DieaiF
aiadSabi

Formation and Stabilization of

Vesiles in Mixed Surfatant Systems

Nina Vlahy

University of Regensburg

Natural Sienes Faulty IV

Chemistry& Pharmay

Ph.D. Supervisor: Prof. Dr. Werner Kunz

Adjudiators: Prof. Dr. Werner Kunz

Prof. Dr. Ksenija Kogej

Prof. Dr. Jörg Heilmann

Chair: Prof. em. Dr. Dr. JosefBarthel

July 2008

Promotionskolloquiumam: 4. Juli 2008

ompetitor, my biggest fan

Contents v

Prefae xi

1 Introdution 1

1.1 Surfae Ative Agents . . . 1

1.1.1 General . . . 1

1.1.2 Surfatant Self-Assembly: Vesiles . . . 3

1.1.3 Catanioni Surfatant Mixtures . . . 4

1.1.4 Appliationof Catanioni Vesiles inCosmeti Formulation. . 7

1.2 Ion Eets . . . 8

1.2.1 Ions in Water . . . 8

1.2.2 Hofmeister Eet . . . 11

1.2.3 Ion Pairingin Water . . . 12

1.2.4 Collins' Theory of Mathing Water Anities . . . 13

1.2.5 Ion-Spei Eets in ColloidalSystems . . . 17

I Salt-InduedMorphologialTransitions inNon-Equimolar Catanioni Systems 19 2 Blastulae Aggregates: Spontaneous Formation of New Catanioni Superstrutures 21 2.1 Abstrat . . . 21

2.2 Introdution . . . 22

2.3 Experimental Proedures . . . 24

2.4 Results . . . 26

2.4.1 Charaterizationof SDS / DTAB Miellar Solution . . . 26

2.4.2 Salt-Indued Mielle-to-Vesile Transition . . . 27

2.5 Disussion . . . 32

2.5.1 Models of the Mielle-to-Vesile Transition . . . 32

2.5.2 Blastulae Vesiles . . . 35

2.5.3 TheOurreneofConvex-ConavePatternsinBiologialSys- tems . . . 37

2.5.4 RaspberryVesiles . . . 38

2.5.5 Blastulae Vesiles: A General Trend inCatanioni Systems? . 38 2.6 Conlusions . . . 39

3 Spei Alkali Cation Eets in the Transition from Mielles to Vesiles Through Salt Addition 41 3.1 Abstrat . . . 41

3.2 Introdution . . . 42

3.3 Experimental Proedures . . . 43

3.4 Results . . . 44

3.4.1 Phase Diagrams . . . 44

3.4.2 Counterion Eets . . . 45

3.4.3 Co-ionEets . . . 51

3.4.4 Nonioni Eets . . . 51

3.4.5 Eets of `Hydrophobi Ions' . . . 52

3.5 Disussion . . . 54

3.5.1 Aggregation Behavior of CatanioniSystems . . . 54

3.5.2 Counterion Properties . . . 55

3.5.3 Collins'`Law of Mathing Water Anities' . . . 56

3.5.4 Counterion Seletivity of Alkyl Sulfates . . . 56

3.5.5 Counterion Seletivity of Alkyl Carboxylates . . . 57

3.5.6 AlkylSulfates vs. AlkylCarboxylates . . . 57

3.5.7 Moleular Dynamis Simulations . . . 58

3.5.8 Generalizationofthe Conept: HofmeisterSeries ofHeadgroups 60 3.5.9 The Anioni Eet . . . 60

3.5.10 The Non-Ioni Eet . . . 60

3.5.11 Eets of `Hydrophobi Ions' . . . 62

3.6 Conlusions . . . 63

4 Anion SpeiityInuening Morphology inCatanioniSurfatant Mixtures with an Exess of Cationi Surfatant 65 4.1 Abstrat . . . 65

4.2 Introdution . . . 66

4.3 ExperimentalProedures . . . 67

4.4 Results and Disussion . . . 68

4.4.1 Ion Binding in CatanioniSurfatant Mixtures . . . 68

4.4.2 Phase Behaviorupon Salt Addition . . . 70

4.4.3 Anion Speiity in Physio-Chemial Properties of Alkyl- trimethylammoniumSystems . . . 72

4.4.4 Inuene of Salt onthe Aggregation Behavior of Surfatants . 73 4.4.5 Explaining Counterion Speiity in Surfatant Systems . . . 76

4.4.6 Dierent self-aggregation behavior of atanioni systems in the atanioni-and anioni-rih regions . . . 77

4.5 Conlusions . . . 78

II Inreasing the Stability of Catanioni Systems 79 5 Inuene of Additives and Cation Chain Length on the Kineti Stabilityof Supersaturated Catanioni Systems 81 5.1 Abstrat . . . 81

5.2 Introdution . . . 82

5.3 ExperimentalProedures . . . 83

5.4 Results and Disussion . . . 85

5.4.1 Shift of Solubility Temperaturewith Time . . . 85

5.4.2 Behavior of the Anioni-Rih Region of the Phase Diagrams

WithoutAdditives . . . 88

5.4.3 Eet of Additives on the Stability and the `Solubility Tem- perature Depression' . . . 92

5.5 Conlusions . . . 101

III Toward Appliation 103 6 Use of Surfatants in Cosmeti Appliation: Determining the Cy- totoxiity of Catanioni Surfatant Mixtures on HeLa Cells 105 6.1 Abstrat . . . 105

6.2 Introdution . . . 106

6.3 Experimental Proedures . . . 107

6.3.1 Materials . . . 107

6.3.2 Growing HeLaCell Cultures . . . 108

6.3.3 HeLaToxiity Test . . . 109

6.3.4 Detetion . . . 111

6.3.5 Evaluationof spetrosopial data . . . 111

6.4 Results and Disussion . . . 111

6.4.1 Single-ChainSurfatants . . . 111

6.4.2 Catanioni Surfatant Systems. . . 113

6.5 Conlusions . . . 116

7 Spontaneous Formation of Bilayers and Vesiles in Mixtures of Single-Chain Alkyl Carboxylates: Eet of pH and Aging 117 7.1 Abstrat . . . 117

7.2 Introdution . . . 117

7.3 Experimental Proedures . . . 120

7.4 Results and Disussion . . . 122

7.4.1 Lowering of the SolubilityTemperature of Fatty Aids . . . . 122

7.4.2 The Eet ofpH on Vesile Formation . . . 123

7.4.3 Cryo-TEM Study ofTime-Dependent Vesile Formation . . . 127

7.5 Conlusions . . . 131

Conlusion 133

Bibliography 137

Aknowledgements 161

List of Publiations 163

List of Oral and Poster Presentations 165

Surfatantsanself-assembleindiluteaqueoussolutionsintoavarietyofmirostru-

tures, inluding mielles, vesiles, and bilayers. In reent years, there has been an

inreasing interest in unilamellar vesiles, whih are omposed of a urved bilayer

that separates aninneraqueous ompartmentfromthe outeraqueousenvironment.

This interest is motivated by their potential to be applied as vehiles for ative

agents in osmetis and pharmay. Ative moleules an be enapsulated in the

bilayer membrane if they are lipophili or in the ore of the vesile if they are

hydrophili. Furthermore atanioni systems an be used as models for biologial

membranes.

When oppositely harged surfatants are mixed, new properties appear due to the

strong eletrostati interations between the harged headgroups. These so-alled

atanioni mixtures exhibit low ritial mielle onentration (m) values and a

non-monotoni hangein the surfatant paking parameter (

P

is varied. Oneadvantage ofatanioni systems,asompared withmore robustgen-

uinely double hained surfatants, are their greater sensitivity to parameters suh

astemperatureorthe preseneofsalts. Tooptimizethe appliationsitisimportant

to have ageneral understanding ofthe interplay ofinterations between the surfa-

tants and of the fators inuening the phase diagram of a mixed system. In this

thesis the formationand stabilization of atanioni vesileswasstudied. The eet

of alohol and salton the morphologialtransitions is desribed, and the potential

of using suh systems for osmetial and pharmaeutial purposes was explored.

A short overview of the phase behavior of atanioni systems is given in the Intro-

dution.

The morphologial transitions ourring in mixed surfatant systems upon the in-

isinvestigated andanintriguingnew intermediatestruture, namedblastulaevesi-

le, is desribed.

Many phenomena in olloid siene that involve eletrolytes show pronouned ion

speiity. In ordertoeluidate the spei ioneets inatanioni surfatant sys-

temswithdierentsurfatantheadgroupswereompared. Usingvariousexperimen-

tal tehniques and MD simulations, and employing a general onept of `mathing

water anities',adetailedstudy ofionspeiityinatanionisystemsisdesribed

in Chapters 3 and 4. A Hofmeister like series for lassifying surfatant headgroups

is established.

Consideringthatontrollingthepreipitationphenomenaisusefulforavastnumber

ofindustrialappliations,theeetofvariousadditivesonthestabilityofatanioni

systems was probed and is reportedin Chapter 5.

Chapter 6introduesan importantparameter that should be onsidered when for-

mulatingnew enapsulatingsystems foreitherosmetiormedialuse: thetoxiity

ofthepartiipatingsurfatantmoleules. Weevaluatedtheytotoxiityofanumber

of ommonlyused surfatants,as wellas that of atanioni surfatant mixtures.

Seeingthat the preseneof onlyasmallamountof aationisurfatantinthe mix-

tureresultsinalargeinrease initstoxiity,wefousedonamixtureoftwoanioni

and in osmeti formulation already ommonly used surfatants. In Chapter 7 we

present a way to formvesiles in suh mixtures at roomtemperatureand at physi-

ologialpH.

Thisthesisomprisesdierentstudiesrangingfromphasediagramdeterminationto

ytotoxiitystudies. Forthis reason,thethesiswaswrittensothateahhapterlay-

out followsthe usualonvention: Abstrat, Introdution, ExperimentalProedures,

Results, Disussion and Conlusions. The bibliography is given at the end of the

thesis in order toavoid repeating. The dierent studies led to several publiations

whih are already published, aepted or submitted, summarized in the following

table. A omplete list of publiations and a list of oral and poster presentations,

whihwere presented at international ongresses, are alsogiven at the end.

2 N. Vlahy, A. Renonourt, M. Drehsler, J.-M. Verbavatz, D. Touraud,

W. Kunz, Blastulae aggregates: New intermediate strutures in the

mielle-to-vesiletransitionofatanionisystems. J. Colloid Interf. Si.

2008 320, 360-363.

3 A.Renonourt, N.Vlahy, P. Bauduin,M. Drehsler, D. Touraud,J.-M.

Verbavatz, M. Dubois, W. Kunz, B. W. Ninham, Spei alkali ation

eets in the transition from mielles to vesiles through salt addition.

Langmuir2007 23, 2376-2381.

3 N. Vlahy, M. Drehsler, J.-M. Verbavatz, D. Touraud, W. Kunz, Role

of the surfatant headgroup onthe ounterion speiity in the mielle-

to-vesile transition through salt addition. J. Colloid Interf. Si. 2008

319, 542-548.

3 N. Vlahy, B. Jagoda-Cwiklik, R. Váha, D. Touraud, P. Jungwirth,

andW.Kunz,Hofmeisterseriesofheadgroupsand speiinterationof

harged headgroupswith ions. J. Phys. Chem. B 2008 (Submitted).

4 N. Vlahy, M. Drehsler, D. Touraud, W. Kunz, Anion speiity in-

uening morphology in atanioni surfatant mixtures with an exess

of ationi surfatant. Comptes rendus Chimie Aadémie des sienes

2008 (Submitted).

5 N.Vlahy,A.F.Arteaga,A.Klaus,D.Touraud,M.Drehsler,W.Kunz,

Inuene of additives and ation hain length on the kineti stability of

supersaturatedatanioni systems. Colloids Surf., A2008 (Aepted).

6 N. Vlahy, D. Touraud, J. Heilmann, W. Kunz, Determining the de-

layed ytotoxiity of atanioni surfatant mixtures on HeLa ells.

Colloids Surf., B 2008 (Submitted).

7 N. Vlahy, C. Merle, D. Touraud, J. Shmidt, Y. Talmon, J. Heilmann,

W. Kunz, Determiningthe delayed ytotoxiity of atanioni surfatant

mixtures onHeLa ells. Langmuir2008 (Submitted).

Introdution

1.1 Surfae Ative Agents

1.1.1 General

Surfae ative agents (a.k.a. surfatants) are moleules with a hemial struture

that makes them partiularly favorable to reside at interfaes. All lassial sur-

fatant moleules onsist of at least two parts, one whih is soluble in water (the

hydrophilipart) and the other whihis insolublein water (the hydrophobi part).

Thehydrophilipart(apolarorionigroup)isreferredtoastheheadgroupandthe

hydrophobipart(alonghydroarbonhain)asthetail(Figure1.1). Thehydropho-

bi part of a surfatant may be branhed orlinear. The degreeof hain branhing,

the position of the polar head group and the length of the hain are parameters

that aet the physio-hemialproperties ofthe surfatant 1 ,2

. The primarylassi-

ation of surfatants is made on the basis of the harge of the polar head group:

anionis, ationis, non-ionis and zwitterionis.

Surfatantmoleulesadsorbatinterfaes,therebyreduingthefreeenergyofthe

system 3

. When surfatants are dissolved in water, the hydrophobi group disrupts

the hydrogen-bonded struture of water and therefore inreases the free energy of

the system. Surfatant moleules therefore onentrate at interfaes, so that their

hydrophobigroupsareremovedordiretedawayfromthewaterandthefreeenergy

of the solution is minimized. The distortion of the water struture an also be de-

inthetheoryofthepakingparameter)andaunilamellarvesile.

reased (andthefreeenergyredued) bythe aggregationof surfae-ativemoleules

into lusters (mielles) with their hydrophobi groups direted toward the interior

of the luster and their hydrophili groups direted toward the water. The proess

of surfatant lustering or miellization is primarily an entropy-driven proess 1,3

.

However, the surfatantmoleules transferred fromthe bulksolution tothe mielle

may experiene some loss of freedom from being onned to the mielle. In addi-

tion, they may experiene an eletrostati repulsion from other similarly harged

surfatant moleulesin thease of surfatantswith ioni headgroups. Thesefores

inrease the free energy of the system and oppose miellization. Hene, mielle

formation depends on the fore balane between the fators favoring miellization

(vanderWaalsandhydrophobifores) andthoseopposingit(kinetienergyofthe

moleules and eletrostati repulsion).

The onentration at whih mielles rst appear in solution is alled the ritial

miellaronentration, abbreviated CMC, and an bedetermined from the dison-

tinuity of the inetion pointin the plot of aphysial property of the solutionas a

funtion of the surfatant onentration 1 ,3

. Above this onentration, almost all of

theaddedsurfatantmoleulesareonsumedinmielleformationandthe monomer

onentration does not inrease appreiably, regardless of the amount of surfatant

added tothe solution. Sine only the surfatant monomers adsorb atthe interfae,

the surfae tension remainsessentially onstant above the CMC. Thus, the surfae

tension an be diretly related to the ativity of monomers in the solution 3

. The

CMC isstronglyaetedby the hemialstrutureof thesurfatant 4,5

,by the tem-

perature andbythepreseneofosolutessuhaseletrolytes oralohols . Mielle

formation, or miellization, an be viewed as an alternative mehanism to adsorp-

tion atinterfaes. In both ases, the driving foreof the proess is the tendeny of

the surfatant toremove their hydrophobi groupsfrom the ontat with water 8

.

1.1.2 Surfatant Self-Assembly: Vesiles

As was mentioned previously, surfatants an self-assemble in dilute aqueous solu-

tions into avariety of mirostrutures, inludingmielles, vesiles, and bilayers. In

reentyears, therehas been aninreasinginterestinunilamellarvesiles, whihare

omposed of a urved bilayer that separates an inner aqueous ompartment from

the outer aqueous environment (Figure 1.1). This is mainly beause of their wide

appliationinbiologyandmediineasmodelellmembranes,aswellastheirstrong

potential as drug arriers and other enapsulating agents of industrial relevane 9

.

Twomajor theoretial approahes have been pursued inthe modelingof surfatant

self-assembly: the urvature-elastiity approah and the moleularapproah.

The urvature-elastiity approah desribes the vesile bilayer as a ontinuous

membraneharaterizedbythespontaneousurvatureand theelastibendingmod-

ulus 10 ,11

. Inthis approah,theformationofnite-sizedvesiles thusdependsonthe

interplay between these two quantities 12 ,13

. The theory providesanelegant,simple

way to desribe the formation of vesiles, however, beause this approah is based

onaurvatureexpansionof thefreeenergyofamembrane,itbreaks downforsmall

vesiles, for whih the urvature isquite pronouned.

ThemoleularapproahwaspioneeredbyIsraelahvili,Mithell,andNinham 1416

who developed a geometri paking argument that allows one topredit the shape

of self-assemblingmirostrutures, inludingspheroidal,ylindrial,ordisoidalmi-

elles, vesiles, and bilayers. Whih aggregates form, is determined primarily by

geometri pakingofamphiphiles,hydroarbonhain stiness,and the hydrophili-

hydrophobi balane. Fordilute solutionsin whih interations between aggregates

are not important,the neessary (geometri) onditions for formation of anaggre-

gateanbedesribedbyaasurfatantpakingparameter

P = v/(l max a

v

a

l max

lengthrelatedtoabout90% ofthe maximumextended length(see Figure1.1). The

optimalstabilityof the dierentaggregates ours as follows: (1)spherialmielles

P ≤ 1/3

1/3 < P ≤ 1/2

1/2 < P ≤ 1

Low paking parameters (around

1/3

with a strongly polar head group. An inrease in the paking parameter an

be obtained by adding a seond hain, therefore doubling the hydroarbon vol-

ume. Toreahthis value, doublehain surfatants 1722

,twosurfatantsof opposite

harge 23 26

, or the assoiation of a surfatant and a o-surfatant 27 33

an be used.

Inthelattertwoases,apseudo-doublehainsurfatantisobtainedbyeitheranion-

pair formationbetween the anioniand ationisurfatant ordue toanassoiation

of the twodierent moleules viahydrogen bounds.

In many ases, the formation of vesiles requires the input of some form of en-

ergy, for example, soniation, injetion or extrusion 34 39

. However, vesiles have

been found to form spontaneously in some aqueous surfatant systems, inlud-

ing solutions ontaining (i) mixtures of leithin and lysoleithin 40

, (ii) mixtures

of long- and short-hain leithins 41

, (iii) mixtures of AOT and holine hloride 42

,

(iv)dialkyldimethylammoniumhydroxidesurfatants

20,21,4345

,(v)ationisiloxane

surfatants 46

, and (vi) mixtures of ationi and anioni surfatants 24,4750

. These

spontaneously-forming vesiles oer advantages overthe more traditionalphospho-

lipid vesiles in being easier to generate and more stable, thus making them more

attrative as enapsulating agents in diverse pratial appliations, inluding the

ontrolled delivery of drugs, ative substanes in osmetis, and funtional food

ingredientssuh asenzymes 51,52

.

1.1.3 Catanioni Surfatant Mixtures

The main thermodynami driving fore for the assoiation of a ationi and an

anionisurfatant mixtureisthe releaseof ounterionsfromthe aggregate surfaes.

Thisresultsinalargeentropyinrease. Sinethetwosurfatantsaresingle-hained,

the resulting atanioni surfatant an be onsidered as a pseudodouble-hained

headgroup. For these systems a non-monotoni hange in

P

pronounedmaximumasthemixingratioisvaried 1,3

. Duetothestrongeletrostati

interationbetween the oppositelyhargedheadgroups,atanionimixturesexhibit

CMC valuesmuhlowerthanthoseofthesurfatantsinvolved. TheCMCisdiretly

orrelatedtotheleaningeieny ofsurfatants,pointingtoyetanotheradvantage

of suh systems.

Mixtures of anioni and ationi surfatants exhibit rih mirostruture phase

behavior in aqueous solutions. Aggregate strutures suh as spherial and rod-like

mielles, vesiles, lamellar phases, and preipitates have all been observed depend-

ing on the onentration and the ratio of the surfatants in solutions

24,26,48,5357

.

Around equimolaritya zone of preipitationis observed. However, when one of the

surfatantsispresent inasmallexess, the ationi-anionisurfatantbilayers usu-

ally spontaneously form losed vesiles. The phase behaviorof atanioni mixtures

is represented in Figure1.2.

Figure 1.2: Shemati phase behavior enountered in atanioni surfatant systems. Phase

notations:

V ⁻

V ⁺

2φ

i.e. mostlydemixingofphasesbetweenavesiularandalamellarphaseoravesileandamiellar

phase;

L ⁻

L ⁺

P: preipitate region;

I ⁻

I ⁺

and ationisurfatants(reproduedfrom Khan 58

).

Yuet and Blankshtein presented a detailedmoleularthermodynami theory

todesribetheformationofatanionivesiles. Theirtheoryrevealsthat(i)thedis-

tribution of surfatant moleules between the two vesile leaets plays an essential

role inminimizingthe vesiulationfree energies ofnite-sized vesiles, (ii)the om-

positionofmixedationi/anionivesilesismainlydeterminedbythreefators: the

transferfreeenergyofthe surfatanttails,theeletrostatiinterationsbetweenthe

harged surfatant heads, and the entropi penalty assoiatedwith the loalization

of the surfatant moleules uponaggregation, and (iii) the entropy assoiated with

mixingnite-sizedvesilesan beanimportantmehanism ofstabilizingvesiles in

solution. The presentmoleular-thermodynamitheory alsohas theabilitytoover

the entire rangeof vesile sizes(or urvatures),thusenablingadesriptionof small,

energetially stabilized, vesiles.

The free energy of vesiulation,

g ves

ing ve ontributions: (1)the transfer free energy,

g tr

g pack

g σ

g steric

eletrostati free energy,

g elec

monomeri state. The transfer free energy,

g tr

eet 60

,whihonstitutesthemajordrivingforeforsurfatantself-assemblyinwa-

ter. Indeed,thetransferfreeenergyistheonlyfavorablefree-energyontributionto

moleularaggregation,withtheotherfourfree-energyontributionsworkingagainst

thisproess. Thehydrophobiregioninavesileisdierentfrombulkhydroarbon.

Inavesile,the surfatanttailsareanhoredatoneend oneitherthe outerorinner

interfaes,whihrestritsthe numberofonformationsthateahsurfatanttailan

adopt while still maintaining a uniform liquid hydroarbon density in the vesile

hydrophobi region. This subtle dierene between a bulk hydroarbon phase and

the hydrophobi region ina vesile is aptured by the pakingfree energy,

g pack

addition, free-energy penalties are imposed, upon aggregation, by the reation of

the outerand innerhydroarbon/water interfaes,aptured in

g σ

repulsions and eletrostati interations between the surfatant heads, aptured in

g steric

g elec

tems: (1)In the `simplemixtures' of ationi and anioni surfatants oratanioni

surfatantsystemswithexesssalt, bothsurfatantsstillbeholdtheirown ounteri-

ons. (2) The `true atanionis' (ion pair amphiphiles) onsist of surfatant systems

where the original ounterions have been removed and replaed by hydroxide and

hydronium ions. The ombination of the ounterions at equimolarity thus forms

watermoleules. Eahsurfatantstandsasounterionforthesurfatantofopposite

harge. The present work will fous on the rst type of atanioni systems (with

ounterions).

1.1.4 Appliation of Catanioni Vesiles in Cosmeti Formu-

lation

Vesilesareommonlyusedinosmetis andpharmayasvehilesforativeagents.

Ative moleules an thus be enapsulated in the bilayer membrane if they are

lipophiliorintheore ofthe vesile iftheyare hydrophili. Enapsulationisuseful

to protet atives in preventing any undesired reation. Vesiles an thus be used

as vetors to deliver drugs to a spei plae, without being destroyed. The phar-

maeutial appliations ontinuously inrease and vesiles are used more and more

in the dermatology for prevention, protetion and therapy.

The rstenapsulationexperimentsonatanionisystems wereperformedbyHarg-

reavesandDeamer 61

ontheetyltrimethylammoniumbromide/sodiumdodeylsul-

fate system. They suessfully entrapped gluose,however, the system was limited

to high temperatures (

> 47 ^◦ C

ilar experiments using vesiles formed from etyltrimethylammoniumtosylate and

sodium dodeylbenzenesulfonate (CTAT/SDBS) mixtures. A more omprehensive

study of the entrapment ability was made by Tondre et al.

62

on the CTAT/SDBS

and by Kondo et al.

50

on the didodeyltrimethylammoniumbromide/ sodium do-

deylsulfate (DDAB / SDS) system. The surfatant onentration as well as the

ratio of the two partiipating surfatant have proved to have a big eet on the

entrapment eieny.

non-ionisurfatant isused (i. e. Triton X-100) 50

. A more elegant solutionfortar-

geteddrugdeliveryisdesigningvesilesthat beomeunstableataneasilytunedpH

value. Itisknownthat,forexample,tumorsandinamedtissuesexhibitadereased

extraellular pH 63 67

. For this reason a large number of groups have foused their

attention onthe preparation of pH-sensitive liposomes 6878

as possible drug arrier

systems. Furthermore,improvingthe bioompatibilityofproduts usedinosmeti

formulation even further is always sought after. For this reason it is important to

identify the skin irritatingproperties of ommeriallyused surfatants. In addition

totheir entrapment abilities,vesiles serve alsoas modelsfor membranesof biolog-

ialells 7981

and as templates 8285

for the synthesis of nanopartiles, extration of

rare earth metal ions 86

, and asgene deliverysystems 87

.

1.2 Ion Eets

1.2.1 Ions in Water

It has long been known that the dissolution of ions bringsabout hangesinsolvent

struture 88

. The region of modied solvent surrounding an ion has been denoted

as a osphere of the ion 89

. The degree and manner in whih ospheres overlap in

the lose-range enounter of two ions depends speially on the nature of both

ions and the primary fores between them. Ion hydration has been studied exten-

sively experimentally 88,9093

as well as theoretially 9498

. Terms suh as `ontat'

pairs and `solvent-separated' pairs have ome into use to distinguish the results of

omplete and partialeliminationof solvent moleulesfrom between twointerating

ions (see Setion 1.2.3.). The basi features responsible for ion-spei short-range

interations are, in the ase of monoatomi ions, their harge, their size, their po-

larizability, and availability of eletrons and/or orbitals for ovalent ontributions.

Additional features of polyatomi ions are the harge density distribution, and, in

some ases, the presene of hydrophobi groups 99

. The ease with whih hydration

eets aompanying assoiation-dissoiation proesses an be observed, depends

more readily with weak eletrolytes and with pairs of multivalent ions, than with

strong 1-1eletrolytes.

Ions have long been lassied as being either kosmotropes (struture makers) or

haotropes (struture breakers) aording to their relative abilities to indue the

struturing of water. The degree of water struturing is determined mainlyby two

quantities: the inrease or derease of visosity in water due to added salt, and

entropies of ion solvation. For example, the visosity

η

typiallyhas the following dependene onion onentration

c

η/η _◦ = 1 + Ac ^{1 / 2} + Bc + · · ·

where

η _◦

A

stant independent of

c

B

B

degree of water struturing.

B

haotropi ions. (One issue in interpreting experimentsis how toseparate the on-

tributions of the anion from the ation. The standard assumption is that

K ⁺

the same

B

Cl ⁻

K ⁺

Cl ⁻

ioni ondutane 100

and beausethe value of

B

KCl

fets of the anion from the ation need to be separated (it is assumed that the

solvation entropies are additive 90

). Furthermore, the ion solvation entropy needs

to be divided into ion and hydration water ontributions. Subtrating the former,

∆S II

the presene of anion 90

. Ions whih are kosmotropi in visosity experiments tend

to have a negative hydration omponent to their solvation entropy, implying that

they order the nearby waters, while haotropi ions have a positive

∆S II

1.3 plots the entropy of water near monovalentions asalulated from the entropy

of hydration of the ion (fromdissolving the ionin water) versus the ioni radius of

the ion 92

. A negative

∆S II

0.5 1.0 1.5 2.0 2.5

-15 -10 -5 0 5 10 15

-S II

r (Å) Li

+

Na +

K +

Rb +

Cs + F

-

Cl -

Br -

I -

Figure1.3: Theentropyofpurewaterminustheentropyofwaternearanionin

calK ^{− 1} mol ^{− 1}

Therystalradiioftheionsin angstromsareplottedalong theabsissa. Positivevaluesof

∆S I I

(lowerportion of gure) indiate water that ismoremobile than bulkwater. Negative valuesof

∆S I I

are in the upper portion of the gure; haotropesare in thelower portion of the gure (Figure

reproduedfromCollins 101

).

water that is less mobile than bulk water, whereas a positive

∆S II

of Figure 1.3) indiates loosely held water that is more mobile than bulk water.

Inreasingionsize (dereasingionharge density) isassoiatedwith inreasing mo-

bility of nearby water moleules. If this mobile, loosely held water is immediately

adjaent tothe ion, assuggested by x-ray and neutron dirationdata 102

, thenthe

horizontal linein Figure 1.3 indiating

∆S II = 0

(above the line) from weakly hydrated ions (below the line). Sine this transition

from weak to strong hydration ours at a larger size for anions than for ations,

the anions must bemore strongly hydrated than the ations,sine anions begin to

immobilizeadjaent watermoleulesatalowerharge density thandoations. The

experiments showthat wateris ordered by smallormultivalentions and disordered

by large monovalent ions. Therefore, water orderinghas generally been interpreted

in termsof ion harge densities 90 ,103

. Chargedensities are high onions that have a

small radius and/ora large harge.

The water-ordering eet of ions is also theoretially extensively studied. Contin-

uum eletrostatismodelssuhasthatofDebyeandHükel utilizeamarosopi

dieletri onstant and assume that all interations involvingions are stritly ele-

trostati, implying the existene of long range eletri elds strong relative to the

strength of water-water interations. In these models, ions are often thought of as

point harges and water as a dipole whih orients in the long-range eletri eld.

Suh models are unable to aurately desribe suh simple ion-spei behaviors

as their tendeny to form ontat ion pairs, whih is a major determinate of the

solubility of spei salts and of the role of spei ions in biologialsystems. For

example, models employing a marosopi dieletri onstant predit that all ions

arestronglyhydratedandwillberepelledfromnonpolarsurfaesbyimagefores. In

fat,weakly hydratedions(e.g., ammonium,hloride,potassium,and thepositively

harged aminoaid side hains) atually adsorb to nonpolar surfaes 105107

and in-

terfaes 108,109

. The driving fore for this adsorption has been to be the release of

weakly bound water tobeomestrongly interating bulk 110

, a proess not inluded

in the alulations utilizing the marosopi dieletri onstant. Sophistiated mi-

rosopi alulations have indiated a role for the polarizability 111,112

of weakly

hydrated ions (as opposed to their dehydration energy) and dispersion fores 113

in

driving them to neutralinterfaes.

1.2.2 Hofmeister Eet

A related property is the Hofmeister eet 114,115

. In 1888, Franz Hofmeister om-

pletedthe rstsystematistudy onspei-ioneets. Hereportedthatsalts aet

the solubility of proteinsin water. Certainions preipitate proteins inwater (`salt-

ing out') while others help solubilize them (`salting in'). This behavior has been

interpretedasamodulationofthe hydrophobi eet bysalts due tothehanges in

the water struture brought about by ions. Suh salt eets orrelate with harge

densities of the salts. Small ions tend to ause `salting out', that is, to redue

hydrophobi solubilities in water, whereas large ions tend to ause `salting-in', in-

reasing nonpolarsolubilities. In Hofmeister'spapers anorderingof salts,and later

an orderingofions, usually denoted as`The Hofmeisterseries' wasdeveloped. `The

Hofmeister series' orders ions as a monotoni funtion of their surfae harge den-

strongly hydrated from weakly hydrated speies. It is most onvenient togenerate

a separate series for anions and forations (. f. Figure 1.4).

SO ₄ ^2- > OH ^- > F ^- > Cl ^- > Br ^- > NO ₃ ^- > I ^- > SCN ^- > ClO ₄ ^- NH ₄ ⁺ > K ⁺ > Na ⁺ > Cs ⁺ > Li ⁺ > Rb ⁺ > Mg ²⁺ > Ca ²⁺ > Ba ²⁺

Cosmotropes Salting-out

“Hard”

Chaotropes Salting-in

“Soft”

Organic

Figure1.4: AtypialHofmeisterseries.

1.2.3 Ion Pairing in Water

Ion pairing desribes the (partial) assoiation of oppositely harged ions in ele-

trolyte solutionsto form distint hemial speies alled ion pairs. If the ionasso-

iation is reasonably strong (the value depends on the harges of the ions and the

relativepermittivityofthesolvent),thereisusuallylittlediultyinseparatingthe

properties of the ion pair from the long-range nonspei ion-ion interations that

exist in all eletrolyte solutions. However, when the ion assoiation is weak, there

is astrong orrelation between these nonspei ion-ion interations (haraterized

in terms of ativity oeients) and ion pair formation (haraterized in terms of

an assoiation onstant). Speies are generally desribed as ion pairs if two oppo-

sitelyharged ionsinsolutionstay together ataseparation r, whihis smallerthan

some speied uto distane R. Ions further apart than R are onsidered `free'.

Various theorieshave been proposed for hoosing the value of Rand for desribing

the propertiesof the ion pairsand free ions that together produe the observed be-

havior of eletrolyte solutions. It is generally aepted that ions an not approah

eahothermorelosely thansome`distane oflosestapproah'adue tothe strong

repulsive fores of the eletron shells of the ions, even if polarizable. The distane

a is understood tobear some relation to the sum of the (rystal ioni) radii of the

oppositely harged ions, generally a

≥ r ⁺ + r ⁻

signare onsideredtoformanionpairiftheir distaneapartisbetween

a

R

a timelongerthanthe timeneeded todiuse oversuh adistane . Oneions are

paired, they are thought to haveno tendeny toassoiate with otherions indilute

solutions, although, at higher eletrolyte onentrations, ion triplets, quadruplets,

or larger aggregates may form. A major role in the assoiation of ions in solution

intopairsisthoughttobeplayedbylong-rangeeletrostatifores betweentheions,

usually modeled as a Coulomb's law attration, attenuated by the solvent permit-

tivity. Veryshort-rangeinterations (hardornearly-hard sphererepulsions)involve

the mutualexlusionofionsatr

<

the onstituentions must be onsidered. On this basisanionpair maybelassied

as a (double) solvent-separated ion pair (2SIP), when the primary solvation shells

of both ions remain essentially intat, asa solvent-shared ionpair (SIP), if asingle

solventlayerexistsinthe spaebetween the ionpartners ofthepair, orasaontat

ion pair (CIP), if no solvent exists between the partners and the ions are in diret

ontat (Figure 1.5).

Figure 1.5: Shematirepresentationofion-pairtypes: (

a

b

shared (SIP), and (

c

displayed. (Reproduedfrom Marusand Hefter 117

)

1.2.4 Collins' Theory of Mathing Water Anities

Takingintoaountexperimentalndingsasimplemodelforthe ion-induedstru-

turing and disordering of water has been proposed 103

. Collins 103

proposed that ion

eets on water struture ould be explained by a ompetition between ion-water

interations, whih are dominated by harge density eets, and water-water inter-

ations, whihare dominatedby hydrogenbonding. Forexample,lithiumisasmall

ionwithhighhargedensity,soitinteratsstronglywiththewaterdipoletostrongly

hargedensityhavealowertendeny toorientwaterintheion's rstsolvation shell

(haotropes). Aordingly, ions with high harge density have ahigh propensity to

order water (kosmotropes). He suggested that anions are stronger than ations at

waterorderingbeauseoftheasymmetryofhargeinawatermoleule: thenegative

end of water's dipoleis nearertothe enter of the water moleulethan the positive

end. Therefore, anions feel alarger eletrostati potentialat the surfae of awater

moleule than ations. The alulations of Kalyuzhnyi et al.

118

indiate that the

solvation modelof Collins yieldsqualitativeagreementwith the experimentaldata.

-60 -40 -20 0 20 40 60

-15 -10 -5 0 5 10

q(kcalmol

-1 )

W A

-- W C

+ (kcal mol -1

) CHAOTROPE-KOSMOTROPE

KOSMOTROPE-CHAOTROPE KOSMOTROPE-KOSMOTROPE

CHAOTROPE-CHAOTROPE

CsF KF

NaF CsCl

CsI

NaCl

NaBr

LiCl

LiBr

LiI

Figure 1.6: Relationshipbetweenthestandardheatof solutionofarystallinealkalihalide (at

innitedilution)in

kcal · mol ^{− 1}

orrespondinggaseousanionandation,alsoin

kcal · mol ^{− 1}

(weakly hydrated) or kosmotropes (strongly hydrated). The enthalpy of solution of haotrope-

haotropeand kosmotrope-kosmotropesalts is positive(takesup heat), whereasthe enthalpy of

solutionofhaotrope-kosmotropeandkosmotrope-haotropesaltsiseithernegative(givesoheat)

orpositive(takesupheat). FiguresreproduedfromCollins 101

.

Collins' law of mathing water anities states that oppositely harged ions in

water anities 103

. Experimental observations of the systemati dependene of the

heats of solutionof simple alkalihalides onthe water anity of the individualions

(absolute free energies of hydration) and the dependene of the solubilities of the

alkalihalideson ionsize ontributed to the realization that asimple law ontrolled

the tendenyofions ofoppositehargetoforminnersphereionpairs. InFigure1.6,

the enthalpy ofsolutionis plottedonthe vertialaxis: those salts learly abovethe

line at

0

0

ions ofthesalt. Weseethatwhentheonstituentionsofasaltaremathedinwater

anity (kosmotrope-kosmotrope and haotrope-haotrope salts), old solutions are

produed, suggesting that no strong interations with water have ourred (whih

would release heat) and that the oppositelyharged ions of the dissolved salttend

to stay together. This istobe expeted: the pointhargeatthe enter of a (small)

kosmotropi ion an get loser to the point harge at the enter of an oppositely

harged (small) kosmotropi ion than it an to the point harge at the enter of

the oppositely harged portion of a medium size zwitterion (water moleule); and,

the point harges atthe enters of the two harges on the medium size zwitterions

an get loser to the harges on other water moleules than it an to the point

harge at the enter of a (large) haotrope. In ontrast, when the onstituent ions

are mismathedinwateranity (kosmotrope-haotropeand haotrope-kosmotrope

salts), hot solutions are often produed, suggesting that a strong interation of

the small ion with water has ourred and that the oppositely harged ions of the

dissolved salt have separated. This is also to be expeted, sine the point harge

at the enter of a (small) kosmotropi ionan get loser to the point harge at the

enteroftheoppositelyhargedportionofamediumsizezwitterionthantothepoint

harge at the enter of the oppositely harged (large) haotrope. The requirement

of a haotrope-kosmotropeor kosmotrope-haotrope saltfor anexothermi heat of

solutionisaneessary butnotsuientonditionsinewhensuhasaltisdissolved

the kosmotropi ion willgenerate heat as it goes from a (large) haotropi partner

heat as it goes from a (small) kosmotropi partner to a medium size zwitterioni)

water moleule. The net eet an be exothermi or endothermi.

+ +

+ +

+ _

_

_

_ + _

+ +

Small-Small

Small Small

Small

Big Big Big-Big

Big

X

Figure 1.7: Ion size ontrols thetendeny of oppositely harged ions to form inner sphere ion

pairs. Smallionsofoppositesign spontaneouslyform innersphereion pairsin aqueoussolution;

largeions ofoppositehargespontaneouslyform inner sphere ionpairsin aqueous solution; and

mismathedionsof opposite hargedonotspontaneouslyform inner sphereionpairsin aqueous

solution.

This is shematially represented in Figure 1.7. Small ions of opposite harge

willtend toome together beausethe pointharges at theirenters an get loser

to eah other than with the pointharges at the enters of the medium size water

moleules. Large ions of opposite harge will ome together beause the released

water moleules an form stronger medium - medium interations. And (small)

kosmotropiions willnot spontaneously dehydrate toform aninnersphere ionpair

with an oppositely harged (large) haotropi ion beause the point harge at the

enter of the kosmotropi ionan get loser tothe point hargeat the enter ofthe

oppositely harged portion of a medium size zwitterions than to the point harge

atthe enter of anoppositelyharged (large)haotrope. Thus, itan be onluded

that oppositely harged ions in free solution spontaneously form inner sphere ion

pairs only whenthey have equal water anities.

Ion-water interations are important throughout biology and hemistry. Ions af-

fet the onformations and ativities of proteins and nulei aids 119 123

. Ion om-

plexation in ells is ruial for the ativities of biomoleules suh as enzymes and

drugs 124,125

. Ionsregulatetheeletrostatipotentials,ondutanes,andpermeabil-

itiesof ellmembranes 126 ,127

,the strutures ofmielles,and the hydrophobi eet,

whihdrivespartitioning,permeation,andfoldingaswellasbindingproesses 107,128

.

In hemistry, ions aet the rates of hemial reations 129 ,130

, rates of gelation 131

(widely used in food appliations), ion-exhange mehanisms 132

(widely used for

hemial separations), and the expansion and ontration of lays, responsible for

environmentalproesses suh asmudslides 133 ,134

.

It has been observed that in assoiation olloids, the hanges in the balane of

fores ontrolling the aggregate struture are reeted in the hanges in interfaial

onentrationsofwaterandotheromponents 135

. Insurfatantsystems,thishange

is reeted inthe dierent self-aggregationmorphologies. This isespeiallytruefor

atanioni mixtures, due to their enhaned sensitivity to outside parameters. For

this reason, atanioni surfatant systems have been hosen to eluidate the ion-

speibehavior and role of the surfatant headgroupsin olloidalhemistry.

Salt-Indued Morphologial

Transitions in Non-Equimolar

Catanioni Systems

Blastulae Aggregates: Spontaneous

Formation of New Catanioni

Superstrutures

2.1 Abstrat

The transition of ioni mielles to vesiles upon the addition of salt was explored.

The atanioni surfatant solution was omprised of sodium dodeylsulfate (SDS)

anddodeyltrimethylammoniumbromide(DTAB)withanexessofSDS.Thehange

in aggregate size an be aommodated by the inrease of ounterion binding and

onsequent dehydration of the surfatant headgroups. A new type of intermediate

struture was found: a symmetrially shaped spherial super-struture, whih we

named blastulae vesile. In ontrast to known raspberry-like or egg-arton stru-

tures, we believe that harge utuations within the bilayers are responsible for

this spontaneous super-aggregationtoourinthe presene of onlya smallamount

of sodium hloride. A possible mehanism for the observed pattern formation is

proposed.

It is known for a long time that double hain surfatants an spontaneously form

vesiles 43

. A similar phenomenon an be observed with mixtures of ationi and

anioni surfatants (here alled atanionis). These systems display a wide variety

of phase behavior and strutures suh as mielles, vesiles, diss and folded bilay-

ers an beobserved

24,136 ,137

. Reently, new self-assembled strutures, suhas onion

phases and iosahedra were found in `true' atanioni solutions with no other ions

than the surfatant moleules 138,139

. Spontaneously formed vesiles are of applia-

tional interest, espeially in atanioni systems, sine they an be tailored at will

by varyingthe anioni/ationisurfatantratio,the size ofthe hainlength orthe

nature of the polarheads 26

.

Fordilutesystems,loalaggregate urvaturedeterminedbygeometrialonstraints

embodiedinasurfatantpakingparameter

P = v/(l max a

v

l max

volumeandlengthofthehydrophobipart,respetively,and

a

at the interfae (headgroup) is a onvenient variable that haraterizes phase dia-

grams 15

. A neessary ondition for the formation of vesiles from either single or

mixed surfatants an be shown to be that the paking parameter is

1/2 < P < 1

Fora singlesurfatant that onditionan besatisedby hoosing adouble hained

surfatant, e.g. didodeyldimethylammoniumbromide (DDAB); for mixed surfa-

tant systems a pseudo-double hain surfatant is obtained by ion-pair formation

between ananioni and aationi surfatant 18 ,45

.

However, this ondition is not suient. While the type of aggregate that forms

with ioni surfatants an be broadly understood in terms of a balane between

foresdue tothe pakingpropertiesofsurfatanttailsandthoseduetodouble-layer

eletrostatiinterations,onditionsfortheformationofsingleorfewwalledvesiles

areverysubtle

16 ,140,141

. Foreets ofglobalpakingonstraintsandinter-aggregate

interations, see André et al.

142

. As the surfatant parameter (intrinsi loal ur-

vature) varies in the range

1/2 <

< 1

addition ortemperature or mixinghain lengths, then, if the surfatant hains are

exible,the system formsvesilesthat growas

P

at

P = 1

If the hains are not exible, the system at rst forms multiwalled vesiles, then

vesiles, then lamellar phases again. At

P = 1

paking onstraints that emerge beause the interior and exterior surfatants of a

urved bilayerexperienevery dierentonstraints. Depending on those hain and

headgroup onstraints the system forms ubi phases at P

≈ 1

of zero average urvature but varying Gaussian urvature. Again, with inrease in

surfatant onentration equivalent to inreased repulsion between aggregates, the

system an form equilibrium states of supra aggregation. In these the interior an

bemielles orubi phases proteted by few walled vesiles.

Theappearaneofsomanydierentstruturesisknownforaverylongtime

20,45,143 149

.

But apart from the system of double hained didodeyldimethylammonium salts

with dierent ounterions studied by Ninham and Evans, they have been littleex-

plored.

Vesiles from atanioni systems are easily prepared. There is an expetation that

they might also be used as vehiles for ontrolled delivery of drugs

62 ,150 ,151

or as

templates for the synthesis of hollow partiles 150152

. One advantage of atanioni

vesiles asomparedwith morerobustgenuinelydoublehainedsurfatantsistheir

greater sensitivity to parameters suh as temperature 153

or the presene of salts 47

used toinduetransitionsfromvesiles tomiellesortopreipitation. Ofpartiular

interestis the direttransitionfrom miellesto vesiles. Suh aphenomenon oers

in priniple an easy way of enapsulating ative agents by dissolving them in the

miellar phase prior to vesile formation. Mielle-to-vesile transition was already

observed when dilutinga miellarsolutionwith water

26,154,155

, hangingthe anioni

/ ationisurfatant ratio 156158

,inreasing temperature 159

,oruponthe additionof

organi additives 160

and salt 161163

.

The eet of ioni strength on atanioni systems was previously studied experi-

mentally by Brasher et al.

47

. Their results show that the addition of monovalent

salt hanges the phasebehavior and aggregate properties of mixed surfatant solu-

tions. Theoretially the eet of salt on the atanionis was desribed by Yuet et

al.

59

, however, due to the onstritions of the model (smeared surfae harges and

In the present hapter, we explore salt-mediated transition of mielles to vesiles

in a well-studied system

47,48,164

. We are onerned with the inuene of salts on a

atanioni system omposed of sodium dodeylsulfate (SDS) and dodeyltrimethy-

lammoniumbromide(DTAB) in aqueoussolution,with anexess ofanionisurfa-

tant. Inreasingamountsofsodium hloridewassuessivelyadded toasolutionof

mixed SDS / DTAB mielles. The miellarsystem was rst haraterized by rheo-

metrimeasurementsandryo-TEM.Wereportonthepartiularmorphologiesthat

arise during the salt-indued mielleto vesile transition. The formation of irregu-

laronvex-onave patterns and aseondary self-assemblyof vesile-like strutures

uponthe additionof sodium hloride ispresented. Inorder tostudy this transition

in detail, the onentration of salt inthe system was inreased insmallinrements

and the eet was studied using dynami lightsattering, ryo-and freeze-frature

transmissioneletron mirosopy. Twodierentmirosopy tehniques were usedin

order to exlude the artifats that might arise during the preparation of samples.

FF-TEM providesadiret visualizationof the three-dimensionalstruture of parti-

les. The frature follows the path of least resistane, and in olloidal dispersions,

the frature surfae propagates along the interfae of two phases. This makes FF-

TEMideal to study membranesurfaes. To observe whether the vesiles are losed

and if the membranes are intat ryo-TEM wasemployed. Furthermore,ryo-TEM

is very appropriate tostudy miellarsolutions.

2.3 Experimental Proedures

Materials Sodium dodeyl sulphate (SDS) (purity: 99%) and sodium hloride

were purhased from Merk, Germany. The ationi surfatant used was 99% do-

deyltrimethylammoniumbromide(DTAB) purhased fromAldrih, Germany. All

hemialsmentioned abovewere used as reeived withoutfurther puriation. Mil-

lipore water wasused as solvent in allases.

amountsof dried substanes inMillipore water. The solutionswere then left for 24

hours toequilibrateat

25 ^◦ C

surfatantstok solutionsto obtaina xed anioni/ ationisurfatant mass ratio

of

70/30

2.5/1

onentration was kept at 1 wt.% at all times. Salts were added to the miellar

solution at inreasing onentrations. The solutions were then stirred and left to

equilibrate for aweek at

25 ^◦ C

Dynami Light Sattering (DLS) Measurements Partile size analysis was

performedusingaZetasizer3000PCS(MalvernInstrumentsLtd.,England),equipped

with a5mWheliumneonlaserwithawavelengthoutputof633nm. Thesattering

angle was

90 ^◦

25 ^◦ C

Rheology RheologialexperimentswereperformedonaBrookeldDV-III+rate

ontrolledrheometer. Aone-and-plategeometryof

48

0.8

deg one angle wasused (spindle modelCP -

40

Cryo-Transmission Eletron Mirosopy (ryo-TEM) Speimens for ryo-

TEMwerepreparedbyplaingasmalldrop(a.

4µ

grid. Immediatelyafterblottingwith lterlmtoobtaina thinliquidlmoverthe

grid, thesampleisplungedintoliquidethane(atitsmeltingtemperature). The vit-

ried lmis thentransferred under liquidnitrogentothe eletron mirosope. The

grid wasexaminedwithaZeissEM922EF TransmissionEletronMirosope(Zeiss

NTS mbH,Oberkohen,Germany). Examinationswere arriedout attemperatures

around

90

ltered images (DE =0 eV) were taken under redued dose onditions (100 - 1000

e/nm

2

). Imageswere registereddigitallybyabottommounted CCDamerasystem

(Ultrasan 1000, Gatan, Munih, Germany) ombined and proessed with a digital

imaging proessing system (Digital Mirograph 3.9 for GMS 1.4, Gatan, Munih,

Germany).

ryoproteted by 30% glyerol and frozen in liquid N

2

formed in a Balzers (Balzers, Switzerland) apparatus at

− 150 ^◦ C

of

10 ^{− 6}

ture surfaes. The replia were examined and photographed with a Philips CM 12

transmission eletron mirosope.

2.4 Results

2.4.1 Charaterization of SDS / DTAB Miellar Solution

Thesystemunderstudyisawell-knownmixtureofationiandanionisingle-hain

surfatants. The total surfatantonentration(1wt.%;

≈ 33

/ ationi molar ratio (

2.5

1

The initialsample was olorlessand isotropi,orresponding tothe miellarregion

of the phase diagram(Figure 2.1).

Figure 2.1: Shemati ternary phasediagram of theSDS /DTAB systemat

25 ^◦ C

rossshowsthestartingpoint(referenesample),towhih sodium hloridewasadded)

DLSmeasurementsonrmamiellarsolutionindiatingahydrodynamiradius

(

R H

10

0.27

shows aryo-TEM image of our referene solution(without added salt), exhibiting

very long rod-or ribbon-likemielles, in equilibriumwith spherial mielles(hene

servedbySANSmeasurementsinsystemssimilartoours 165

. Resultsfromrheometry

experiments show that the visosity dereases with applied strain rate (Figure 2.2

(right))thereforeexhibitingpropertiesofnon-Newtonianshear-thinninguids. This

kindofbehaviorisommonforsolutionsontaininglargenon-spherialmoleulesin

a solvent with smaller moleules. It is generally supposed that the large moleular

hainstumbleatrandomand aetlarge volumesof uidunderlowshear, butthat

they graduallyalignthemselvesinthediretionofinreasingshearand produeless

resistane. This behavior onrms the presene of rod-like mielles. No enthalpy

hangeouldbedetetedbydierentialsanningalorimetrysothatnoinformation

ould be dedued about possible phase transitions ourring inthe system between

10

80 ^◦ C

detetion with our equipment.

200 400 600 800 1000

3 4 5 6 7 8 9 10 11 12 13

/ Pas

/ s -1

Figure2.2: Left: Cryo-TEMimageofaSDS/DTABaqueoussolutionatthemolarratioof2.5/1

and atotalsurfatantonentrationof 1 wt.% (reprodued from ref.

169

); right: visosity of the

samesampleasafuntionofshearrate.

2.4.2 Salt-Indued Mielle-to-Vesile Transition

Upon the addition of sodium hloride, the solutions exhibited a transition from a

olorless to a blue solution, the blue olor being typial of the presene of large

objets. Sampleswith dierentsalt onentrationswere analyzedby dynami light

0.00 0.01 0.02 0.03 0.04 0

10 20 30 40 50 60 70 80 90

R H

/nm

c NaCl

/ molL -1

Figure2.3: Inreaseinthemeanhydrodynamiradiusofatanioniaggregatesupontheaddition

ofsodiumhloride.

sattering. The additionof hloride salts auses aninrease inaverage partilesize

and a ertain turbidity of the solution (Figure 2.3). DLS indiated a signiant

inrease in the mean hydrodynami radius (

R H

10

70

nm. It an be expeted that the salt sreens the eletrostati interations, whih

leads to smallerheadgroups and therefore ahigher paking parameter.

Freeze-frature and ryo-TEM onrm the formation of lamellar sheets in the

sample(Figure2.4). However, byarefullyobservingthe ryo-TEM imagesonean

see that at lowest onentration of added salt (10 mM) the long rod-like mielles

presentinthe startingsolutionstarttobreak-upandlustertogether,seeFigure2.5

(left). Other images fromthe same solution show that these lusters seem to form

small pieesof lamellae,whih eventuallylose toformround vesiles. The urving

of membranes is represented by the presene of darker, sti-looking edges, due to

the higher eletron density in these points. Figure 2.5 (right) shows some urved

piees oflamellarsheets aswell assome omplete vesiles. The vesilesrepresented

inryo-TEM appeartobeperforated;suh perforatedvesileshavepreviouslybeen

multi-lamellarsheets upon theaddition of

10

observe the unraveling of ribbon-like mielles into lamellarsheets. The molarity in the ase of

FF-TEM experimentsorrespondsto theonentrationofthesolutionspriorto ryo-proteting.

observed invarious surfatant systems 170174

. Surprisingly,FF-TEM images donot

onrm suhperforations.

The addition of salt produes dramati eets deteted by freeze-frature. At

20

tioned previously, FF-TEM exploits the property that surfaes frature along the

area of least resistane. In the ase of vesiles this is within the bilayer. There-

fore, only 3-dimensional objets an be observed. The size range of the partiles

is from

150

500

depends onthe regionwhere the samplesare fratured. Some of the aggregates are

fratured lose to the middle; Figure 2.6 (right)shows a ring of vesiles. Sine the

aggregates pitured in Figure 2.6 are observed in high amounts, they most likely

represent the same objet, fratured in dierent plaes (lose to the `poles' of the

blastulae vesiles, as opposed to the middle of the vesile). The images suggest

that the inside of these partilesare hollowand lled with the same solvent as the

surrounding (water). Due tothe similaritiesinappearane we named the observed

strutureblastulae,takingthenamefrombiologialorigin;theblastulaeareanearly

onsequentlustering ofthepiees. Clustersofelongated aggregatesareindiatedbyblak(left),

whereas individual aggregates are designated by white arrows. The image on the right shows

lustersofsmalleraggregates(blakarrows)andvesileswithperforatedsurfaes(white arrows).

Figure 2.6: FF-TEM photographs representing the formation of blastulae-like lusters upon

the addition of 20mM NaCl ut near the surfae(left); ut through the middle (right), learly

representingthesolventlledavity. Thearrowshowsanindividualvesilewithitsownmembrane.

stage of embryoni development onsisting of a spherial layer of ells surrounding

auid-lled avity. Onthe basis ofthe present pitures weannotsay,whether the

blastula vesile is an aggregate onsisting of one individual membrane or a luster

of elongated mielles as observed in ryo-TEM. Both possibilities will be explored

vidual vesiles are also present. Interestingly, the average diameter of the vesiles

(a. 60 nm) is of the same size as the bulges forming the blastulaestruture. This

might speak for the possibility that the blastula is atually a luster of individual

smallvesiles. These,however, arenot deformedinawaythatitisusuallyobserved

in aggregates. A mehanism for this type of lustering will be disussed later on,

pointing to the similarity with spei-site (or ligand-reeptor) binding. It should

be noted that similar aggregates have been observed in another ontext 175,176

and

will alsobe disussed later.

Figure2.7: FF-TEMimageofindividualunilamellarvesilesupontheadditionof

30

Thebarrepresents

100

Asmoresaltisaddedtothesystemthelustersbegintodisaggregate. Atsodium

hloride onentrations of

30

Finally,at NaClonentrationsof

40

45

not spherially symmetrialand the individual vesiles forming the aggregates are

45

200

deformed.

In summary, two features are new in this system: i) the appearane of blastulae

strutures, andii)theseries ofdierentstruturesthat areindued by saltaddition

only, withoutany further additives.

2.5 Disussion

2.5.1 Models of the Mielle-to-Vesile Transition

Inthefollowingweproposeapossiblemehanismfortheobservedpatternformation.

The dierentsteps are represented inFigure 2.9.

`Lamellar Model' It is well known that, as an eletrolyte is added to a mixed

miellar solution, the eetive surfae area of the surfatant headgroups beomes

smaller. This eet ismainlydue toounterion onentrationand sreening aswell

as onsequent dehydration of the neutralized heads. This eet favors a lamellar