Rapid Spontaneous Assembly of Single Component Liposomes

6.3 MB·English

by  P. Sunthar

RapidSpontaneousAssemblyofSingleComponentLiposomes
P. Sunthar and Sopan M. Phapal
∗
DepartmentofChemicalEngineering,IndianInstituteofTechnologyBombay(IITB),Powai,Mumbai400076,India.
Wepresentamechanismandshowtwovariantsofamethodwheretheaveragediameterofspontaneously
(barrier-free)assembledsinglecomponentunilamellarliposomesisintrinsic,inagreementwithHelfrich’sthe-
ory.Itdependsonlyonthetemperatureandthelipidtype,eliminatingkineticeffectsorexternalforcingnormally
observed. Thisprovidesthefirstpuresystemtostudytheself-assemblyofvesicleformingcomponents, and
withanaturallengthscaleitmayhaveanimplicationforvesiclesizeselectionunderpre-bioticconditions.
Current hypotheses for the formation of enclosing mem- scale, this diameter has not been observed by a spontaneous
5
branesofprotocells(pre-bioticvesicles),aremainlyonpossi- processstartingfromasolutionphaseofasinglecomponent
1
blesurfactants,theiroriginsorsynthesisroutes,autocatalysis, lipidsystem.
0
2 and their chemical evolution [1–4]. While these chemistries Stronghydrophobicinteractionofdoubletailedsurfactants
arethemostcrucialelementsinsolvingthepuzzleoforiginof and the high energy state of the intrinsic diameter are the
y
a cellularlife,thephysicsofself-assemblyhasnotseenanysig- chief reasons behind the difficulty of observing such a spon-
M nificantattentioninthiscontext. Helfrichsfreeenergymodel taneous assembly [6, 16]. The small critical aggregation
[5]providesthesimplestdescriptionofvesicleself-assembly concentrations of phospholipids (typically less than a nM),

8 from solution, which has a natural length scale for its aver- does not permit efficient dispersion and mixing of the sur-
age diameter. We show how this can be achieved through a factants. Conventionalmethodsthereforerequireexternalen-

] truly spontaneous process (barrier free, unlike the externally ergy (such as sonication or extrusion) to be dissipated in the
t
f drivensystemsknownthusfar)forthesimplestbilayerform- system [12], using multi-component systems (such as catan-
o
s ingsystem(singlecomponentlipid). Ifhavingareproducible ionic, lecithin+bile, etc.) [17–21], or by inducing a sponta-
. diameter is important to protocells, then Helfrichs theory is neouscurvaturethroughcertainmolecules[22,23](formulti-
t
a the simplest physical model that provides one, and the sta- component systems, spontaneous formation occurs when ki-
m tionary phase interdiffusion (SPI) mechanism proposed here neticeffectsofothersurfactantsplaysaroleinmodifyingthe
- isonewaytoattainitspontaneously. Thisisapplicabletoany edgeenergy[20]).
d
vesicleformingsystem,withsuitablychosensolvents.
n Weconceptualiseamechanismtoobservethespontaneous
Asuspensionofunilamellarvesiclesiskineticallytrapped
o assembly. Considerauniformlydispersedsolutionofbilayer
in high energy states. The vesicle diameter is usually deter-
c formingmolecules. Ifthebulk(good)solventissuddenlyre-
[ mined by external parameters from the sample preparation placedbyan aqueous(poor) solvent, the changedconditions

3 processwhichleadstooneoftheseenergytraps(seeSupple- favouraggregationofthemoleculestoformcirculardiscs(bi-
mentaryMaterialSectionAforthethermodynamicsofvesi-
v layermembranesorbicelles).Thesystemthengetstrappedin
cles)[6,7]. Nevertheless,thereisonecharacteristicdiameter,
1 itsintrinsicsizeasthediscsgrow. Suchasituationof“mag-
4 forvesiclesspontaneouslyassembledfromasolutionphaseof ically” replacing the surroundings can be easily achieved in
5 lipids, obtained from a energy balance on a disc micelle ag-
computersimulationsofmolecules[11,24,25],butthechal-
0 gregate (bilayer disc membrane) [5, 8–10]. This diameter D
lengeistorealiseitinreality.
0 isgivenby
. Instead of a bulk replacement, the solvents can be diffu-
1
(2κ+κ¯) sivelyreplacedacrossaninterface,providedtheyaremiscible.
0
D 4 (1) When the interdiffusion coefficient of the solvents is much
5 ≈ γ
1 largecomparedtothatoftheamphiphilemoleculeintheorig-
: whereκistheelasticbendingmodulusofthemembrane,κ¯ is inalsolvent(lipidslengthsareabout10timesthediameterof
v
itsGaussiancurvaturemodulus[11], andγ istheline(edge) thewatermolecules,andhaveacorrespondinglysmallerdif-
i
X tension of the membrane with the surrounding solvent. This fusivity),thesemoleculeswillbelocalisedinspacewhilethe
r diameter has also been known as the “minimum” diameter, original solvent is replaced by the aqueous solvent by inter-
a obtainablebyintensesonication[5,12]. However,wechoose diffusion, as illustrated in the schematic shown in Figure 1.
to call it as an intrinsic size of the vesicle, as κ, κ¯, and γ are This results in a condition analogous to the sudden bulk re-
thermodynamicparametersofthevesicleformingamphiphile placementdiscussedabove,albeitinanearlytwo-dimensional
inthesurroundingsolvent(seeSupplementaryMaterial Sec- region. This region of the solution is essentially subjected
tion E for an elaboration on this point). For phosphotidyl- to a “quench” from a dilute or “gaseous” phase (of the am-
cholinelipidvesiclesinwaterthistheoreticaldiametercanbe phiphiles) to a spinodal region leading to a bulk phase sepa-
estimatedtobe13nm(seeSupplementaryMaterialSectionD ration to the bilayer phase. The assembly of the free surfac-
for the calculation) [13–15]. Despite being a natural length tantshappensspontaneouslywithoutanybarriertoformdiscs.
These discs, above the critical diameter given by Equation 1
againtransition,withoutanybarriers,toalowerenergystate
ofavesicle(seeSupplementaryMaterialSectionA).Thesize
∗[email protected] ofthevesiclewouldbeintrinsic,butatavaluedeterminedby
2
observations. (i) The concentration of lipids does not have
aninfluenceonthediameterofvesiclesasseeninFigure3a.
Higher concentration only leads to higher number density of
vesicles of the same average size, as evidenced by the same
number counts from scattering measurements achieved at a
higherdilutionandvisuallybyamoreturbidsuspension(see
Elementary Bilayer Supplementary Material Section H for calculations support-
forming Compound
ing this claim). (ii) the proportion of ethanol and water on
eithersideoftheinterfacedoesnotinfluencethesizesolong
FIG.1: Mechanismofspontaneousassemblyinanearly
asthevesiclesareformed,asseeninFigure3b.Weinferfrom
two-dimensionalplaneHeretheelementarybilayerforming
thisthatthelipidassemblyhappenswhenthebulkconcentra-
compound(lipidsforexample)isgenericallyrepresented
tionofethanolintheinterfacialregionisbetween25and50%
schematicallybyahydrophobicbody(green)andtwopolar (v/v).
ends(purple). Therearetwosolventsthataremisciblewith
Another important inference from Figure 3 is that in this
eachotherandtheelementarycompoundishighlysolublein
method,kineticeffectsdonotseemtoplayaroleindetermin-
one(top;magenta)andpoorlysolubleintheother(bottom;
cyan). Attheinterfaceasthesolventsinterdiffuse,the ing the average size of the vesicles. Otherwise the concen-
tration of lipids or the magnitude of the gradient of ethanol
environmentofthecompoundisrapidlyreplacedbythepoor
across the interface would have altered the size, as may be
solventwhichfavoursself-assemblyandformationofclosed
expectedinadetergentdepletionmethod[29].
vesicles. Note: Therepresentationoftwolipidstogetherasa
The intrinsic size is expected to change when the modulii
singleunitinthegoodsolventisonlyforillustratinga
κ,κ¯ortheedgetensionγisaltered. Thetemperatureandlipid
genericprinciple;inrealitythelipidcoordinateswillbe
different. chemistry(chainlengthandunsaturation)aretwohandlesthat
canreadilyaffectachangeinthediameterbythisalteration.
Liposomes are formed only at temperatures higher than the
maintransitiontemperatureofthephospholipid(T = 24 C
m ◦
thethermodynamicsofthelipidinthemixtureofsolvents. for DMPC), and has a significant effect on the size of lipo-
To achieve this mode of assembly, three components are somes as seen in Figure 4a. In the temperature range stud-
essential: bilayer forming amphiphile (lipid or a block co- ied, thesizeoftheDMPCliposomesshowsanincreasewith
polymer), agoodsolventandapoorsolventwhichareinter- temperature. In the temperature range 30–40 C the bending
◦
miscible. Wefoundaclassicalsystem—phosphotidylcholine modulus is nearly constant at κ 1.3 0.1 10 19 J [30].
−
(PC)lipid,ethanol,andwater—wellsuitedtocapturethephe- However,itisexpectedthattheed≈geten±siond×ecreasesdueto
nomenon described. Here, the lipids dissolved in ethanol is aknowndecreaseintheinterfacialtensionasthetemperature
broughtintocontactwithwateracrossastationaryinterface. increases. Lipids of various chain lengths and unsaturation
Owing to the high miscibility of water and ethanol the in- alsoformmono-disperse,unilamellarvesiclesasseeninFig-
terfaces need to be brought into contact in a nearly station- ure4b. Wefindthatlipidswithshorteralkylchainlengthand
ary manner, taking care to avoid any convective (advective) thosewithlargernumberofunsaturatedbondsformlargerli-
mixing,thatcaninfluencetheenergeticsofself-assembly[26] posomes, as shown in Figure 4b, in agreement with an ex-
(seeSupplementaryMaterialforadescriptionofthesetupand pected reduction in the bilayer thickness and hence the edge
the method). Achieving the stationary contact sets this apart tension. Accurate measurements of the edge tension values
from the broad class of ethanol injection methods using the and the bending modulus for different lipids in the presence
same components [26–28]. As the interdiffusion proceeds, a ofethanolisrequiredtofurthervalidateourclaim.
turbid front is seen to move upwards, indicating the forma- A remark about the time of synthesis is now in order. In
tionofliposomes(seeSupplementaryMaterialforadetailed thecuvetteassemblyconsideredhere,thevesiclesynthesisis
descriptionandavideo). completedbyabout6hours(seeSupplementaryMaterial).An
The liposome suspension obtained shows a monodisperse evenfastersynthesistimeof15minutesisachievedinahori-
population with a mean hydrodynamic diameter equal to zontalcontactofstationaryphasesinsideacapillary(seeSup-
530 25nmforthedimyristoylphosphotidylcholine(DMPC) plementary Material). However, these are not normalised by
±
lipid as seen in Figure 2(a). High resolution transmission theamountofliposomessynthesized. Insteadweestimatethe
electron microscope (HRTEM) images under negative stain- timeittakesfortheformationofasinglevesicletobeabout
ingshowthattheliposomesareunilamellarforvarioustypes t D2/4 5 10 5 s, for water to diffusively replace
ves −
∼ D ≈ ×
oflipidsused(Figure2(b-d)). Anindependentestimateofthe ethanoloverthelengthscaleofavesicle. Here, D 500nm
intrinsic diameter using Equation 1 can be obtained. In the is the diameter of the vesicle, 1.3 10 9 m≈2/s is the
−
presence of ethanol we find this to be about 300 nm for the diffusivity of water in ethanol. DEsti≈mates×of the time it takes
DMPC liposome, which is a reasonable order of magnitude byothermethodsisgivenintheSupplementaryMaterialSec-
estimateoftheexperimentallyobservedhydrodynamicdiam- tion G. For a method that yields monodisperse vesicles by a
eterof530nm,giventheapproximateestimatesinvolved(see spontaneousprocess,thisispossiblythefastestknownvalue,
SupplementaryMaterial SectionDforthecalculations). andcanthereforebequalifiedas“rapid”.
That the diameter is intrinsic is reinforced by two crucial WeconcludethatSPImechanismdoesprovideatrulyspon-
3
(b)DimyristoylPC
(c)DipalmitoylPC(DPPC) (d)SoyPC
(DMPC)
(a)DLS
FIG.2: Liposomecharacterisation(a)MonodispersesizedistributionofDMPCliposomesasintensityofscatteredlight
obtainedbydynamiclightscattering(DLS),shownincomparisonwithpolystyrenestandardsofmonodispersepopulation.
Here,theaveragesizeofliposomes,determinedfromtheintensitydistribution,is530 25nm. (b-d)Negativestaining
±
micrographsfromHRTEMshowingalargeunilamellarliposomesfromvariouslipids.
(a)Effectoflipidconcentration (b)Effectofethanol/waterratio
FIG.3: Liposomesizeisintrinsic(a)Thereisnoeffectofthelipid(DMPC)concentrationinitiallydissolvedinethanolonthe
diameteroftheliposomesformed. (b)Evidencethatshowsthatconcentrationofethanoloritsgradientsacrosstheinterface
doesnotinfluencethediameterofliposomes. Thepointsontheleft(squaresinblue)wereobtainedbythepre-mixingethanol
totheaqueousphaseinthecuvette,whereasthoseontheright(circlesinred)wereobtainedbypre-mixingwaterinthe
lipid+ethanolsolutioninthesyringetube. Exceptforasmallregioninethanolfraction(between25–50%v/v),whereno
liposomesarestablyformed,thediameteroftheliposomesisnearlyconstantat510 50nm.
±
taneous(barrier-free)assemblyoflipidstoformvesicles,and time-resolvedscattering, whichhasbeenstudiedonlyforki-
the observed diameter of the liposomes is quantitatively ex- netically controlled mixed surfactant systems [29, 32]. The
plained by Helfrich’s model [5]. It is too early to claim that proposed mechanism could be combined with hydrodynam-
SPIcouldbeapossiblemechanismfortheformationofvesi- ics to understand vesicle size selection in ethanol injection
cles of a uniform size under prebiotic conditions, pending methods [26–28]. Our preliminary investigations also reveal
identification of possible surfactants [31], suitable solvents, that using the SPI process it is also possible to achieve a
andconditionsfavouringainterdiffusivemixing. However,it highdegreeofencapsulation(morethan80%)ofhydrophilic
doesprovideadirectiontowardsthis,groundedinthefactthat molecules(ordrugs)bydissolvingitintheethanolphase.
thethereisanaturallengthscaledictatedbythermodynamics
[5].
Nevertheless, the method itself could have fundamental ACKNOWLEDGMENTS
and applied implications. Due to the stationarity of the SPI
method it may now be possible to infer the dynamics of mi- ThefinancialsupportofDepartmentofScienceandTech-
celletovesicletransitioninasinglecomponentsystemusing nology,GovernmentofIndia,throughaspecialIRHPAgrant,
4
1400
m)
n 1200
(
er
et
m 1000
a
Di
e
m
800
o
s
o
p
Li
600
400
26 28 30 32 34 36 38 40
Temperature( C)
◦
(a)Effectoftemperature
(b)Effectoflipidtype
FIG.4: Temperatureandlipidtypeinfluencesthediameter(a)DiameterofDMPCliposomesshowsanincreasewith
increaseintemperature. (b)Liposomesizealsodependsonthelipidtype,at27.5 C. Shownarefourlipidswithvariousvalues
◦
ofacylchainlength:unsaturation: DMPC—14:0,DPPC—16:0,SoyPC—mixture-16:0(14.9%),18:0(3.7%),18:1(11.4%),
18:2(63%),18:3(5.7%)andDOPC—18:1.
is gratefully acknowledged. Facilities in SAIF and CRNTS, forhelpfulcomments,andKITPC,ChineseAcademyofSci-
IIT Bombay were also used in this work. PS also wishes to encesfortheMembraneBiophysicsMeetingthatprovidedan
thankIRCCfortheSeedGrant,DuPontfortheYoungProfes- opportunitytodiscussthisproblematlengthwiththeseveral
sor fellowship that supported a part of this work; colleagues otherparticipants.
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D=2 . (A6)
γ
SupplementaryMaterialA:Thermodynamicsofvesicles
For diameters larger than this, the sphere has a lower free
energy; however, there is an energy barrier for the transition
Here, we briefly recall important results in the thermo-
fromthedisktothesphere,whichvanishesat
dynamics of vesicle formation [5, 8, 10, 11]. The analysis
presentedhereisrestrictedtozero-temperature(ignoringen- (2κ+κ¯)
tropic effects). Consider a curved patch (in the form of a D=4 γ . (A7)
spherical cap) of bilayer membrane (which is also known as
bicelleorabilayerfragment)ofareaA. Therearetwoimpor- Thisdiameteristhereforethemostlikelyvaluefortheclosed
tant contributions to its free energy density (per unit area of vesicle known as the vesiculation diameter [8], and called
thepatch). Firstistheelasticbendingenergy in the present work as the intrinsic diameter. Earlier works
haveneglectedtheGaussiancurvaturemodulus[8,9],butre-
g =(2κ+κ¯) (cid:32)1(cid:33)2, (A1) cent coarse grained simulations [11] have shown its impor-
el R tance and a way to obtain it accurately. It was found that
κ¯/κ = 0.95 0.1. Approximating κ¯ κ, we get from
where,κistheelasticbendingmodulus(splay),κ¯istheGaus- − ± ≈ −
EquationA7
sian curvature modulus (saddle-splay) and R is the radius of
curvature of the spherical cap. The second contribution is κ
D 4 . (A8)
fromtheedgeofthefragmentwhichhashydrophobicgroups ≈ γ
ofthelipidexposedtowater;theedgeenergyperunitareaof
thepatchis
1. Suspensionofvesicles
Lγ
g = , (A2)
ed A
We now consider the free energy of a suspension of vesi-
where L is the contour length of the edge, and γ is the line cles. FromEquationA4,weseethattheenergydoesnotde-
tension. Thetotalfreeenergycanbeshowntobeafunction pendonthediameter. Considerasuspensionof Mnumberof
ofthefragmentareaAandtheradiusofcurvatureR[8]as: unilamellarvesicles,allatitsintrinsicdiameter D ,givenby
0
EquationA8.ThetotalfreeenergyisthereforeM4πκ,substi-
G =(g +g )A
el ed tutingtheaboveapproximationforκ¯.Lettheoverallenergyof
A (cid:32)A A2 (cid:33)1/2 (A3) thissuspensionbenormalisedto1bythisvalue(seeFigure5).
=(2κ+κ¯) +2πγ .
R2 π − 4πR2 Nowconsiderthesamesuspensionwhereeverytwovesicles
fusetoformonelargervesicleofdiameterD = √2D (con-
1 0
The interpretation of this equation is that for a given area A
servingthesurfacearea). Thetotalnumberofvesicleswould
(and a lipid-solvent system) the total free energy is a func- now be M = M/2, and the normalised suspension free en-
1
tion of R. The minimum of this free energy determines the
ergy would now be 1/2, since the energy per vesicle is still
likelyshape(intermsofthemeancurvature1/R)tofindthe
the same. This logic can be inductively taken for further fu-
fragment in. While this form of the energy provides precise sions to give D = √2D = 2D with M = M /2 = M/4.
2 1 0 2 1
expressions for the diameter of the spontaneously assembled
ThisisschematicallyillustratedinFigure5.
vesicle,andthefree-energybarriersforbetweenthetwostates
Fromtheaboveanalysisitisclearthattheintrinsicsizeis
ofasphereandadisk,asimplerderivationisalsopossible.
the highest energy state for vesicles (other higher states for
The total energy of a close sphere (without an open hy-
thelipidsolutionarethediscsandfreesurfactants,asshown
drophobicedge)is
in Figure 5). These SUVs (small unilamellar vesicles), over
G =4π(2κ+κ¯). (A4) time,willtendtowardsformingLUVs(largeunilamellarvesi-
sph
cles). However,thistransitionwouldneedtocrossafreeen-
Wenotethatthefreeenergyofasphereisnotdependentonits ergy barrier for fusion, of the order of 10 kT [7]. Further
diameter; the free-energy density is dependent on the curva- loweringoffreeenergyisalsopossiblebyformationofmulti-
ture,butthisdependencevanishesfortheenergyitself,when lamellarvesicles,whichhaveanattractiveinteractionbetween
integratedoverthesphericalsurface, thebilayers[33](calculationsnotshownhere).
6
on the glass surface of the RBF. The RBF is then kept in a
Free Surfactants vacuumdesiccatorovernighttoremoveallthetracesofchlo-
roform.AnappropriatevolumeofethanolisaddedtotheRBF
Disc aggregates
tomakevariousconcentrationsofphospholipidinethanol(5,
10,20,40,80mg/ml).
b. Devicesetup TheexperimentalsetupisshowninFig-
y 1.0
erg ure6. Thekeycomponentsofthedeviceare: acontainerfor
n
bbs E 0.75 Intrinsic Size acqounetaoiunserp(hcaustes(ysrqinugaeretucbueve4t.t8em10m×4in0nmermdi)aamnedteorr)g.aAniscsphhoawsne
Gi
0.5 inthesetup,aholeismadeinthecuvettecapandthecuttube
isinsertedthroughit. Thephospholipidsolutioninethanolis
filled in the tube (held by the vacuum of the syringe piston).
0.25
Theaqueousphaseisthengentlyfilledinthecuvettethrough
Unilamellar
Vesicles a syringe needle pierced through the cuvette cap. The two
phases—water and ethanol—are held in stable contact. The
mixingoccurssolelyduetomoleculardiffusionofonephase
Reaction Coordinate
into the other. Adequate care is taken not to introduce con-
FIG.5: Schematicrepresentationofthefree-energyfora vectivemixinginthetwophasesneartheinterfaceduringthe
suspensionofvesicles. Thehighestenergystateisthatofthe process.
freesurfactantssubjecttosuddenchangeintheenvironment c. Dynamic light scattering (DLS)/Photon correlation
thatleadstoaspontaneousaggregationtoformdiscs. The spectroscopy (PCS) The liposomes were characterized by
discs,beyondacriticaldiameter,spontaneouslycloseupto DLS or PCS (Zetasizer nano ZS, Malvern, UK) for size and
formvesicles. Vesiclesoflargerdiameterareformedby sizedistribution(polydispersity),withoutanyfurtherprocess-
fusionofsmallervesiclesreducingthefreeenergyfurther. ing,exceptaqueousdilution,whenrequired,forreliablemea-
Butthisprocesshasanenergybarrieroftheorderof10kT. surements. The laser used internally is He-Ne laser with
633 nm wavelength and 4 mW power and the scattered light
is detected at a single angle of 173 . The size and size dis-
◦
Therefore, we can conclude that uni-lamellar vesicles tributionwascalculatedbyasoftware(DTS6.1,suppliedby
are meta-stable structures kinetically trapped in high energy Malvern).Thepolydispersityandaveragediameterwereeval-
states. Thekineticnatureofthevesicularstateisunlikeother uatedusingmono-modalorcumulantanalysismethodandthe
surfactantaggregatessuchasmicelleswhichareinadynamic intensity-weighteddistributionofdiameterwasevaluatedus-
equilibrium with the dilute (solution) phase. The vesicle di- ing multimodal/distribution analysis. Usual care is taken to
ameterisusuallydeterminedbyexternalparametersfromthe preventdustandothercontaminantsinthesystem.
samplepreparationprocesswhichleadstooneoftheseenergy d. TEM using Negative staining High resolution trans-
traps (by dissipating energy into the system) [6]. The anal- mission electron microscopy (Jeol, JEM-2100F) was used to
ysis shown here is minimalistic in that it does not consider visualizetheliposomelamellarity. Negativestainingmethod
any other shape transformation other than a simple fusion to was used to observe the liposomes under the HR-TEM.
conserve area, which is expected to be valid only for dilute An electron dense material—phosphotungstic acid (PTA)
mono-disperseunilamellarsuspensions,intheinitialtimes. 2%w/v—isusedasthenegativestainingagent. PTAisacidic
innatureandneedstobeneutralized;thepHisadjustedbya
diluteNaOHsolution. Adropofliposomesolution(10µl)is
SupplementaryMaterialB:Materialsandmethods placedcarefullyonacarboncoatedTEMgridwiththehelpof
amicropipetteandkeptforairdryingfor10minutes,whichis
followedbyaplacingdropofPTA.Thesamplewasairdried
a. Preparation of phospholipid solution. The phos-
beforeobserving. Theoperatingvoltageiskeptat120kV.
pholipids used to synthesize the liposomes are: 1,2-
dimyristoyl-sn-glycero-3-phosphocholine(14:0-DMPC);1,2-
dipalmitoyl-sn-glycero-3-phosphocholine (16:0-DPPC); and
L-α-phosphatidylcholine(SoyPC,amixtureofvariouslipids: SupplementaryMaterialC:StagesofLiposomeformationin
SPI
18:2, 16:0, 18:1, 18:3, 18:0 phosphatidylcholine), all pur-
chasedfromAvantiPolarLipids,wasusedwithoutfurtherpu-
rification. 1,2-dioleoyl-sn-glycero-3-phosphocholine (18:1- The phospholipid dissolved in ethanol is taken in an in-
DOPC) was purchased from Sigma Aldrich. Ethanol (AR vertedtubeandisincontactwithanaqueousphase(cuvette)
grade)waspurchasedfromMerckandMilliQwaterwasused asshown inFigure6. Assoon astheinterfacecontact ises-
for the aqueous medium. All the lipids from Avanti Polar tablished, the progress of liposome formation is clearly visi-
Lipids were in solution form in chloroform (20 mg/ml). An bleintheformofaturbidfrontthatmovesupwardthroughthe
appropriate volume of lipid solution is taken out in a round ethanolsection.Timelapsephotographsofthevisualchanges
bottomflask(RBF)andthechloroformisevaporatedfromthe observedareshowninFigure7.
lipidsolutionusingarotaryevaporator. Thisformsathinfilm Acopyofthetime-lapsevideooftheformationofliposome
7
FIG.6: ExperimentalsetupStationaryphaseinter-diffusionapparatuscomprisestwocomponents—acontainerforaqueous
phase(cuvette)andacutsyringetubecontainingphospholipid-ethanolphaseheldbythesyringepiston. Theneedleisusedto
gentlyaddtheaqueousphasetogenerateastationarycontactwiththeethanolphase.
0 5 10 15 20 25 30 36 hrs
FIG.7: StagesofliposomeformationTime-lapsephotographsofliposomeformation;eachphotographistakeninatime
intervalof5hours,showinginterdiffusionandturbidityofliposomeformation. Aftertheinitialcontact,aturbidfrontmoves
upwardintheethanolmedium,indicatingliposomeformationaswaterdiffusesin. Ataround6hourstheentiresectionofthe
phaseinsidethecut-syringebecomesuniformlyturbid. Afterabout15hours,stringsofsuspensionmovedownwards,which
laterallydiffusetoformauniformsuspensionbyabout36hours. Thetimestampislabelledatthetopofeachframeinthe
formatDD:MM:HH(denotingday,minute,andhourrespectively).
suspensionisavailablefrom: goesuptowardsthewater-airinterface,duetothelowerden-
sity of ethanol that is diffusing into the aqueous phase. This
http://goo.gl/KOW3VN isfollowedbyadownwardmovementof“strings”ofsuspen-
siontowardsthebottomofthecuvette(atasteadyvelocityof
The video has been made from photographs taken in 10 min about1µmpersec). (c)Finallythestringsdiffuseouttoform
timeintervals(thetimestampislabelledonthetopofthecu-
auniformlyturbidmilkywhitesuspension.
vette, intheformatDD:HH:MM).Threedistinctphenomena
areobserved—(a)Initially,boththelipidandaqueousphases Thetimeof6hoursforthefirststageroughlyagreeswith
areclear,andsoonafter,aturbidlayerisobservedinsidethe thetimescaleofdiffusionofwaterthroughethanolthrougha
invertedtubewhichmovesupwards,indicatingthatliposomes distanceof1cm,thelengthoftheethanolsectioninthecut-
arebeingformed. Thisiscompletedbyabout6hours(b)As syringe (see Section G). In the second stage, we believe, the
the white region moves vertically, some amount of liposome stringsformbecauseofaviscousfingeringinstabilityoccur-
suspensionalsocomesoutintheaqueousphase(cuvette)and ring due to a mean motion generated by diffusiophoresis of
8
the liposomes in a gradient of ethanol. Further experiments tension[15]forvariouslipidsinthepresenceofethanolmay
andanalysesareunderwaytoconfirmthisclaim. confirmourhypothesisoftheintrinsicsize.
SupplementaryMaterialD:EstimateoftheIntrinsicDiameter SupplementaryMaterialE:“Minimum”vs“Intrinsic”diameter
The intrinsic diameter of the vesicle is given by Equa- It has been customary to refer to the diameter given in
tion A8. In the SPI method, the hydrodynamic diameter of Equation A8 as the minimum diameter of vesicles, because
theliposomeswasfoundtobe530nmfortheDMPClipids. uponsubjectingtoexternaldriving(sonicationorturbulence)
Our claim that this is the intrinsic diameter can be validated theaveragediameterofliposomesinanaqueouslipidsolution
byusingindependentmeasurements. willbegivenbyEquationA8; alsoseeFigure5. Theenergy
Thelinetensionγcanbeestimatedbyamoleculargeome- dissipatedintothesystembyexternalmeans,whichcanbreak
trymodeloftheofarrangementoflipidsalongtheedge[13]: larger and multilamellar vesicles, leads to the reassembly by
aggregation (of discs and free lipids). The qualifier ‘mini-
γ= 5πσb σb , (D1) mum’isusedbecausevesiclesofadiametersmallerthanthe
16 0 ≈ 0 diameter given by Equation A8 is highly improbable, owing
tothehighcostofbendingadiscofsmalldiameters.
where σ is the interfacial tension between the hydrophobic
However, we choose to call it as the ‘intrinsic’ diameter
tailandthesurroundingsolvent,andb isthelengthofthehy-
0 since it is more generic. In the present system the observed
drophobictailofthelipid. ForDMPClipidsthelengthofthe diameter of 530 nm is intrinsic for the lipid+ethanol+water
hydrophobic tail b = 2.54/2 1.27 nm [34]. We note that
0 ≈ system. Itisthe‘minimum’onlyinthesurroundingswhereit
recentmeasurementsofthelinetensionbyobservingthedy-
forms(inthepresenceofasignificantconcentrationofethanol
namicsofporeclosure[15],findthelinetensiontobedepen-
(25–50%) in water). However, as water completely replaces
dentonthemanufacturingsource(possiblybecauseofsmall
ethanol, the overall concentration of ethanol in the final sur-
amountsofimpurities). Intheabsenceofmeasurementsofγ
roundingcanbeverylow(lessthan1%dependingonthetotal
for our sample, we choose to use the above theoretical esti-
volumes of mixtures taken initially). In such a situation, the
mateconsistentlyforthetwocasesbelow.
minimum diameter obtained by sonication (of aqueous sus-
pensionoflipids)wouldbemuchsmaller,around20nm(see
Section D). It would thus be misleading to call a 500 nm
1. Intrinsicdiameterinwater
vesicle to be in its minimum diameter. The nomenclature
of ‘intrinsic’ vs ‘minimum’ is only one of semantics. Using
When the surrounding solvent is water, we use an water- ‘intrinsic’ also connotes a physical basis to a spontaneously
oil interfacial tension of σ = 35 mN/m. This gives the line formed vesicle, whereas ‘minimum’ can also refer to some-
tension to be γw = 44 pN. For DMPC bilayer in water the thingachievedbyexternalforce.
bending modulus is κ = 1.5 0.06 10 19 J at 27 C [30].
− ◦
Substitutingtheseestimatesw±efind D× = 13nm. Experi-
water
mentally,itisobservedthatmorethantwohoursofsonication SupplementaryMaterialF:CapillarySetupforStationary
resultsinadiameterof17 6nm[12]. PhaseInter-diffusion
±
Thecuvetteandsyringemethod(Figure6)takes 6hours
2. Intrinsicdiameterinethanol-watermixture to form the liposomes (1 cm height and 4.8 mm∼diameter
tube). The formation time is observed to be considerably
In the method employed in Section B the self-assembly smaller when capillaries are used. Glass capillaries 10 cm
takes place in the presence of ethanol, and the estimates of longand1mmininnerdiameterisusedwithoneendsealed.
thephysicalpropertiesandhencethediameterwilldiffer.The The procedure in short is—the aqueous phase is filled up to
bending modulus of lipid membranes is reduced in the pres- 8cmwiththehelpofaglasssyringeattachedtoa12cmlong
enceofethanolbyabout30%at20%v/vofethanolforSOPC 30GSSneedle;Thephospholipid-ethanolsolutionistakenin
membranevesicles[13]. ThisimpliesforDMPCwecanex- another syringe and filled up to 1.5 cm by keeping a 1/2 cm
pectκ 1 10 19 Jinthepresenceofethanol. Moresignifi- airgapbetweenthephases. Thenthetwomisciblesolutions
−
≈ ×
cantisthereductioninthelinetensionalcoholcaninduceto are brought in contact by sucking out the air bubble through
anoilwaterinterface.Thelowestmeasuredinterfacialtension another needle. Care is taken to avoid any convective mix-
forthissystemisaboutσ 1mN/m[14]. Thereforetheline ing at the interface otherwise a multi-modal size distribution
≈
tension can be estimated from Equation D1 as γ 1.3 pN. ofliposomesisobserved. Thetotaltimerequiredtoformli-
≈
This gives an estimated vesicle diameter of 300 nm, which posomesislessthanan45minutesasseeninFigure8. This
agreestowithinanorderofmagnitudeoftheexperimentally canbesignificantlyreducedevenlesstoabout15minutesby
observed value of 530 nm, and is reasonable given approxi- gentlytappingthecapillary,toreleasevapourbubblesthatoc-
mate nature of the property estimation we have used. More clude the motion of the turbid front. We believe the reason
accuratemeasurementsoftheeithertheinterfacialortheedge forthefastersynthesisinacapillarysetupisduetoaphoretic
9
motionoftheliposomesdrivenbyaconcentrationgradientof taken. WecanalsoinferthatsincetheSPImethodisinterface
ethanol in water (diffusiophoresis, [35]) or because of a vis- driven,alargerinterfacialcontactwillproduceagreateryield
cositygradient, whichinturnleadstofastermixingofwater ofliposomesinthesametimeforagiveninitialconcentration
in ethanol in the opposite direction, furthering the assembly oflipids.
ofliposomes.
SupplementaryMaterialH:ConcentrationofLiposome
SupplementaryMaterialG:Timeforvesicleformation Suspension
As shown in Section C, the entire process in the cuvette As shown in the main communication, in the SPI process
cell configuration takes about 36 hours. However, samples theconcentrationoflipidsdoesnotinfluencethesizeoflipo-
takenassoonastheturbidfrontreachesthetopofthesyringe somes. Increasing the concentration of lipids only results in
section at about 6 hours, also show the same monodisperse morenumberofliposomesformed. Weobserveasuspension
distributionofliposomes,implyingthecompletionofvesicle ofaveryhighliposomefraction,asvisuallyseenbyahighly
synthesis. turbidmassinsidethecut-syringesection. However,wewere
Thetimeittakesforwatertodiffusivelytransportalongthe unabletoexactlyquantifytheconcentration;asshowninFig-
lengthofthesectioninsidethesyringecanbeestimatedtobe ure7,apartoftheliposomesuspensioncomesoutofthecut
t L2/4 5hrs,whereL = 1cmsectionthatinitially syringesectionastheturbidfrontmovesupwards.Thismakes
water
contai∼nsthelDipi≈dsolutioninethanol,and 1.3 10 9m2/s it difficult obtain accurate measurements. However, an esti-
−
isthediffusivityofwaterinethanol.ThisDisi≈nnear×agreement mate of the volume fraction of the liposome suspension can
withtheaboveobservationoncompletionofliposomesynthe- be obtained based on the average liposome diameter and the
sis,andinlinewiththehypothesisofthediffusivemechanism amountoflipidsused.
proposed(maintext). Thoughthe6hoursinthecuvettesetup Assuming that all of the lipids dissolved in ethanol form
and 15 minutes in the capillary setup (Section F) seems fast liposomes of the same size, and all of them are contained in
for a method that yields monodisperse liposomes in a single the cut-syringe section, we can estimate the volume fraction
step,thisneedsadditionalqualification. oftheliposomesuspensioninthecut-syringesection:
We now provide detailed comparison of the time it takes
cN Da
forliposomesynthesisinthismethodwithothermethods. It φ= A 0. (H1)
12M
will be inappropriate to simply compare the overall time of
anexperiment,becausethisalsoinvolvesthetotalamountof Here,cisthemassconcentrationoflipids,N istheAvogadro
A
liposomesformed,whichcouldvaryfromonemethodtoan- number,Distheaveragediameteroftheliposomes,a isthe
0
other, given that there has not been any standardised study. areaperheadgroupoflipid,andMisthemolecularweightof
Insteaditisplausibletocomparetheexpectedtimeforforma-
lipid.
tion of a single vesicle in each of the methods. Table I lists ForDMPClipid,M =678g/mol,anda =0.71nm2.From
0
the order of magnitude time taken in various methods. The
the DLS measurements we obtain the diameter of liposomes
estimateshavebeenobtainedbyusingthetimespentonlyin D 500nm. For a concentration of lipids at c = 20 mg/ml,
the actual liposome formation step. For the present method, ≈
weobtainthevolumefractiontobe
we use the approximate time it takes for water to diffusively
replace ethanol over a length scale of a liposome diameter: φ 0.5 (H2)
t D2/4 5 10 5 s. where D 500 nm is the li- ≈
SPI −
poso∼mediamDete≈r, an×d 1.3 10 9 m≈2/sisthediffusivity which is close to the maximum volume fraction of φ = 0.64
−
D ≈ ×
ofwaterinethanol. Thisshowsthataliposomeisformedin forrandompackingofspheres. Higherconcentrationsleadto
about50microseconds. even higher volume fractions. For the highest used concen-
For methods which use shear forces to drive assembly (or tration, c = 80mg/ml, we get φ 2, which is higher than
≈
breakupliposomes),therearenodetailedstudiesofthetime theclosepackingfraction. Inthiscase, asmentionedabove,
taken;inlieuofwhich,wehaveusedatypicalturbulent(Kol- theliposomesuspensionnolongerremainsentirelyconfined
mogorov) time scale t Re 1/2d/U 1 10 5 s for a to the cut-syringe section, and the estimate is not a correct
turb − −
∼ ≈ ×
Reynolds number Re of 10000, generated in a macroscopic reflectionoftheactualconcentration.
lengthofd =1mmwithabulkfluidvelocityU 1m/s. Ahighconcentrationliposomesuspensioncanbeharvested
≈
It can be concluded from analysing Table I that the time bycarefullyremovingthecut-syringeassemblyfromthecu-
taken for the synthesis of a single vesicle in SPI is among vette, as soon as the turbid front reaches the top of the cut-
the fastest in comparison with other methods that result in syringe. As indicated in the main communication, the lipo-
monodispersedistributionbyaspontaneousprocess. Evenin someformationiscompletedbythistime(ataboutsixhours)
comparisonwiththeotherexternallydrivenmethodstheSPI and a mono-disperse distribution is obtained even without
method is comparable in the order of magnitude of the time waitingfor36hoursfortheentireprocesstocomplete.
10
FIG.8: CapillarysetupofSPImechanism. LiposomesynthesisinacapillarybytheSPImethod,showscompletionof
liposomeformationinlessthan45minutes. Herethedistancemovedbytheturbidfrontisplottedagainsttime. Alsoshown
belowaresampleimagesofthevesicledispersionforminginsidetheglasscapillaryatdifferenttimes.
TABLEI:Estimateoforderofmagnitudetimetakenfortheformationofasinglevesicleinvariousmethodsagainstthesize
distributionandthedrivingforceforsizeselection.
Method Distribution DrivingForceforsizeselec- Time Taken one liposome
tion (orderofmagnitude)
Ethanolinjection Polydisperse Fluidshear 10microsecond
Reversephaseevaporation Polydisperse Fluid shear (mixing upon Minute
sonication)
Detergentdepletion(extem- Monodisperse Rateofdetergentremoval Minute
poraneous)
Electroformation Monodisperse Electricfield Minute
Sonicationofaqueoussolu- Monodisperse Fluidshear 10microsecond
tionoflipids
Frenchpress Monodisperse Fluidshear 10microsecond
Thinfilmhydration Polydisperse,MLV Spontaneous (or gentle Hour
shear)
Bilayerhydration Monodisperse Spontaneous Minute
SPI Monodisperse Spontaneous 10microsecond