines[51,52].Wefurthersuggestedaparticularprobedesign, usinglongalkylchainsandbulkyhydrophobiccounterions,thatcan increasethedyeencapsulationtogetherwithpreservationofthe highlyefficientfluorescence[53,54].Moreover,recentFRET-based studiesrevealedtheirhighstabilityinvitroandinvivoandcapacity toentertumorsintheintactform[55,56].Inparticular,weshowed thattheuseofhighlyhydrophobicsubstituentsofNileRedenables tosignificantlyincreasedyeloading,preservegoodfluorescence efficiencyanddrasticallydecreasethedyeleakage[53].However, thequestionremained:canastrongilluminationofalargequantity ofdyesconfinedonthenanoscopicspacedestabilizelipiddroplets? Thispoint wasnotstudieduptonowowingtothedifficultyto encapsulatealargeamountofdyesinthesenanoscalereservoirs. Takingrecentlyproposedapproaches[53,54],thedye concentra-tioncan beincreasedup to5 wt%along withconserving their fluorescentproperties.Thisispreciselythepointthatcanopenthe doortophoto-triggeredproperties.AlthoughNileRed photosensi-tizingpropertieswerenotdescribedyet,wehaveshownearlierthat largeconcentrationsofNileRed-basedmembraneprobe(NR12S) canproducesomephoto-damageoncellmembranes[57],which suggeststhatNileRedasphoto-activemoleculetodestabilizelipid nanocarriers.
Inthepresentwork,wedescribeanunexpectedphenomenon, whichisthelight-triggeredreleaseofnano-dropletcontent,when encapsulatingahydrophobicNileRedanalogue.Weshowthatthe dyereleasecanbeobservedbothincellcultureandinvivoona zebrafishmodel.Thelight-induceddyereleasewasvalidatedwith
differentacceptormedia,suchasblanknanocarriersand10%serum anditcanbeinhibitedinthepresenceofoxygenscavenger.Finally, the light-drivenrelease of a co-encapsulatedmolecule (second encapsulateddye)alongwiththeNileRedderivativewasvalidated, whichproposestheroutetolight-controlleddrugreleasefromlipid nano-droplets.
2. Resultsanddiscussion
Theideaoflight-triggeredrelease fromnano-carrieris sum-marizedinFig.1.Thenano-emulsiondropletscanbeconsidered asreservoirwithhighlocalconcentrationofdyes.Our question waswhetherilluminationofthislargeensembleofdyes within thesmallvolumeoflipidnanocarriercouldproducereleaseofits contentduetophotophysicalorphotochemicalphenomena.Asa dyeforencapsulation,weselectedaderivativeofNileRed bear-inglongalkylchains(NR668,Fig.1).Althoughthisdyeisexpected tobecellpermeant(likeparentNileRed),itshigh hydrophobic-ityensuresencapsulationintonano-dropletswithminimalleakage effects,whichpreventsitsfastaccumulationinthecells[53].
Wefirstpreparednanocarriersbyspontaneousemulsification ofSolutol,Labrafacand NR668dyeinwater,which gave32nm particlesoflowpolydispersityindex(<0.1)accordingtodynamic lightscattering.Thephenomenonofdyereleaseunderlight illu-minationwasfirststudiedincellculture.Solutol®(Kolliphor®)HS 15/Labrafac®WLnanoemulsionsloadedwithhydrophobicNileRed derivativeNR668wasincubatedwithcellsandstudiedby wide-fieldfluorescencemicroscopy.Ourearlierworksshowedthatno internalizationwasobservedforthesamesystemevenafter2hof incubationwithHeLacells[53],whichshowedthatthese nanocar-rierscannotenterthecellsonthistimescale.Therefore,thissystem isidealfortestingtheeffectoflight,asnoartifactsduetoparticle internalizationordyeleakageintothecells wasobserved.After 30minofincubationofthecellswiththeemulsionofnanocarriers containing1wt%ofNR668,thecellsremainedpoorlyfluorescent, asthefluorescenceofthemediumwasmoreintensethanthatof thecells.Moreover,afterwashingofthecells,nosignificant fluores-cencewasdetectedinsidethecells(Fig.S1),whichconfirmedthat NR668nano-dropletsdidnotspontaneouslyinternalizewithinthis timescale.Then,weilluminatedthecellssurroundedby NR668-loadednanocarriersfor30sat535nm(absorptionmaximumof NR668innano-dropletsis526nm[53])usingthemaximallamp power(∼14W/cm2powerdensityatthesamplelevel).Thelight actionresultedinastrongphotobleachingoftheareaof observa-tion.Indeed,itwasobservedthatinitiallyverybrightfluorescence fieldbecamenon-fluorescentaftertheillumination(Fig.2).Then,
Fig.2.Releaseofdyemoleculesinvitro.Brightfield(AandC)andfluorescence(BandD)imagesofHelacellsinthepresenceofnanocarrierloadedwithat1%(A,B)or 5%(C,D)ofNR668.Thefirstimagesontheleftcorrespondtosamplesbeforeillumination,whileallothersareafterilluminationfor30sat535nmwith2mindelayfor eachconsecutiveimage.Alltheexperimentsweredoneat37◦C.(F):differencebetweenintracellularandextracellularfluorescenceintensity,Iint-Iext,vstimedelayafter
illumination.Theerrorbarsrepresentthestandarddeviationofthemeanfromthreeindependentilluminationcycles(n=3).(G-H)Fluorescenceimagesafterwashing(from nanocarriersat5wt%NR668)showingnon-illuminatedandilluminatedregions.
withtime,thefluorescenceofthemediumstartedtorecover,but surprisinglythefluorescenceofthecellsincreasedwithtime.This phenomenonwasobserved alreadyat1wt%ofdyeloadingand tookabout10mintoachieveaclearintracellularstaining(Fig.2A andB),characterizedbyasignificantdifferencebetween intracel-lularandextracellularfluorescenceintensity(Fig.2F).At5wt%dye loading(Fig.2CandD),thecellstainingafterilluminationwasmuch stronger,sothatthedifferencebetweenintracellularand extracel-lularfluorescenceintensitywas>3-foldlargercomparedtothatfor 1wt%loading.Aftercellwashingfromthenanocarriers,wecould findtheinitiallyilluminatedregions,wherethefluorescenceinside thecellswasmany-foldlargerthanthatforthenon-illuminated regions(Fig.2Gand H).These experimentsclearlyshowedthat nanocarriers,unabletoenterthecellsinthedark,candeliverthe dyeafterillumination.ItshouldbenotedthatwhenNR668dye withoutlipidnanocarrierwasaddeddirectlytothecellsfromits DMSOstocksolution(finalDMSOconcentrationwas1%),the intra-cellularfluorescence of this dyewas observed(Fig. S1). Earlier worksshowedthatitsparentanalogueNileRedcouldalso spon-taneouslyinternalize[58],whichimpliesthatplasmamembrane isnotreallya barrierforthesehydrophobicdyes.Theseresults highlighttheroleofthenanocarrier,whichencapsulatesNR668
preventingitsleakageintothecells,butensuringitsreleaseafter theillumination.
Then,weinvestigatedwhetherthelight-inducedreleasecanbe alsorealizedinvivoonzebrafish.Our earlierworkshowedthat NR668-loadednanocarriersremainedinthebloodcirculationfor atleast30minwithoutdyeleakageintotheendothelialcellsofthe vesselsandsurroundingtissues[53].Here,weinjected nanocarri-erscontaining5wt%ofNR668andperformedmicroscopyimaging as a function of time without and with illumination (through 450–490nmbandpassfilter).Withoutillumination,thered fluores-cenceofnano-dropletswasobservedexclusivelyinsidetheblood vesselsforatleast60min(Fig.3A,B),indicatingthatthe nanocar-riers remain in the blood circulation without dye leakage into thetissues.Bycontrast,aftertheillumination,wecouldobserve aprogressiveincreaseofthefluorescenceinsurroundingtissues betweenthebloodvessels(Fig.3C-F).Thefluorescenceintensity, recordedinthetissueareasshowedcontinuousincreaseintime fortheobservationperiodof60min(Fig.3G).Thus,the illumina-tiontriggeredreleaseoftheencapsulatedNR668dye,whichthen diffusedfreelyintothesurroundingtissues.
To understand better the observed phenomenon of release, weperformedfluorescencecorrelationspectroscopy(FCS)ofour nanocarriersindifferentmodelbiologicalmedia.FCSisapowerful
Fig.3. Thephoto-inducedreleaseoftheNR688dyefromthenanoemulsionsinthevasculatureoflivingzebrafishembryo.Fluorescenceimagesoftailvasculature(green) containingNR668nanoemulsions(red).(A,B)Controlnon-illuminatedembryo,showingnoleakageofthedye.(C-F)Embryosilluminatedfor5minthrough450–490nm bandpassfilter,showingaredfluorescenceinthetissuessurroundingthevessels.Scalebaris100m.(G)TheincreaseinthefluorescenceintensityaveragedintheROIs showninpanel(C)withtime.Theerrorbarsrepresentthestandarddeviationofthemeanfromtwodifferentregionsoftheembryo(n=2).(Forinterpretationofthereferences tocolourinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.)
techniquetocharacterizenanoparticlesinsitu,whichcanprovide informationaboutsize,concentrationandbrightnessofparticles
[53,59,60].AccordingtoourFCSdata,theparticlesize,measured fromthediffusioncorrelationtime oftheemissivespecieswas 33nm(TableS1).Thisvalueisremarkablyclosetothe hydrody-namicdiameterobtainedforthesenanocarriersbyDLS(32nm). Moreover,theparticlesconcentrationforthissampleat1:10000 dilution,evaluatedfromthenumberofemissivespeciesperfocal volume,was1.7nM.Basiccalculationbasedonknowntotallipid concentrationintheusednanoemulsion(1.1×10−3%)andthemass ofasingle33-nmnano-droplet(1.9×10−17g,assumingdensityof thedropletof1g/ml),wecouldestimatethattheconcentrationof nanocarriersshouldbearound1.0nM.Thecorrespondenceofthe theoreticalandexperimentalvaluesoftheparticleconcentration andthematchoftheparticlesizeobtainedfromDLSandFCS sug-gestthatafterthis10000-folddilutionthenanocarriersremained practicallyintact.
Inordertomonitorthephoto-releaseofthedye,westudied thenumberofemissivespecies,which, accordingtoourearlier studies,isanimportantindicatorofthedyerelease[53].Indeed, ifthedyesarereleasedintotheacceptormedium(blankparticles orserum),thenumberofemissivespeciesshouldincrease(Fig.4A). Forexample,incontrolexperimentswithoutillumination,NileRed encapsulatedintothenanocarriersshowedalownumberof emis-sivespeciesinwater and Opti-MEM,wherepoorly solubleNile Redcannotreallyescape(Fig.4B).Ontheotherhand,inthe pres-enceofblanknanocarriersorfetalbovineserum(FBS),thenumber ofemissivespecies increaseddrastically, showingthatNile Red leakedfromnanocarriersanddistributedintherecipientmedium. Bycontrast,thenumberofemissivespeciesfortheNR668-loaded nanocarrierswaslowforallfourstudiedmedia(Fig.4C)andthe sizeofemissivespeciesremainedstable(TableS1).Theseresults confirmedourearlierdataonthehighstabilityofNR668-loaded nanocarriersagainstleakage[53].Afterilluminationwiththelaser at532nm(powerdensity 60mW/cm2)thenumber ofemissive speciesgrewdrasticallyinthepresenceblanknanocarriersandFBS, butdidnotchangeinwaterandOpti-MEM.Thenumberofemissive speciesinthemediawithblanknanocarriersorFBSgrew gradu-allyonincreaseinthetimeofillumination(Fig.4D).Theseresults suggestthat thedyephoto-releaseis light-dosedependentand itrequiresabiologicalmediumcontaininghydrophobic
microen-vironment. This wasexpected, because NR668 is highly apolar (logP=9.22[53])andpracticallyinsolubleinwater.Moreover,as thenumberandthesize(TableS1)ofemissivespeciesinwaterdid notchangeafterillumination,wecouldconcludethatillumination didnotsplitthenanocarriersintomultiplespecies.Therefore,the observedphoto-releaseinthepresenceoftheacceptormedium aswellasincellandzebrafishembryoisprobablyrelatedtothe light-triggered dyeleakage from thenanocarriers into the sur-roundinghydrophobicmicroenvironment.Theillumination also producedadropinthetotalfluorescenceintensity(Fig.4E), indicat-ingthephotobleachingofthedye.ThelatterindicatesthatNR668 underwentphotochemicaltransformation,whichcouldexplainthe destabilizationofthenanocarrierwithfurtherreleaseofthedyes intothemedium.Photobleachingofdyesiscommonlyassociated withgenerationofsingletoxygenthatfurtheroxidizethedyeor othercomponentsofthenanocarrier.Therefore,werepeatedour FCSexperimentsin thepresence ofsodiumsulfite.Thelatteris areductivedeoxygenatingagent,whichdepletesoxygenthrough oxidationintoasulfateanion[61–63].Remarkably,sodiumsulfite drasticallydecreasedthereleaseofthedyeunderilluminationin thepresenceofblanknanocarriersandserum,asitcanbeseen fromonlysmallchangeinthenumberofemissivespecies(Fig.4C). Moreover,sodiumsulfitedidnotshowasignificantinfluenceon thefluorescenceintensityofthesamplesbeforeillumination(Table S1), indicatingthat it didnot actas quencherthroughelectron transferorothermechanisms.Overall,theseresultssuggestthat thekey mechanismthat drivesthedyerelease is linkedtothe photo-oxidationthatcoulddestabilizethenanocarriers.
TheimportantquestioniswhetherilluminationofNR668can triggerreleaseofothermoleculesformthenanocarriers.Tothis end,weencapsulatedintoourlipidnano-dropletsaseconddye, F888,which was alsoreportedpreviously toremain inside the dropletswithoutdyeleakage[53],similarlytoNR668.Importantly, themaximaofabsorptionandemissionofF888innano-droplets at1wt%loading(388and455nm,respectively)arewellseparated fromthoseofNR668(526and592nm)[53],whichshouldallow selectiveexcitationand detectionofthree twodyes by fluores-cencemicroscopy.Weincubatedtheduallylabelednanocarriers withcellsfor15minandthenilluminatedthemfor30sat535nm. Beforeillumination,theintracellularfluorescencewasweak con-firmingnoleakageoftheencapsulateddyesinthepresenceofcells
Fig.4.Fluorescencecorrelationspectroscopy(FCS)studiesofthelight-induceddyereleaseintomodelbiologicalmedia.(A)Schemeexplainingtheeffectofthedyerelease onthenumberoftheemissivespecies.(B)ControlexperimentshowingNileReddyereleasefromnanocarriersintoblanknanocarriers(NCs)and10%serum(FBS),which wasrecordedasnumberofemissivespecies.(C)Theeffectof30minilluminationby532-nmlaser(powerdensity60mW/cm2)onlipidnanocarriersloadedwithNR668
indifferentmedia:water,Opti-MEM,blanknanocarriersandFBS.Presenceofoxygen-depletingagentsodiumsulfitewasalsotested.(D)Effectoflaserilluminationtime onthenumberofemissivespeciesandthetotalfluorescenceintensityofNR668-loadedlipidnanocarriersindifferentmedia.Theerrorbarsin(B-E)representthestandard deviationofthemean(n=20recordedcorrelationcurves).
(Fig.5AandB).Afterillumination,thefluorescencerecordedatthe NR668channelshowedexpectedbehavior,wherethetotal fluores-cencedecreasedsignificantlyafterillumination,andthenincreased insidethecellsinthetimeperiodof10min(Fig.5B).Importantly, theintracellularfluorescencerecordedatthechannel correspond-ingtoF888dye(showningreen)increasedafterilluminationand continuedincreasingtogetherwiththatofNR668(Fig.5A). Impor-tantly,thebackgroundfluorescenceintheF888channeldidnot changeafterillumination,becausethisilluminationcannotexcite F888dye.Inthecontrolexperiment,weincubatedthecellswith onlyF888-loadednanocarriers.After30silluminationat535nm, wefoundnochangesinfluorescenceinsidethecells(Fig.S2),sothat NR668wasabsolutelyrequiredtoinducereleaseofF888dye.These resultsshowedthatilluminationthatexcitesselectivelyNR668dye insidenano-dropletscantriggerthereleaseofthesecond encap-sulateddyeF888.Thus,aprototypeofaphoto-releasesystemis developed,whereilluminationof nanocarrierscouldinducethe releaseofamoleculeofinterest,forinstanceinthedrugdelivery applications.
Finally,toclarifywhetherthewholenanocarrierisdeliveredor justitscontent,weincubatedthecellswitha mixtureof nano-droplets containingseparately NR668 and F888. In this case, if thewhole nanocarriers enter after theillumination, then both NR668-andF888-loadednanocarriersshouldbedetectedinside thecells.However,itwasfoundthatafterillumination,onlythe NR668signalwasobservedinsidethecells,whereastheF888signal remainedlow(Fig.5CandD).Thissuggestedthatlight illumina-tiontriggeredthereleaseofthedyecontentfromNR668-loaded
nanocarriersintothecells,but couldnotreallyinduce internal-ization of the wholenanocarrier. Moreover,the fact that F888 didnotenterthecellsafterilluminationalsosuggestedthatthe photo-releaseprocessdidnotdisruptthecellplasmamembranes. Thisfeatureisimportantinthedevelopmentofphoto-release sys-temswithminimalphoto-damagingeffects.TogetherwiththeFCS data,theseobservations suggest thatthelight actsat thelevel ofthenano-droplet,producingdestabilizationandfurthercargo releaseintothecells.Inthisrespectthedescribedphenomenon isclose tothelight-triggeredrelease fromlipidvesicles, which wasexplainedbyphoto-thermal orphotochemicalmechanisms
[38–42].Thedescribedphenomenonisalsorelevantto photochem-icalinternalization[34–37],however,inthelattercaselightacts mainlyatthelevelofcellmembranes.
3. Conclusions
Inthepresent work,aphenomenon oflight-induced release fromlipidnano-dropletsundervisiblelightconditionsisdescribed forthefirsttime. It wasobserved usingnanoemulsiondroplets of 32nm size encapsulating hydrophobic the analogue of Nile Red,NR668.Thesenanocarriersdidnotenterthecellsafter sev-eralhoursofincubationat37◦C,however,afterilluminationfor 30sunderthemicroscopeatthewavelengthof NR668 absorp-tion(535nm),fastaccumulationofthereleaseddyesinsidethe cellswasobserved.Thelight-triggeredreleasewasalsovalidated invivoonzebrafish,wherenano-dropletsshowingstableemission inthebloodcirculation,stainedsurroundingtissuesafter
illumina-Fig.5.FluorescenceimagingofHeLacellsincubatedfor15minat37◦Cwithnanocarrierbeforeandafterilluminationat535nmfor30s.Imagesweretakenevery2min.
(AandB)NanocarriersencapsulatingbothdyesF888-NR668(1wt%each).(CandG)MixtureofnanocarriersencapsulatingseparatelyF888(1wt%)andNR668(1wt%)dyes (1:1,v:v).F888channel:excitation360/40nmandemission470/40nm.NR668channel:excitation535/50nmandemission610/75nm.
tion.Usingfluorescencecorrelationspectroscopy,weshowedthat laserilluminationatrelativelylowpower(60mW/cm2)triggered dyereleaseinto10%serumorblanklipidnanocarriers.Asthis pro-cessisinhibitedbythedeoxygenatingagentsodiumsulfite,wecan concludethatthemechanismislinkedtooxygen-dependent pho-tochemistry,whichdestabilizesthenanocarrier.Nevertheless,we cannotexcludethataphoto-thermaleffectcouldalsotakepart,as proposedinsomerecentliteratureforNIRdyes.Finally,weshowed thatilluminationofNR668caninducethereleaseofanotherhighly hydrophobicdyefromthelipidnanocarriers,whichwasobserved asaccumulationofbothdyesinside livingcells.Based onthese results,dye-loadedlipidnano-dropletsemergeasapromising plat-formforlight-triggeredreleaseofactivemoleculesinbiomedical applications.Weexpectthatthisconceptcouldbereadilyextended tootherdyes,whichcouldinduceunderlightstimulustherelease ofdrugsfromnano-droplets.
4. Materialsandmethods
4.1. Materials
AllchemicalsandsolventswerepurchasedfromSigma-Aldrich. Kolliphor® HS15 non-ionic surfactant (mixture of polyethy-lene glycol 660 hydroxystearate and free polyethylene glycol 660 and) obtained from BASF (Ludwigshafen, Germany) was a kind gift from Laserson (Etampes, France), Labrafac® WL (medium chain triglycerides) was obtained from Gattefossé (Saint-Priest, France). MilliQ-water (Millipore) was used in all experiments. Culture reagents were obtained from Sigma (St. Louis,USA),Lonza(Verviers,Belgium)andGibco-Invitrogen(Grand Island, USA). Opti-MEM® reduced serum medium was from Gibco. Dihexylamino-2-(2-ethyl-hexyloxy)-benzo[a]phenoxazin-5- one (NR668) and 4-Dioctylamino-3-octyloxyflavone (F888) weresynthesizedasdescribedbefore[53].
4.2. Formulationandcharacterizationoflipidnanoparticle
Lipid nanocarriers were prepared by spontaneous nano-emulsification.Briefly,NR668orF888wassolubilisedinLabrafac® WL.Then,Kolliphor®HS15wasaddedandthemixturewas homog-enizedundermagneticstirringat90◦ C.Nano-dropletsof32nm diameterwerepreparedbyadding230mgofultrapurewatertoa mixtureof35mgofLabrafac® WLand65mgofKolliphor®HS15 underintensestirring.
Thesizedistributionofthenanoemulsionswasdeterminedby dynamic lightscattering ona Zetasizer® Nano seriesDTS 1060 (MalvernInstrumentsS.A.,Worcestershire,UK)andbyFCS (home-builtsetup, seebelow). In the DLS measurements statistics by volumewasused.
4.3. Cellculture
HeLacellswereculturedinDulbecco’smodifiedEaglemedium (D-MEM,highglucose,Gibco-invitrogen)supplementedwith10% (v/v) fetal bovine serum (FBS, Lonza), 1% antibiotic solution (penicillin-streptomycin,Gibco-invitrogen)inahumidified incu-bator with 5% CO2 at 37◦C. Cells plated on a 75cm2 flask at a density of 106 cells/flask were harvested at 80% confluence withtrypsin–EDTA(Sigma)andseededontoachambered cover-glass(IBiDi)ata densityof 0.1×106cells/IBiDi.After24h,cells in theIBiDi dishes were washed 2 times withPBS (phosphate buffersaline)(Lonza).Then,asolutionofdye-loadednano-droplets dilutedat1:1000in Opti-MEMwasadded.Microscopyimages weretakenafter30-minincubationat37◦C,unlessindicated. 4.4. Microscopy
Fluorescenceimagesweretakenona LeicaDMIRE2inverted microscopeequippedwithaLeicaDC350FXCCDcameraanda ther-mostatedchamber(LifeImagingServices,BaselSwitzerland)for maintainingthetemperatureat37◦C.Cy3filtercube(excitation 535/50nm,emission610/75nm)andDAPIfiltercube(excitation 360/40nm,emission470/40nm)wereusedfordetectionofNR668 andF888,respectively.A63×HCXPLAPO(1.32NA)wasusedasan objective.Inthephoto-releasestudies,theilluminationfor30swas doneusingthesameCy3filtercubeat∼14W/cm2powerdensity oflight.
4.5. Fluorescencecorrelationspectroscopy(FCS)anddyerelease FCSmeasurementswereperformedonatwo-photonplatform includinganOlympusIX70invertedmicroscope.Two-photon exci-tationat830nm(10mWlaseroutputpower)wasprovidedbya mode-lockedTsunamiTi:sapphirelaserpumpedbyaMilleniaV solidstatelaser(SpectraPhysics).Themeasurementswere real-izedinan96wellplate,usinga50uLvolumeperwell.Thefocal spotwassetabout20mabovetheplate.Thenormalized auto-correlationfunction,G(t)wascalculatedonlinebyanALV-5000E correlator(ALV,Germany)fromthefluorescencefluctuations,␦F(t), byG()=<␦F(t)␦F(t+)>/<F(t)>2where<F(t)>isthemean fluores-cencesignal,andisthelagtime.Assumingthatlipidnanoparticle diffusefreelyinaGaussianexcitationvolume,thecorrelation func-tion,G(),calculatedfromthefluorescencefluctuationswasfitted accordingtoThompson[64]: G () = 1 N
1+ d −1 1+ 1 s2 d −1/2 (1) wheredisthediffusion(correlation)time,Nisthemeannumber offluorescentspecieswithinthetwo-photonexcitationvolume, andSistheratiobetweentheaxialandlateralradiiofthe excita-tionvolume.Theexcitationvolumeisabout0.34fLandSisabout3–4.Typical datarecordingtimeswere5min,usingdye-loaded lipid nanoparticle diluted 1: 10 000 from the originally pre-parednanoparticle.Using6-carboxytetramethylrhodamine(TMR from Sigma-Aldrich) in water as a reference, the hydrody-namic diameter, d, of nanocarriers (NCs) was calculated as: dNCs=d(NCs)/d(TMR)×dTMR,wheredTMRisahydrodynamic diam-eterofTMR(1.0nm).ConcentrationofNCswascalculatedfromthe numberofspeciesby:CNCs=NNCs/NTMR×CTMR,usingaTMR con-centrationof50nM.Thedatawereobtainedbasedon20recorded correlationcurves;therecordingtimeforeachcurvewas10sfor NCsand30sforTMR.
ForthedyereleasestudiesbyFCS,dye-loadednano-droplets were diluted10 000 fold tofour differentmedia: water, Opti-mem,blanknanocarriersat1000-folddilution(10-foldexcesswith respecttodye-loadeddroplets)and10%ofFBSinwater(serum). Eachsamplewasplacedintospecial50lcuvetteandthewhole samplewasilluminatedwith532-nmlaserat∼60mW/cm2power densityfordifferenttime(10and30s,10,2030and60min)and wemeasurenumberofspeciesandtotalcountrate.
4.6. Invivostudiesinzebrafish
Zebrafishwerekeptat28◦Candbredunderstandardconditions. Thetransgenicline,Tg(flk1:eGFP)[65],expressingeGFP specifi-callyintheendothelialcells, wasusedin ordertovisualizethe vasculature.Fortheangiography,theembryos,3daysafter fertil-ization,wereanaesthetizedineggwatercontaining0.04%tricaine and0.05%phenylthiourea(Sigma-Aldrich)andimmobilizedin0.8% lowmeltingpointagarose(Sigma).Theinjectionswereperformed usingaNanojectmicroinjector(DrummondScientific,Broomal,PA, USA).TheglasscapillarywasfilledwithNR668-loaded nanocar-riers(5wt%inoil)in5mMHEPES(1000-folddilution)and2.3nL wereinjectedinthesinusvenosusoftheembryos.Thenanocarriers wereimmediatelydistributedinallthevasculature.Theinjected embryoswereplacedonthemicroscopestageandimagedwithin 5minafterinjection.Intravitalconfocalmicroscopywasperformed onaLeicaSP5fixedstagedirectmicroscopewitha25×(NA0.95) and10×(NA0.3)waterimmersionobjectives.488nmargonlaser linewasusedtoexcitebotheGFPandtheNR668dyes,andtheir emissionwasdetectedbytwoseparatePMTsinthespectralrange 500–530nmand620–650nm,respectively.Attheseconditions,no cross-talkbetweenthechannelswasobserved.Theilluminations forthelight-drivenreleasewereperformedwithaxenonlampin epifluorescencemodeusinga450–490nmbandpassfilter. Con-focalz-stacksandtimelapseswererecordedandtreatedbythe ImageJsoftware(rsbweb.nih.gov/ij/).Theembryoswereimaged duringonehourpostinjectionandnotoxicityduetotheinjectionor totheilluminationwasobserved.Allexperimentsperformedwith zebrafishcompliedwiththeEuropeandirective86/609/CEEand IGBMCguidelinesvalidatedbytheregionalcommitteeofethics.
Acknowledgements
ThisworkwassupportedbyERCConsolidatorgrantBrightSens 648528.R.BouchaalawassupportedbyMinistryofHigher Educa-tionandScientificResearchofAlgeria.WethankIevgenShulovfor re-synthesizingNR668,L.Richert,P.DidierandF.Przybillaforhelp withFCS,wethankR.VauchellesfromPIQimagingplatformfor helpwithfluorescenceimaging.Wealsoacknowledgehelpfrom theIGBMCimagingandzebrafishfacilities.
AppendixA. Supplementarydata
Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.colsurfb.2017.05. 035.
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