Aqueous dispersions of DMPG in low salt contain leaky vesicles

ContentslistsavailableatSciVerseScienceDirect

Chemistry

and

Physics

of

Lipids

jo u r n al 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 h e m p h y s l i p

Aqueous

dispersions

of

DMPG

in

low

salt

contain

leaky

vesicles

Rafael

P.

Barroso

_{, Katia}

_Regina

_Perez

_{, Iolanda}

_M.

_Cuccovia

_{, M.}

_Teresa

_Lamy

a_{InstitutodeFísica,UniversidadedeSãoPaulo,SãoPaulo,Brazil}

b_{DepartamentodeBiofísica,UniversidadeFederaldeSãoPaulo,SãoPaulo,Brazil} c_{InstitutodeQuímica,UniversidadedeSãoPaulo,SãoPaulo,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received25November2011

Accepted13December2011

Available online 20 December 2011

Keywords:

DMPG

Leakyvesicles

Spinlabels

[14_{C]sucroseentrapment}

Intrinsicviscosity

a

b

s

t

r

a

c

t

Aqueousdispersionsofdimyristoylphosphatidylglycerol(DMPG),atlowionicstrength,display uncom-monthermalbehavior.Modelsforsuchbehaviorneedtoassignaformtothelipidaggregate.Although moststudiesacceptthepresenceoflipidvesiclesinthelipidgelandfluidphases,thisisstillcontroversial. Withelectronspinresonance(ESR)spectraofspinlabelsincorporatedintoDMPGaggregates, quantifi-cationof[14_{C]sucroseentrappedbytheaggregates,andviscositymeasurements,wedemonstratethe} existenceofleakyvesiclesindispersionsofDMPGatlowionicstrength,inbothgelandfluidphasesof thelipid.Asacontrolsystem,theubiquitouslipiddimyristoylphosphatidylcholine(DMPC)wasused.For DMPGinthegelphase,spinlabelingonlyindicatedthepresenceoflipidbilayers,stronglysuggestingthat DMPGmoleculesareorganizedasvesiclesandnotmicellesorbilayerfragments(bicelles),asthelatter hasanon-bilayerstructureattheedges.Quantificationof[14_{C]sucroseentrappingbyDMPGaggregates} revealedthepresenceofhighlyleakyvesicles.Duetotheshorthydrocarbonchains(14_Catoms),DMPC vesicleswerealsofoundtobepartiallypermeabletosucrose,butnotasmuchasDMPGvesicles.Viscosity measurements,withthecalculationoftheintrinsicviscosityofthelipidaggregate,showedthatDMPG vesiclesarerathersimilarinthegelandfluidphases,andquitedifferentfromaggregatesobservedalong thegel–fluidtransition.Takentogether,ourdatastronglysupportsthatDMPGformsleakyvesiclesat bothgelandfluidphases.

© 2011 Elsevier Ireland Ltd.

1. Introduction

Dimyristoylphosphatidylglycerol(DMPG),ananionicsaturated phospholipidwith two 14_{C atoms chains, is themajor}

compo-nent ofmembranes of gram-positive bacteria(Umeyamaet al., 2006;Paradis-Bleauetal.,2007).DMPG,mixedwithzwitterionic phospholipids, hasbeenused tomimic membranesof bacteria (Chiaetal.,2002).DMPGhasalsobeenwidelyusedtostudythe interactionsofcationicpeptideswithlipidbilayers(Biaggietal., 1997;Prenneretal.,1999;Mozsolitsetal.,2001;Turchielloetal., 2002;Amonetal.,2008;BroemstrupandReuter,2010),sinceitis expectedthatpeptidescomposedofbasicaminoacidshavehigh affinityforanioniclipiddomainsinmembranes.

Inhighionicstrengthaqueoussolutions,DMPGexhibitsahighly cooperative gel–fluid phase transition, around 23◦_{C, similarto}

thetransitionofthezwitterioniclipiddimirystoyl phosphatidyl-choline (DMPC), which has also two 14_{C atoms chains. At low}

ionicstrength,DMPGdispersionsexhibitanuncommon thermo-structuralbehavior.Indistilledwater,DMPGwasreportedtoform smallbilayer“discsorshells”(EpandandHui,1986),orvesicles

∗_{Correspondingauthor.Tel.:+551130916829;fax:+541138134334.}

E-mailaddress:[email protected](M.TeresaLamy).

whichwouldfuseinasponge-likephasebelow∼31◦_C(Gershfeld

etal.,1986;Koshinumaetal.,1999).Clearly,theelectrostatic repul-sionbetweenchargedPG−_{groupsatlowionicstrengthdetermines}

thearchitectureofDMPGaggregates.

FreshlypreparedDMPGdispersionsatlowionicstrength(Hepes buffer, 0.01M,pH7.4) showa wide gel–fluidtransition region, betweenapproximately18◦_C(T

mon;theonsetofthetransition)and 35◦_C(T

moff;theoffsetofthetransition)(see,forinstance,Barroso etal.,2010;Alakoskelaetal.,2010,andreferencestherein).Along thistransitionregionseveralheatabsorptionpeakscanbedetected (Salonenetal.,1989;HeimburgandBiltonen,1994;Riskeetal., 1997,1999), andthedispersionshowslow turbidity(Heimburg andBiltonen,1994),highelectricalconductivity(Riskeetal.,1997; Barrosoetal.,2010)andhighviscosity(HeimburgandBiltonen, 1994;Schneideret al.,1999;Duarteet al.,2008;Barrosoetal., 2010).Thestructure ofDMPGaggregates alongthis remarkable transitionregionisstillamatterofdebate.Becauseofthehigh vis-cosityofDMPGdispersionatthetransitionregion,andonthebasis ofcryo-transmissionand freeze-fractureelectronmicroscopy,it hasbeenproposedthatDMPGatlowionicstrengthformsalipid network,similartotheso-calledspongephase(Schneideretal., 1999).However, this possibility was ruled out on the basis of experimentsthatdiscardedlipidexchangebetweenDMPG struc-turesalongthetransitionregion(Lamy-FreundandRiske,2003;

0009-3084© 2011 Elsevier Ireland Ltd.

doi:10.1016/j.chemphyslip.2011.12.007

Open access under the Elsevier OA license.

170 165 (2012) 169–177

Alakoskelaand Kinnunen,2007).Itwasshown thatthere isno fusion between DMPG aggregates along temperature variation, hencewhateverlipidaggregateispresentatacertaintemperature (forinstanceatthegelorfluidlipidphase)itwillbepresentalong thewholerangeofstudiedtemperatures,from5to50◦_C,though

itsformcanchange.

Thepurposeofthepresentworkistoexaminethestructure ofDMPGaggregatesinthegelandfluidphases(attemperatures belowTmonandaboveTmoff),atlowionicstrength.Evenoutside thetransitionregionthereisnoconsensusabouthowDMPG is organizedin aqueousdispersions.While Schneideretal.(1999) observedvesicularstructuresforDMPGatthegelandfluidphases, usingcryo-transmissionandfreeze-fractureelectronmicroscopy, Kinoshita et al. (2008), with freeze-etch electron microscopy, detectedDMPGvesiclesonlyinthefluidphase.

Threecomplementarytechniqueswereusedhere.Electronspin resonance(ESR)ofspinlabelsincorporatedintoDMPGaggregates inthelipidgelphaseclearlyindicatedthatlipidswereorganizedin bilayersandnotinmicellesorbicelles(bilayerfragments). Quan-tificationof[14_{C]sucroseentrapmentbytheaggregatesshowedthe}

presenceofhighlyleakyvesicles,andviscositydataindicatedthat DMPGaggregatespresentinthegelandfluidphaseswere simi-lar.Takentogether,ourdatademonstratethatDMPGinlowionic strengthaqueoussolutionsformsleakyvesicles,bothinthegeland fluidphases.

2. Materialsandmethods

2.1. Materials

DMPG (1,2-dimyristoyl-sn-glycero-3-[phospho-rac-glycerol]

sodium salt), DMPC

(1,2-dimyristoyl-sn-glycero-3-[phosphocholine]), 5-PCSL(1-palmitoyl-2-(5-doxyl stearoyl)-sn-glycero-3-phosphocholine), and 16-PCSL (1-palmitoyl-2-(l6-doxylstearoyl)-sn-glycero-3-phosphocholine) werefrom Avanti Polar Lipids (Birmingham, AL, USA) and used without further purification. The buffer used throughout was Hepes (4-(2-hydroxyethyl)-1-piperizineethanesulfonic acid), obtained from Sigma–Aldrich (St Louis, MO, USA). Deionized water (Milli-Q Plus,Millipore).[14_{C]sucrose,fromAmershanLifeScience,hasa}

specificactivityof612mCi/mmolandaradioactiveconcentration of 200Ci/ml. Biodegradable Counting Scintillant Cocktail (BCS) wasfromAmershamLifeScience.

2.2. Lipiddispersionpreparation

ForDMPGandDMPCdispersions,alipidfilmwasformedfrom achloroformsolution,driedunderastreamofN2andleftunder

reducedpressure fora minimumof2h, toremovealltraces of organic solvent. Dispersions were prepared by the addition of Hepesbuffer(10mMadjustedwithNaOHtopH7.4,+2mMNaCl, final[Na+_{]=6mM)followedbyvortexingfor2minabovethelipid}

gel–fluidtransitiontemperatureforDMPC(Tm=23◦C)orTmofffor DMPG(theendofthegel–fluidtransition,Tmoff∼35◦C).AsDMPC formsverylargemultilamellarvesicles,thelipiddispersionwas extruded(11times)throughpolycarbonatefilters(100nmpores) toyieldlargeunilamellarvesicles(LUV)(Hopeetal.,1993).For ESRmeasurements,spinlabels(5-PCSLor16-PCSL)wereaddedto thelipidchloroformsolutionat0.2mol%relativetothelipid con-centration(10mM),hencenospin–spininteractionwasobserved. Sampleswerekeptatroomtemperatureandusedimmediately afterpreparation.Forchromatography,25mMDMPGandDMPC wereprepared.Viscositymeasurementswereperformedwith dif-ferent DMPGconcentrations (see Fig.7), preparedas described above.

2.3. Chromatography

Toobtainvesicleswith[14_{C]sucroseentrappedinsideand}

out-side, (In/Out), radioactive sugar wasadded tothe Hepes buffer to a final radioactive count of 40×103_{cpm/␮l. To maintain}

[14_{C]sucroseonlyoutside(Out),thelipidwasfirstsuspendedwith}

Hepesbufferasdescribedabove(DMPCwentthroughthe extru-sionprocess),thesamplewaskeptatthedesiredtemperature(6or 45◦_{C)and,afterthermalequilibration,the[}14_{C]sucrosewasadded.}

Sampleswerekeptatthedesiredtemperaturebeforeandduring thechromatographicprocedure,hencenotgoingthroughthelipid transitiontemperature.

Both preparations, containing [14_{C]sucrose (In/Out) or}

(Out), were passed through a Sephadex G-25 medium col-umn(1.2cm×28.6cm),previouslysaturatedwithDMPGorDMPC dispersions,atthetemperatureoftheexperiment,andelutedwith Hepesbuffer.Thesamplevolumewas250␮landthephospholipid

concentration was 25mM. The chromatographic system was

keptatthedesiredtemperature(6◦_Cor45◦_{C).Aliquotsof0.5ml}

werecollected.Forthedeterminationoflipidconcentration,the phosphate content of each tube was assayed. Each fraction of thecolumnwasradio-assayedbyaddinganaliquotof0.4–10ml of BCS, and countingthe [14_{C]sucrose ␤-radiation activity in a}

PackardLiquidScintillationSpectrometer1600TR.

2.4. Determinationoftrapped[14_C]sucrose

Thepercentageofentrappedsucrosewascalculatedas

p= 100R0

R (1)

where R is the total amount of radioactivity obtained fromall collectedfractions,andR0thetotalradioactivityfromonlythose

thatalsopresentedphosphorouscontent.Theexperimental appar-entinternalvolumebymoloflipids(Vexp),inunitsofl/mol,was

expressedas

Vexp= p

100[lipid]_eff (2)

where [lipid]eff=f[lipid]In is defined as an effective

concentra-tionin mol/l, i.e., thelipid concentration added tothe column ([lipid]In=25mM)correctedbyafactorf,whichistheratiobetween

thenumberofmolesoflipidcollectedatVo(voidvolume)andthe totalnumberofmolesoflipidaddedtothecolumn.Then(1−f) indicateshowmuchlipidwasretainedatthetopofthecolumn.

Assuming spherical vesicles, with external radius R, bilayer thicknessdandthesameareaperlipidheadgroup(a)intheinternal andexternalmonolayers,theinternalvolume,VT,canbe theoreti-callycalculated:

VT=1000×

aNA(R−d)3

3[R2_+(R−d)2_] (3)

whereNAistheAvogadro’snumber,aisexpressedinm2andR anddinm.Inanexperimentwithentrapmentof[14_{C]sucroseinthe}

internalvolumeofthevesicles,andnoleak,itwouldbeexpected thatVexp=VT,Vexpmeasuredattheendofthecolumn.Hence,the

trapefficiencyattheendofthecolumn,Eff,canbedefinedas

Eff=

Vexp

VT

(4)

2.5. Determinationofphospholipidconcentration

165 (2012) 169–177 171

DMPG DMPG

DMPC 45 Co

5-PCSL

16-PCSL 5 Co

DMPC

Fig.1. ESRspectraofspinlabels(5-PCSLand16-PCSL)incorporatedinto10mM

DMPCandDMPGat5◦_{C(leftcolumn)and45}◦_{C(rightcolumn).Totalspectrawidth}

100G.

wereassayed,andtheamountofphospholipidineachfractionwas determined.Theconcentrationofphosphateineachsamplewas calculatedusingastandardcurve(from10to100␮MofNaH2PO4)

determinedwithastandardsolutionofNaH2PO4(1×10−3M).

2.6. ESRspectroscopy

ESRmeasurementswereperformedwithaBrukerEMX spec-trometer.Thesampletemperaturewascontrolledwithin0.1◦_Cby

aBrukerBVT-2000variable-temperaturedevice.Toensurethermal equilibrium,beforeeachscan,thesamplewasleftatthedesired temperatureforatleast10min.TheESRdatawereacquired imme-diatelyaftersamplepreparation. Field-modulationamplitudeof 1Gandmicrowavepowerof10mWwereused.

2.7. Viscositymeasurements

ViscositiesweremeasuredwithanOstwald viscometer (Vis-coClockUnitfromSCHOTTInstruments,Germany)coupledtoa thermostatbath(SCHOTTInstruments,Germany).The tempera-turewasmeasuredwithaFluke51K/Jthermometerimmersedin thebath(precisionof0.1◦_{C)andallowedtoequilibratefor10min}

beforedataacquisition.

3. Resultsanddiscussion

3.1. ESRofspinlabels

ESRspectraofspinlabelsincorporatedintolipidmembranescan monitorthebilayergel–fluidtransition,yieldingtypical,and dis-tinctESRsignalsforthegelandfluidphasesofthebilayer(see, for instance,McConnell,1976).Fig.1shows theESR spectraof twophospholipidspinprobes,whichmonitordifferentdepthsof abilayer:5-PCSLmonitorsthebilayermicro-environmentaround the5thC-atomofthehydrocarbonchain,and16-PCSLislocatedat thebilayercore.DifferencesbetweenESRspectraof5-and16-PCSL areduetothewell-knownflexibilitygradienttowardthebilayer core(McConnellandMcFarland,1972).Fig.1showsthatspectraof 5-or16-PCSLincorporatedintoDMPCandDMPGdomainsare sim-ilar,indicatingcomparablestructuresforthetwolipids,i.e.,at5◦_C,

ESRspectraaretypicalofspinprobesingelphasebilayersandat 45◦_{CESRspectraindicatefluidmembranes.Thepresenceoftypical}

gelbilayerspectrainDMPGlowionicstrengthdispersions,similar tothoseofthewell-studiedDMPC(Fig.1),isstrongevidencethat DMPGismostlyorganizedasbilayers,andnotmicelles.

A+B B A

(a)

(b)

Fig.2. (a)ESRspectraof16-PCSLincorporatedintoDMPGatthegel(B,5◦_C)and

fluid(A,35◦_{C)phases,normalizedtorelativedoublyintegratedintensitiesof0.99}

and0.1,respectively.(b)Thesumofthetwosignals(A+B),witharrowsindicating

thefeaturesofthespinlabelincorporatedinfluidlipids(A).Totalspectrawidth

100G.

The presence of significant amounts of bilayer fragments (bicelles)inDMPGdispersioncanalsobediscarded,asthe coex-istenceofevena smallpercentageoffluidlipids, corresponding tothe bicelle edge,can beclearly detectedcoexisting withgel bilayersasdiscussedbelow.Spinlabeledsonicateddispersionsof thecationicamphiphiledioctadecyldimethylammoniumbromide (DODAB)(Benattietal.,2011;Oliveiraetal.,2011),which form bicelles(Pansuetal.,1990;Cocquytetal.,2004)exhibitboth gel-andfluid-likespectra.Toillustratethefactthatevensmallamounts ofdisorganized lipids(fluid) coexistingwithgelbilayerscanbe detectedasshowninFig.2atheESRsignalof16-PCSLingel(5◦_C,

B)and fluid DMPG(35◦_{C, A).Spin labelrelative concentrations}

are99:1,gel:fluid,bythecalculationofthedoubleintegral,which quantifiestheamountofresonatingspins.In Fig.2b,where the additionofthetwosignalsisshown(A+B),itcanbeseenthateven 1%ofspinlabelsmonitoringafluiddomain(seearrowsinFig.2b) canbedetectedinthepresenceof99%oflabelsincorporatedingel bilayers.Eveninaverylargelipidbicelle,with50nmofradius,the fractionoflipidsattheedgewillbeabout1mol%.

Hence,theobservationthatspinlabelsincorporatedintoDMPG aggregatesbelow17◦_{Cindicatesthepresenceofgelbilayersonlyis}

astrongindicationthatDMPG,in10mMHEPESbuffer+2mMNaCl, pH7.4,ismostly organizedasbilayers,makingvesiclesandnot micellesorbicelles.AsimilarconclusioncannotbedrawnforDMPG athighertemperaturesfromESRspectroscopy.Abovethegel–fluid transitionofthelipid,itisnotpossibletodistinguishthetwoESR components(bilayerandbicelleborder)eitherduetothesimilarity ofthetwosignalsortothequickexchangeoflabelsbetweenthe twodomainsintheESRtimescale(Benattietal.,2011;Oliveira etal.,2011).

3.2. Entrapmentof[14_C]sucrose

Vesiclesarecharacterizedbythepresenceofaninternalwater compartment.Onemethodtodemonstratethepresenceof vesi-clesina lipiddispersionistheincorporation ofa labelin their internalaqueousvolume.Radioactivemoleculessuchassucrose ([14_{C]sucrose)havebeenwidelyusedtomeasure theefficiency}

172 165 (2012) 169–177

50 40 30 20 10 0 0 5 10 15 20 25

0.0 0.2 0.4 0.6

0.0 0.2 0.4 0.6

0.0 0.2 0.4 0.6

0.0 0.2 0.4 0.6

(b)

(a)

% of total radioactivity

Fraction number

Absorbance at 797 nm

50 40 30 20 10 0 0 5 10 15 20 25

% of total radioactivity

Fraction number

25 20

15 10

0.0 0.5 1.0

(c)

% of total radioactivity

Fraction number

25 20

15 10

0.0 0.5 1.0

(d)

% of total radioactivity

Fraction number

Absorbance at 797 nm Absorbance at 797 nm

Absorbance at 797 nm

Out

In/Out Gel phase

Fig.3. ElutionprofilesthroughSephadexG-25columnsofDMPCvesicles(25mM)atthegelphase(6◦

C),preparedwith[14_{C]sucrose(a)In/Out,(b)Outofthevesicles,(c)}

and(d)areenlargementsofthedatainphospholipidscontainingfractionsin(a)and(b),respectively.

whetherDMPGaggregatescanbedemonstratedtohaveaninternal volumeinthegel(6◦_{C)andfluid(45}◦_{C)phases.Itwasnotpossible}

toperformthechromatographyalongthetransitionregion,asthe sampleisveryviscous,asmentionedinSection1.

AsdescribedinSection2,vesicleswerepreparedeither(a)inthe presenceof[14_{C]sucrose,aggregatesIn/Out;or(b)[}14_C]sucrosewas

addedaftervesiclepreparation,aggregatesOut.Freeandvesicle entrapped[14_{C]sucrosewereseparatedbycolumnfiltration(see}

Section2).Consideringthatfree[14_{C]sucroseelutesthroughthe}

columnslowerthanlipidaggregates,free[14_C]sucroseand

vesi-cletrapped[14_{C]sucroseareexpectedtobedetectedindifferent}

fractions.

Asacontrolweentrapped[14_{C]sucroseinDMPCdispersions.}

Figs.3and4showelutionprofilesofDMPCvesiclespreparedwith [14_{C]sucroseIn/Out(a)andonlyOut(b),inthegel(6}◦_C,Fig.3)and

fluid(45◦_{C,Fig.4)phasesofthelipidmembrane.}

Inthegelphase,withsucroseIn/OutDMPCvesicles,weobserved tworadioactivity peaks.The firstpeak (fractions15–20,Fig.3a andc),coincidedwiththephospholipid,stronglysuggestingthat the[14_{C]sucrosewasentrappedinvesicles.Thesecond}

radioactiv-itypeak(fractions25–35),correspondstofree[14_{C]sucrose.The}

percentageofentrapped[14_{C]sucrose,calculatedbyEq.(1),was}

2.3±0.1%.With[14_{C]sucroseOut,theelutionprofileshowedonly}

oneradioactivitypeak(Fig.3bandd),intheelutionpositionof free[14_{C]sucrose.Enlargingthescales(Fig.3d)showedno}

signifi-cantradioactivityinthefractionscontainingphospholipidinadded Out,demonstratingthatnosignificantamountofsucrosewaseither incorporatedintogelDMPCvesiclesoradsorbedfromsolution.

Inthefluid phase ofDMPC, withsucroseIn/Out,theelution profilewassimilar tothat obtainedin thegel phase (compare Figs. 3 and 4a and c). Both gel and fluid phases incorporated 2.5±0.1%[14_{C]sucroseinthephospholipid-containingpeak.}

Inter-estingly, with [14_{C]sucrose Out of the vesicles, we observed a}

smallentrapmentof[14_{C]sucroseintoDMPCvesicles(Fig.4band}

d, compare withFig. 3b and d), corresponding to 0.12±0.01% (seeTable1).Thisimpliesthat asmallinfluxof[14_C]sucroseto

the internal compartment of DMPC occurs in the time of the experiment.

InexperimentswheresucrosewasaddedoutsideLUVsofDMPC andDMPG,vesicleswerepreviouslytemperatureequilibratedand sampleswereappliedtothecolumnimmediatelyafter[14_C]sucrose

addition.Aspermeationistime-dependent,theincubationtimeof vesicleswithsucroseisimportantinordertoobtainincorporation oftheprobefromoutside.Theseexperimentsdemonstratedthat therewasnoadsorptionof[14_{C]sucrosetoDMPCvesiclesbecause}

inthegelphasewefoundnoradioactivityinthevesicle-containing peak,henceasmallincorporationofsucroseinthefluidphaseof DMPCoccurredinthetimeoftheexperiment.

Taking the diameter of DMPC vesicles as 100nm

(R=50×10−9_{m in Eq. (3), 0.53 and 0.65×10}−18_m2 _{area per}

lipidheadgroup(avaluesinEq.(3)),and5.02and4.48×10−9_m

bilayerthickness(dvalues inEq. (3))(Marsh,1990),at geland fluid phases, respectively, we calculate an internal volume of 2.1l/mol forthegelphase and2.7l/mol forthefluidphase(Eq. (3)).Experimentally determinedencapsulated volumes(see Eq. (2))were0.9±0.1and1.0±0.1l/mol,forthegelandfluidphases, respectively, with[14_{C]sucrose In/Out the vesicles. Thus, DMPC}

165 (2012) 169–177 173

50 40 30 20 10 0 0 5 10 15 20 25 30

0.0 0.5 1.0 1.5 2.0

0.0 0.5 1.0 1.5

0.0 0.5 1.0 1.5 2.0

0.0 0.5 1.0 1.5

(b)

(a)

% of total radioactivity

Fraction number

Absorbance at 797 nm

50 40 30 20 10 0 0 5 10 15

Out

% of total radioactivity

Fraction number

18 16 14 12 10 8 6 0.0 0.5 1.0

(c)

% of total radioactivity

Fraction number

18 16 14 12 10 8 6 0.0 0.5 1.0

(d)

% of total radioactivity

Fraction number

Absorbance at 797 nm Absorbance at 797 nm

Absorbance at 797 nm

In/Out

Fluid phase

Fig.4. ElutionprofilesthroughSephadexG-25columnsofDMPCvesicles(25mM)atthefluidphase(45◦

C),preparedwithsucrose(a)In/Outor(b)Outthevesicles,(c)and

(d)aretheamplificationofthedatainfractionscontainingphospholipidsin(a)and(b),respectively.

entrapmentofsucroseinDMPCLUVs,asmeasuredhere,areto beexpected,duetotheknownpermeabilityofDMPCvesiclesto sucrose,eventhroughmultibilayers.

The incorporation experiments described above with DMPC demonstratedtheexistenceofinternalvolumeinasystemwell characterized as containing vesicles. To monitor the vesicles presentinDMPGdispersions,wemeasured[14_C]sucrose

incorpo-rationinDMPGdispersions.

Fig.5 shows elutionprofiles of DMPG aggregates in thegel phase, preparedwith[14_{C]sucroseIn/Outand with[}14_C]sucrose

Out.Inordertodemonstratethereproducibilityofourexperiments weshowtheresultsof twodifferentpreparations.Noevidence ofincorporationof[14_{C]sucroseintoDMPGaggregatesinthegel}

phasewasfound,sincenosignificantradioactivitywasdetected inthefractionsthatcontainedphospholipids:forthefoursamples theonlyradioactivitypeakdetectedwasthatoffree[14_C]sucrose.

Forthefluidphaseofthelipid,elutionprofilesofDMPG aggre-gates are shown in Fig. 6. Differently from the gel phase, we observedasignificant,radioactivitypeakassociatedtoentrapped [14_{C]sucroseinsamplespreparedwith[}14_{C]sucroseIn/Outvesicles}

(Fig.6,profilesontheleftside).Itisimportanttonotethatthe recoveriesoflipid,afterpassingDMPGvesiclesthroughthe col-umn,are80%and90%whentheexperimentaredonebelowand aboveTm,respectively.Thissmallamountoflipidstuckatthetop ofthecolumnwastakenintoaccountinEq.(2),inthecalculation ofVexp.

Thepercentageofentrapmentof[14_{C]sucrosewas0.8±0.1%in}

onesample(Fig.6aandc)and0.5±0.1%inanothersample(Fig.6e andg).Inoneofthesamplespreparedwith[14_{C]sucroseOut(Fig.6b}

and d),some[14_{C]sucrose seemstoelutewiththelipid,which}

wouldbeanindicationof[14_{C]sucrosepenetratingDMPGvesicles.}

However,itisnotpossibletoquantifysuchentrapmentduetothe

Table1

Entrapped[14_{C]sucrose(pinEq.(1)),apparentinternalvolume(V}

expinEq.(2)),andtrapefficiency(EffinEq.(4))ofDMPCandDMPGdispersions,inthegelandfluidphases,

withsucroseIn/OutandOutoflipidaggregates.

DMPC DMPG

p(%) Vexp(L/mol) Eff(%) p(%) Vexp(l/mol) Eff(%)

Gelphase

In/Out 2.3±0.1 0.9±0.1 42±5 0 0 0

0 0 0

Out 0 0 0 0 0 0

0 0 0

Fluidphase

In/Out 2.5±0.1 1.0±0.1 37±4 0.8±0.1 0.35±0.04 11±5

0.5±0.1 0.22±0.04 7±3

Out 0.12±0.01 0.05±0.01 1.9±0.4 a_{– – –}

0 0 0

a_{ForoneoftheDMPGsamples,atthelipidfluidphase,itwasnotpossibletocalculatereliablevaluesofentrappedsucrose,duetotheoverlapofthevesicleentrapped}

174 165 (2012) 169–177 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 10 20 30 40 50

0 5 10 15 20 25 (a)

Fraction number

% of t ot al r adi oa c ti v it y Abs o rb a nc e at 79 7 nm

10 20 30 40 50

0 5 10 15 20 25 (b) % of t ot al r adi oa c ti v it y

Fraction number

14 16 18 20 22

0.00 0.05 0.10 0.15 (c)

Gel phase

Fraction number

14 16 18 20 22

0.00 0.05 0.10 0.15 (d) % of t o ta l ra di oa c ti v it y

Fraction number

0 10 20 30 40 50

0 2 4 6 8 10 12 (e) % o f to ta l r a d io a ct iv it y

Fraction number

0 5 10 15 20 25 30 35 40 0 2 4 6 8 10 12 14 _(f) % o f to ta l r a d io a ct iv it y

Fraction number

8 10 12 14 16 18

0.00 0.05 0.10 0.15 (g) % of t o ta l r a di oa c ti v it y

Fraction number

8 10 12 14 16 18

0.00 0.05 0.10 0.15 (h) % of t o ta l r a di oa c ti v it y

Fraction number

Ab s o rba nc e at 79 7 nm Abs or ban c e at 797 nm Abs or b a nc e at 79 7 nm Abs o rb a nc e at 79 7 nm Ab s o rba n c e at 79 7 nm Abs or ban c e at 797 nm Out In/Out Abs or b a nc e at 79 7 nm

Fig.5.ElutionprofilesthroughSephadexG-25columnsofDMPGaggregates(25mM)atthegelphase(6◦

C),preparedwith[14_{C]sucroseIn/Out((a)and(e)aretwoidentically}

preparedexperiments),orOut((b)and(f)aretwoidenticallypreparedexperiments)ofthevesicles.(c),(d),(g)and(h)areamplificationsofthedatainfractionscontaining

phospholipidsin(a),(b),(e)and(f),respectively.

overlapofthevesicleentrappedsucrosepeakandthepeakoffree sucrose.

DifferentfromDMPCdispersions,DMPGdispersionswerenot extruded,sincenolargemultilamellarstructuresarepresentatlow ionicstrength(Riskeetal.,2001;Fernandezetal.,2008). Consider-ingthatthez-averageradiusobtainedbydynamiclightscattering forDMPGaggregatesinthegelandfluidphases,R∼70×10−9_m

(Alakoskela and Kinnunen,2007),is probablymuch largerthan thenumber-averageradiusofthevesicles(see,forinstance,Berne andPecora,2000),VTvalueswerecalculatedassumingRvarying from70to50×10−9_{m,andthisvariationwasconsideredinthe}

calculationofanerrorintheVTvalue.Otherparametersusedfor thecalculationofVT were0.6×10−18m2 and4.48×10−9m, for theareaperlipidheadgroup(avalueinEq.(3))andbilayer thick-ness(dvalueinEq.(3)),respectively(Marsh,1990).Accordingly, VTwasfoundtobe3.7±1.2l/mol,comparedwith0.35±0.04l/mol (Fig.6aand c)and0.22±0.04l/mol(Fig.6eandg), pointingto the fact that DMPG aggregates are constituted byrather leaky vesicles.

DatadiscussedabovearesummarizedinTable1:thepercentage ofvesicleentrapped [14_{C]sucrose(pin Eq.(1)), the}

165 (2012) 169–177 175 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2

0 10 20 30 40 50

0 5 10 15 20 Out

In/Out

Fluid phas

e

(a)

Fraction number

% o f to ta l r a d io a cti v it y A bs o rb an c e at 79 7 nm

0 10 20 30 40 50

0 5 10 15 (b) % o f to ta l r a d io a cti v it y

Fraction number

10 12 14 16 18 20 0.0 0.1 0.2 0.3 0.4 (d) (c) (e) % o f to ta l r a d io a cti vi ty

Fraction number

10 12 14 16 18 20 0.0 0.1 0.2 0.3 0.4 % o f to ta l r a d io a cti vi ty

Fraction number

0 10 20 30 40 50

0 5 10 15 (f) % of t o ta l ra di oa c ti v it y

Fraction number

0 10 20 30 40 50

0 5 10 15 % o f to ta l r a d io a c ti v it y

Fraction number

8 10 12 14 16 18 20 0.0 0.1 0.2 0.3 0.4 0.5 0.6 (g) % of tot a l r a d io a ct ivit y

Fraction number

8 10 12 14 16 18 20 0.0 0.1 0.2 0.3 0.4 0.5 0.6 (h) % of tot a l r a d io a ct ivit y

Fraction number

A b s o rb an c e at 79 7 nm Ab s o rb anc e a t 7 97 n m Ab s o rb anc e a t 7 9 7 n m A bs o rb an c e at 79 7 nm A b s or ban c e at 79 7 nm Ab s o rb anc e a t 7 97 n m Ab s o rb anc e a t 7 9 7 n m

Fig.6. ElutionprofilesthroughSephadexG-25columnsofDMPGaggregates(25mM)atthefluidphase(45◦

C),preparedwith[14_{C]sucroseIn/Out((a)and(e)aretwo}

identicallypreparedexperiments),orOut((b)and(f)aretwoidenticallypreparedexperiments)ofthevesicles.(c),(d),(g)and(h)areamplificationsofthedatainfractions

containingphospholipidsin(a),(b),(e)and(f),respectively.

efficiency(EffinEq.(4)).ClearlyDMPGvesiclesarepartially

per-meable tosucrose, and this permeability is much greater than thatexhibitedbyDMPCvesicles,sincetheentrappingefficiency ofDMPGvesiclesismuchlowerthanthatofDMPCvesicles.The presenceofthenegativechargeinDMPGcouldberesponsiblefor thehigherpermeabilityofDMPGLUVswhencomparedtoLUVs ofDMPC.Electrostaticrepulsionbetweenchargedheadgroupsin thebilayercouldincreasethedistancebetweenmonomersand this couldleadtoan increasein thepermeability ofthe mem-brane. However, it is important to have in mind that ESR of spinlabelsincorporatedintothebilayerhydrophobicacylchains

indicatessimilarpackingstructuresforDMPGandDMPC,atgeland fluidphases(Fig.1).

Itisnoteworthythattheflowinthecolumnathigher temper-ature(45◦_{C),forfluidbilayervesicles,wasaroundtwofoldthatat}

lowertemperature,6◦_{C (gelphase lipids).Therefore,vesiclesin}

176 165 (2012) 169–177

of [14_{C]sucrose for fluid and gel bilayer vesicles, was similar}

(Table1).

ESRspectraofspinprobesinDMPGaggregatesinthegelphase clearly indicated that DMPG is organized as vesicles. In addi-tion,experimentswith[14_{C]sucroseentrapmentdemonstratedthe}

formationofleakyDMPGvesicles.Experimentswithspinand flu-orescent probes show nofusion of DMPG aggregates over the rangeoftemperaturestudiedhere,includingthetransitionregion (Riskeetal.,1999;Lamy-FreundandRiske,2003;Alakoskelaand Kinnunen,2007).Furthermoreconsideringthatonlyasmall per-centageof[14_{C]sucrosewasencapsulatedbyfluidDMPG,wehave}

astrongindicationthatDMPGleakyvesiclesarepresentinthegel andfluidphasesofthelipid.Viscosityexperimentsdiscussedbelow supportthishypothesis.

3.3. IntrinsicviscosityofDMPG

Asmentionedbefore,oneoftheunusualcharacteristicsofthe transitionregionofDMPGatlowionicstrengthisthehighviscosity ofthedispersion(HeimburgandBiltonen,1994;Duarteetal.,2008; Barrosoetal.,2010).Theincreaseintheviscosityofthedispersion hasbeenassociatedtostructuralchangesofDMPGaggregatesalong thetransitionregion(Schneideretal.,1999;Barrosoetal.,2010).

The relative viscosity of a dispersion, r, is defined as r=/buffer,wherebufferisthepuresolventviscosityandisthe dispersionviscosity.Atlowconcentrations,therelativeviscosityis afunctionofthelipidconcentration,c,andcanbeexpressedas:

r=1+[]c+k1[]2c2+k2[]3c3+... (5)

where[],theintrinsicviscosity,isthemainparameterusedto obtaininformationaboutthesizeandshapeofcolloidalparticles

0.014 0.012 0.010 0.008 0.006 0.004 0.002 0.000 1.0 1.1 1.2 1.3 1.4 1.5

45 oC

ηr

[DMPG] (g/mL)

0.014 0.012 0.010 0.008 0.006 0.004 0.002 0.000 0 2 4 6 8 10 12 14

22 oC

ηr

[DMPG] (g/mL)

0.014 0.012 0.010 0.008 0.006 0.004 0.002 0.000 1.0 1.1 1.2 1.3 1.4

7 oC

ηr

[DMPG] (g/mL)

Fig.7. Relativeviscosity(r=/buffer)asafunctionofDMPGconcentration,atthe

gel(7◦

C)andfluid(45◦

C)phases,andatthetransitionregion(22◦

C).Curvesarethe

bestfitsobtainedusingEq.(5),truncatedatthirdterm.Dataareaveragevaluesofat

leastthreemeasurementswithdifferentsamples.Whennotshown,theuncertainty

(standarddeviation)wasfoundtobesmallerthanthesymbolinthegraph.

Table2

IntrinsicviscositiesofDMPGdispersions,obtainedbythebestfitsofthedatain

Fig.5.

Temperature(◦

C) [](ml/g)

7 13±1

22 522±137

45 13±1

(see,forinstance,Larson,1999).Thisisanextensionofthe funda-mentalworkofEinsteinonspheres(Einstein,1905,1906).

InFig.7,thedependenceofthedispersionrelativeviscositywith DMPGconcentration(atlowionicstrength)isshownatthree tem-peratures,representativeofthegelandfluidphases(7and45◦_C,

respectively)andthetransitionregion(22◦_{C)(datafromBarroso}

etal.,2010).

Theadjustmentof second-degreepolynomialstothe experi-mentaldataallowedthecalculationoftheintrinsicviscosity,[], accordingtoEq.(5),forthethreelipidphases(showninTable2).

Identicalvaluesobtainedforthegelandfluidphasesindicate thatDMPG aggregates havesimilarstructure atthesetwo con-ditions,supportingourpreviousdiscussionaboutthepresenceof DMPGleakyvesiclesatbothlipidphases.

Though the DMPG transition region is not the focus of the presentwork,itisinterestingtopointoutthatalongthisanomalous phaseofthelipid,theintrinsicviscosityofthedispersionismuch higher,consistentwithnon-sphericalshapefortheaggregates,the presenceofbilayerdeformations,includingbilayerpores(Riske etal.,2004),ortatteredbilayersheets(AlakoskelaandKinnunen, 2007),and/ortheincreaseontheelectricalchargeoftheDMPG aggregate(Barrosoetal.,2010).

4. Conclusions

Complementary techniques were used here to show that

DMPG,in low ionicstrengthaqueous dispersion(10mM Hepes buffer+2mM NaCl, pH7.4),is organized asvesicles, in thegel and fluid phasesof thelipid.At low temperatures, ESRof spin labelsclearlyindicated thepresenceofgelbilayersonly, show-ingthat DMPG isorganizedas vesiclesfor temperaturesbelow thegel–fluidtransitionofthelipid.TheanalysisoftheESRsignal excludesthepresenceofasignificantamountofbicelles,where agelbilayercoexistswithafluidbilayeredge.Therefore, consid-eringthepresenceofDMPGvesicles,theabsenceof[14_C]sucrose

entrappedbygelphaseDMPGvesiclesrevealedthatthevesicles areratherleaky.Forhighertemperatures,thesmallpercentageof entrapped[14_{C]sucrosebyfluidDMPGaggregates,andthe}

simi-laritybetweenintrinsicviscosityvaluesmeasuredforDMPGatgel andfluidphases,stronglyindicatedthatDMPGwasalsoorganized asleakyvesicles.

Acknowledgments

ThisworkwassupportedbyUSP,FAPESP,Capes(RPBandKRP, PhDfellowships)andCNPq(MTLandIMC,researchfellowships). WearegratefultoProf.P.S.Araujoforkindlygivingthe[14_C]sucrose

andscintillationliquidusedintheexperimentsofthiswork,andto Dr.E.L.DuartefortheESRdata.WethankM.A.Silvafortechnical assistanceinsomeexperiments.

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