Thesis Nina Vlachy

Dissertation

Formation and Stabilization of Vesi les in Mixed Surfa tant Systems Nina Vla hy

University of Regensburg Natural S ien es Fa ulty IV Chemistry & Pharma y

Ph.D. Supervisor:

Prof. Dr. Werner Kunz

Adjudi ators:

Prof. Dr. Werner Kunz Prof. Dr. Ksenija Kogej Prof. Dr. Jörg Heilmann

Chair:

Prof. em. Dr. Dr. Josef Barthel

July 2008

Promotionsgesu ht eingerei ht am:

20. April 2008

Promotionskolloquium am:

4. Juli 2008

For Katko: my sister, my toughest

ompetitor, my biggest fan

iv

Contents Contents

v

Prefa e

xi

1 Introdu tion

1

1.1

1.2

Surfa e A tive Agents

. . . . . . . . . . . . . . . . . . . . . . . . . .

1

1.1.1

General

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

1.1.2

Surfa tant Self-Assembly: Vesi les . . . . . . . . . . . . . . . .

3

1.1.3

Catanioni Surfa tant Mixtures

. . . . . . . . . . . . . . . . .

4

1.1.4

Appli ation of Catanioni Vesi les in Cosmeti Formulation . .

7

Ion Ee ts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8

1.2.1

Ions in Water

. . . . . . . . . . . . . . . . . . . . . . . . . . .

8

1.2.2

Hofmeister Ee t . . . . . . . . . . . . . . . . . . . . . . . . .

11

1.2.3

Ion Pairing in Water

. . . . . . . . . . . . . . . . . . . . . . .

12

1.2.4

Collins' Theory of Mat hing Water Anities . . . . . . . . . .

13

1.2.5

Ion-Spe i Ee ts in Colloidal Systems

17

. . . . . . . . . . . .

I Salt-Indu ed Morphologi al Transitions in Non-Equimolar Catanioni Systems 19 2 Blastulae Aggregates: Spontaneous Formation of New Catanioni Superstru tures 21 2.1

Abstra t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

21

2.2

Introdu tion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

22

v

CONTENTS

vi

2.3

Experimental Pro edures . . . . . . . . . . . . . . . . . . . . . . . . .

24

2.4

Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

26

2.4.1

Chara terization of SDS / DTAB Mi ellar Solution

. . . . . .

26

2.4.2

Salt-Indu ed Mi elle-to-Vesi le Transition

. . . . . . . . . . .

27

Dis ussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

32

2.5.1

Models of the Mi elle-to-Vesi le Transition . . . . . . . . . . .

32

2.5.2

Blastulae Vesi les . . . . . . . . . . . . . . . . . . . . . . . . .

35

2.5.3

The O

urren e of Convex-Con ave Patterns in Biologi al Sys-

2.5

2.6

tems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

37

2.5.4

Raspberry Vesi les

38

2.5.5

Blastulae Vesi les: A General Trend in Catanioni Systems?

Con lusions

. . . . . . . . . . . . . . . . . . . . . . . . .

38

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

39

3 Spe i Alkali Cation Ee ts in the Transition from Mi elles to Vesi les Through Salt Addition 41 3.1

Abstra t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

41

3.2

Introdu tion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

42

3.3

Experimental Pro edures . . . . . . . . . . . . . . . . . . . . . . . . .

43

3.4

Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

44

3.4.1

Phase Diagrams . . . . . . . . . . . . . . . . . . . . . . . . . .

44

3.4.2

Counterion Ee ts

. . . . . . . . . . . . . . . . . . . . . . . .

45

3.4.3

Co-ion Ee ts . . . . . . . . . . . . . . . . . . . . . . . . . . .

51

3.4.4

Nonioni Ee ts . . . . . . . . . . . . . . . . . . . . . . . . . .

51

3.4.5

Ee ts of `Hydrophobi Ions'

. . . . . . . . . . . . . . . . . .

52

Dis ussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

54

3.5.1

Aggregation Behavior of Catanioni Systems . . . . . . . . . .

54

3.5.2

Counterion Properties

. . . . . . . . . . . . . . . . . . . . . .

55

3.5.3

Collins' `Law of Mat hing Water Anities' . . . . . . . . . . .

56

3.5.4

Counterion Sele tivity of Alkyl Sulfates . . . . . . . . . . . . .

56

3.5.5

Counterion Sele tivity of Alkyl Carboxylates . . . . . . . . . .

57

3.5.6

Alkyl Sulfates vs. Alkyl Carboxylates . . . . . . . . . . . . . .

57

3.5

CONTENTS

3.6

vii

3.5.7

Mole ular Dynami s Simulations

. . . . . . . . . . . . . . . .

58

3.5.8

Generalization of the Con ept: Hofmeister Series of Headgroups 60

3.5.9

The Anioni Ee t

. . . . . . . . . . . . . . . . . . . . . . . .

60

3.5.10 The Non-Ioni Ee t . . . . . . . . . . . . . . . . . . . . . . .

60

3.5.11 Ee ts of `Hydrophobi Ions'

. . . . . . . . . . . . . . . . . .

62

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

63

Con lusions

4 Anion Spe i ity Inuen ing Morphology in Catanioni Surfa tant Mixtures with an Ex ess of Cationi Surfa tant 65 4.1

Abstra t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

65

4.2

Introdu tion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

66

4.3

Experimental Pro edures . . . . . . . . . . . . . . . . . . . . . . . . .

67

4.4

Results and Dis ussion . . . . . . . . . . . . . . . . . . . . . . . . . .

68

4.4.1

Ion Binding in Catanioni Surfa tant Mixtures . . . . . . . . .

68

4.4.2

Phase Behavior upon Salt Addition . . . . . . . . . . . . . . .

70

4.4.3

Anion Spe i ity in Physi o-Chemi al Properties of Alkyltrimethylammonium Systems

. . . . . . . . . . . . . . . . . .

72

4.4.4

Inuen e of Salt on the Aggregation Behavior of Surfa tants .

73

4.4.5

Explaining Counterion Spe i ity in Surfa tant Systems

76

4.4.6

Dierent self-aggregation behavior of atanioni systems in

. . .

the atanioni - and anioni -ri h regions . . . . . . . . . . . . . 4.5

Con lusions

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

II In reasing the Stability of Catanioni Systems

77 78

79

5 Inuen e of Additives and Cation Chain Length on the Kineti Stability of Supersaturated Catanioni Systems 81 5.1

Abstra t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

81

5.2

Introdu tion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

82

5.3

Experimental Pro edures . . . . . . . . . . . . . . . . . . . . . . . . .

83

5.4

Results and Dis ussion . . . . . . . . . . . . . . . . . . . . . . . . . .

85

5.4.1

85

Shift of Solubility Temperature with Time

. . . . . . . . . . .

CONTENTS

viii

5.4.2

Behavior of the Anioni -Ri h Region of the Phase Diagrams Without Additives

5.4.3

88

Ee t of Additives on the Stability and the `Solubility Temperature Depression'

5.5

. . . . . . . . . . . . . . . . . . . . . . . .

Con lusions

. . . . . . . . . . . . . . . . . . . . . . .

92

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

III Toward Appli ation

103

6 Use of Surfa tants in Cosmeti Appli ation: Determining the Cytotoxi ity of Catanioni Surfa tant Mixtures on HeLa Cells 105 6.1

Abstra t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

6.2

Introdu tion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106

6.3

Experimental Pro edures . . . . . . . . . . . . . . . . . . . . . . . . . 107

6.4

6.5

6.3.1

Materials

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

6.3.2

Growing HeLa Cell Cultures . . . . . . . . . . . . . . . . . . . 108

6.3.3

HeLa Toxi ity Test . . . . . . . . . . . . . . . . . . . . . . . . 109

6.3.4

Dete tion

6.3.5

Evaluation of spe tros opi al data . . . . . . . . . . . . . . . . 111

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

Results and Dis ussion . . . . . . . . . . . . . . . . . . . . . . . . . . 111 6.4.1

Single-Chain Surfa tants . . . . . . . . . . . . . . . . . . . . . 111

6.4.2

Catanioni Surfa tant Systems . . . . . . . . . . . . . . . . . . 113

Con lusions

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

7 Spontaneous Formation of Bilayers and Vesi les in Mixtures of Single-Chain Alkyl Carboxylates: Ee t of pH and Aging 117 7.1

Abstra t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

7.2

Introdu tion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

7.3

Experimental Pro edures . . . . . . . . . . . . . . . . . . . . . . . . . 120

7.4

Results and Dis ussion . . . . . . . . . . . . . . . . . . . . . . . . . . 122 7.4.1

Lowering of the Solubility Temperature of Fatty A ids

7.4.2

The Ee t of pH on Vesi le Formation

7.4.3

Cryo-TEM Study of Time-Dependent Vesi le Formation

. . . . 122

. . . . . . . . . . . . . 123 . . . 127

7.4.4 7.5

Cytotoxi ity Potential of Surfa tant Mixtures

Con lusions

. . . . . . . . . 130

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

Con lusion

133

Bibliography

137

A knowledgements

161

List of Publi ations

163

List of Oral and Poster Presentations

165

ix

x

Prefa e Surfa tants an self-assemble in dilute aqueous solutions into a variety of mi rostru tures, in luding mi elles, vesi les, and bilayers. In re ent years, there has been an in reasing interest in unilamellar vesi les, whi h are omposed of a urved bilayer that separates an inner aqueous ompartment from the outer aqueous environment. This interest is motivated by their potential to be applied as vehi les for a tive agents in osmeti s and pharma y.

A tive mole ules an be en apsulated in the

bilayer membrane if they are lipophili or in the ore of the vesi le if they are hydrophili . Furthermore atanioni systems an be used as models for biologi al membranes. When oppositely harged surfa tants are mixed, new properties appear due to the strong ele trostati intera tions between the harged headgroups. These so- alled

atanioni mixtures exhibit low riti al mi elle on entration ( m ) values and a non-monotoni hange in the surfa tant pa king parameter (P ) as the mixing ratio is varied. One advantage of atanioni systems, as ompared with more robust genuinely double hained surfa tants, are their greater sensitivity to parameters su h as temperature or the presen e of salts. To optimize the appli ations it is important to have a general understanding of the interplay of intera tions between the surfa tants and of the fa tors inuen ing the phase diagram of a mixed system. In this thesis the formation and stabilization of atanioni vesi les was studied. The ee t of al ohol and salt on the morphologi al transitions is des ribed, and the potential of using su h systems for osmeti al and pharma euti al purposes was explored. A short overview of the phase behavior of atanioni systems is given in the Introdu tion. The morphologi al transitions o

urring in mixed surfa tant systems upon the inxi

rease of the ioni strength are reported in Chapter 2. A mi elle-to-vesi le transition is investigated and an intriguing new intermediate stru ture, named blastulae vesi le, is des ribed. Many phenomena in olloid s ien e that involve ele trolytes show pronoun ed ion spe i ity. In order to elu idate the spe i ion ee ts in atanioni surfa tant systems with dierent surfa tant headgroups were ompared. Using various experimental te hniques and MD simulations, and employing a general on ept of `mat hing water anities', a detailed study of ion spe i ity in atanioni systems is des ribed in Chapters 3 and 4. A Hofmeister like series for lassifying surfa tant headgroups is established. Considering that ontrolling the pre ipitation phenomena is useful for a vast number of industrial appli ations, the ee t of various additives on the stability of atanioni systems was probed and is reported in Chapter 5. Chapter 6 introdu es an important parameter that should be onsidered when formulating new en apsulating systems for either osmeti or medi al use: the toxi ity of the parti ipating surfa tant mole ules. We evaluated the ytotoxi ity of a number of ommonly used surfa tants, as well as that of atanioni surfa tant mixtures. Seeing that the presen e of only a small amount of a ationi surfa tant in the mixture results in a large in rease in its toxi ity, we fo used on a mixture of two anioni and in osmeti formulation already ommonly used surfa tants. In Chapter 7 we present a way to form vesi les in su h mixtures at room temperature and at physiologi al pH.

This thesis omprises dierent studies ranging from phase diagram determination to

ytotoxi ity studies. For this reason, the thesis was written so that ea h hapter layout follows the usual onvention: Abstra t, Introdu tion, Experimental Pro edures, Results, Dis ussion and Con lusions. The bibliography is given at the end of the thesis in order to avoid repeating. The dierent studies led to several publi ations whi h are already published, a

epted or submitted, summarized in the following table. A omplete list of publi ations and a list of oral and poster presentations, whi h were presented at international ongresses, are also given at the end.

xii

Chapter 2

Publi ation N. Vla hy, A. Renon ourt, M. Dre hsler, J.-M. Verbavatz, D. Touraud, W. Kunz, Blastulae aggregates:

New intermediate stru tures in the

mi elle-to-vesi le transition of atanioni systems.

J. Colloid Interf. S i.

2008 320, 360-363. 3

A. Renon ourt, N. Vla hy, P. Bauduin, M. Dre hsler, D. Touraud, J.-M. Verbavatz, M. Dubois, W. Kunz, B. W. Ninham, Spe i alkali ation ee ts in the transition from mi elles to vesi les through salt addition.

Langmuir 2007 23, 2376-2381. 3

N. Vla hy, M. Dre hsler, J.-M. Verbavatz, D. Touraud, W. Kunz, Role of the surfa tant headgroup on the ounterion spe i ity in the mi elleto-vesi le transition through salt addition.

J. Colloid Interf. S i. 2008

319, 542-548. 3

N. Vla hy, B. Jagoda-Cwiklik, R. Vá ha, D. Touraud, P. Jungwirth, and W. Kunz, Hofmeister series of headgroups and spe i intera tion of

harged headgroups with ions.

4

J. Phys. Chem. B 2008 (Submitted).

N. Vla hy, M. Dre hsler, D. Touraud, W. Kunz, Anion spe i ity inuen ing morphology in atanioni surfa tant mixtures with an ex ess of ationi surfa tant.

Comptes rendus Chimie A adémie des s ien es

2008 (Submitted). 5

N. Vla hy, A. F. Arteaga, A. Klaus, D. Touraud, M. Dre hsler, W. Kunz, Inuen e of additives and ation hain length on the kineti stability of supersaturated atanioni systems.

6

Colloids Surf., A 2008 (A

epted).

N. Vla hy, D. Touraud, J. Heilmann, W. Kunz, Determining the delayed ytotoxi ity of atanioni surfa tant mixtures on HeLa ells.

Colloids Surf., B 2008 (Submitted). 7

N. Vla hy, C. Merle, D. Touraud, J. S hmidt, Y. Talmon, J. Heilmann, W. Kunz, Determining the delayed ytotoxi ity of atanioni surfa tant mixtures on HeLa ells.

Langmuir 2008 (Submitted).

xiii

xiv

Chapter 1 Introdu tion 1.1 Surfa e A tive Agents 1.1.1 General Surfa e a tive agents (a.k.a. surfa tants) are mole ules with a hemi al stru ture that makes them parti ularly favorable to reside at interfa es.

All lassi al sur-

fa tant mole ules onsist of at least two parts, one whi h is soluble in water (the hydrophili part) and the other whi h is insoluble in water (the hydrophobi part). The hydrophili part (a polar or ioni group) is referred to as the head group and the hydrophobi part (a long hydro arbon hain) as the tail (Figure 1.1). The hydrophobi part of a surfa tant may be bran hed or linear. The degree of hain bran hing, the position of the polar head group and the length of the hain are parameters that ae t the physi o- hemi al properties of the surfa tant

1,2

. The primary lassi-

 ation of surfa tants is made on the basis of the harge of the polar head group: anioni s, ationi s, non-ioni s and zwitterioni s. Surfa tant mole ules adsorb at interfa es, thereby redu ing the free energy of the

3

system . When surfa tants are dissolved in water, the hydrophobi group disrupts the hydrogen-bonded stru ture of water and therefore in reases the free energy of the system. Surfa tant mole ules therefore on entrate at interfa es, so that their hydrophobi groups are removed or dire ted away from the water and the free energy of the solution is minimized. The distortion of the water stru ture an also be de1

Figure 1.1:

S hemati representation of a surfa tant mole ule (with all the parameters involved

in the theory of the pa king parameter) and a unilamellar vesi le.

reased (and the free energy redu ed) by the aggregation of surfa e-a tive mole ules into lusters (mi elles) with their hydrophobi groups dire ted toward the interior of the luster and their hydrophili groups dire ted toward the water. The pro ess of surfa tant lustering or mi ellization is primarily an entropy-driven pro ess

1,3

.

However, the surfa tant mole ules transferred from the bulk solution to the mi elle may experien e some loss of freedom from being onned to the mi elle. In addition, they may experien e an ele trostati repulsion from other similarly harged surfa tant mole ules in the ase of surfa tants with ioni head groups. These for es in rease the free energy of the system and oppose mi ellization.

Hen e, mi elle

formation depends on the for e balan e between the fa tors favoring mi ellization (van der Waals and hydrophobi for es) and those opposing it (kineti energy of the mole ules and ele trostati repulsion). The on entration at whi h mi elles rst appear in solution is alled the riti al mi ellar on entration, abbreviated CMC, and an be determined from the dis ontinuity of the ine tion point in the plot of a physi al property of the solution as a fun tion of the surfa tant on entration

1,3

. Above this on entration, almost all of

the added surfa tant mole ules are onsumed in mi elle formation and the monomer

on entration does not in rease appre iably, regardless of the amount of surfa tant added to the solution. Sin e only the surfa tant monomers adsorb at the interfa e, the surfa e tension remains essentially onstant above the CMC. Thus, the surfa e

3

tension an be dire tly related to the a tivity of monomers in the solution . The CMC is strongly ae ted by the hemi al stru ture of the surfa tant 2

4,5

, by the tem-

perature

6

and by the presen e of osolutes su h as ele trolytes

7

6

or al ohols . Mi elle

formation, or mi ellization, an be viewed as an alternative me hanism to adsorption at interfa es. In both ases, the driving for e of the pro ess is the tenden y of

8

the surfa tant to remove their hydrophobi groups from the onta t with water .

1.1.2 Surfa tant Self-Assembly: Vesi les As was mentioned previously, surfa tants an self-assemble in dilute aqueous solutions into a variety of mi rostru tures, in luding mi elles, vesi les, and bilayers. In re ent years, there has been an in reasing interest in unilamellar vesi les, whi h are

omposed of a urved bilayer that separates an inner aqueous ompartment from the outer aqueous environment (Figure 1.1). This is mainly be ause of their wide appli ation in biology and medi ine as model ell membranes, as well as their strong

9

potential as drug arriers and other en apsulating agents of industrial relevan e . Two major theoreti al approa hes have been pursued in the modeling of surfa tant self-assembly: the urvature-elasti ity approa h and the mole ular approa h. The urvature-elasti ity approa h des ribes the vesi le bilayer as a ontinuous membrane hara terized by the spontaneous urvature and the elasti bending modulus

10,11

. In this approa h, the formation of nite-sized vesi les thus depends on the

interplay between these two quantities

12,13

. The theory provides an elegant, simple

way to des ribe the formation of vesi les, however, be ause this approa h is based on a urvature expansion of the free energy of a membrane, it breaks down for small vesi les, for whi h the urvature is quite pronoun ed. The mole ular approa h was pioneered by Israela hvili, Mit hell, and Ninham

1416

who developed a geometri pa king argument that allows one to predi t the shape of self-assembling mi rostru tures, in luding spheroidal, ylindri al, or dis oidal mi elles, vesi les, and bilayers.

Whi h aggregates form, is determined primarily by

geometri pa king of amphiphiles, hydro arbon hain stiness, and the hydrophili hydrophobi balan e. For dilute solutions in whi h intera tions between aggregates are not important, the ne essary (geometri ) onditions for formation of an aggregate an be des ribed by a a surfa tant pa king parameter

v

P = v/(lmax a) 1416 , where

is the volume per hydro arbon hain, or the hydrophobi region of the surfa tant, 3

a is the a tual headgroup area in the lm, and lmax

is an optimal hydro arbon hain

length related to about 90% of the maximum extended length (see Figure 1.1). The optimal stability of the dierent aggregates o

urs as follows: (1) spheri al mi elles

P ≤ 1/3;

(2) globular or ylindri al mi elles

1/3 < P ≤ 1/2

(3) vesi les or bilayers

1/2 < P ≤ 1. Low pa king parameters (around with a strongly polar head group.

1/3)

are found for single hained surfa tants

An in rease in the pa king parameter an

be obtained by adding a se ond hain, therefore doubling the hydro arbon volume. To rea h this value, double hain surfa tants

harge

2326

1722

, two surfa tants of opposite

, or the asso iation of a surfa tant and a o-surfa tant

2733

an be used.

In the latter two ases, a pseudo-double hain surfa tant is obtained by either an ionpair formation between the anioni and ationi surfa tant or due to an asso iation of the two dierent mole ules via hydrogen bounds. In many ases, the formation of vesi les requires the input of some form of energy, for example, soni ation, inje tion or extrusion

3439

.

However, vesi les have

been found to form spontaneously in some aqueous surfa tant systems, in luding solutions ontaining (i) mixtures of le ithin and lysole ithin of long- and short- hain le ithins

41

46

, (ii) mixtures

, (iii) mixtures of AOT and holine hloride

(iv) dialkyldimethylammonium hydroxide surfa tants surfa tants

40

20,21,4345

42

,

, (v) ationi siloxane

, and (vi) mixtures of ationi and anioni surfa tants

24,4750

.

These

spontaneously-forming vesi les oer advantages over the more traditional phospholipid vesi les in being easier to generate and more stable, thus making them more attra tive as en apsulating agents in diverse pra ti al appli ations, in luding the

ontrolled delivery of drugs, a tive substan es in osmeti s, and fun tional food ingredients su h as enzymes

51,52

.

1.1.3 Catanioni Surfa tant Mixtures The main thermodynami driving for e for the asso iation of a ationi and an anioni surfa tant mixture is the release of ounterions from the aggregate surfa es. This results in a large entropy in rease. Sin e the two surfa tants are single- hained, the resulting atanioni surfa tant an be onsidered as a pseudodouble- hained 4

surfa tant, in the sense that the two hains are not ovalently bound to the same headgroup.

For these systems a non-monotoni hange in

pronoun ed maximum as the mixing ratio is varied

1,3

P

is observed, with a

. Due to the strong ele trostati

intera tion between the oppositely harged headgroups, atanioni mixtures exhibit CMC values mu h lower than those of the surfa tants involved. The CMC is dire tly

orrelated to the leaning e ien y of surfa tants, pointing to yet another advantage of su h systems. Mixtures of anioni and ationi surfa tants exhibit ri h mi rostru ture phase behavior in aqueous solutions. Aggregate stru tures su h as spheri al and rod-like mi elles, vesi les, lamellar phases, and pre ipitates have all been observed depending on the on entration and the ratio of the surfa tants in solutions

24,26,48,5357

.

Around equimolarity a zone of pre ipitation is observed. However, when one of the surfa tants is present in a small ex ess, the ationi -anioni surfa tant bilayers usually spontaneously form losed vesi les. The phase behavior of atanioni mixtures is represented in Figure1.2.

Figure 1.2:

S hemati phase behavior en ountered in atanioni surfa tant systems. Phase

notations: V − and V + : regions of negatively and positively harged vesi les; 2φ: two-phase regions, i.e. mostly demixing of phases between a vesi ular and a lamellar phase or a vesi le and a mi ellar phase; L− and L+ : lamellar phase with an ex ess of respe tively anioni and ationi surfa tants; P: pre ipitate region; I − and I + : mixed mi ellar solutions with an ex ess of respe tively anioni and ationi surfa tants (reprodu ed from Khan 58 ). 5

Yuet and Blanks htein

59

presented a detailed mole ular thermodynami theory

to des ribe the formation of atanioni vesi les. Their theory reveals that (i) the distribution of surfa tant mole ules between the two vesi le leaets plays an essential role in minimizing the vesi ulation free energies of nite-sized vesi les, (ii) the omposition of mixed ationi /anioni vesi les is mainly determined by three fa tors: the transfer free energy of the surfa tant tails, the ele trostati intera tions between the

harged surfa tant heads, and the entropi penalty asso iated with the lo alization of the surfa tant mole ules upon aggregation, and (iii) the entropy asso iated with mixing nite-sized vesi les an be an important me hanism of stabilizing vesi les in solution. The present mole ular-thermodynami theory also has the ability to over the entire range of vesi le sizes (or urvatures), thus enabling a des ription of small, energeti ally stabilized, vesi les.

The free energy of vesi ulation,

gves ,

an be viewed as omposed of the follow-

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

gpack ,

(3) the interfa ial free energy,

ele trostati free energy,

gelec .

gσ ,

gtr ,

(2) the pa king free energy,

(4) the steri free energy,

gsteric ,

and (5) the

These ve free-energy ontributions a

ount for the

essential features that dierentiate a surfa tant mole ule in the vesi le and in the monomeri state. ee t

60

The transfer free energy,

gtr ,

ree ts the so- alled hydrophobi

, whi h onstitutes the major driving for e for surfa tant self-assembly in wa-

ter. Indeed, the transfer free energy is the only favorable free-energy ontribution to mole ular aggregation, with the other four free-energy ontributions working against this pro ess. The hydrophobi region in a vesi le is dierent from bulk hydro arbon. In a vesi le, the surfa tant tails are an hored at one end on either the outer or inner interfa es, whi h restri ts the number of onformations that ea h surfa tant tail an adopt while still maintaining a uniform liquid hydro arbon density in the vesi le hydrophobi region. This subtle dieren e between a bulk hydro arbon phase and the hydrophobi region in a vesi le is aptured by the pa king free energy,

gpack .

In

addition, free-energy penalties are imposed, upon aggregation, by the reation of the outer and inner hydro arbon/water interfa es, aptured in

gσ ,

and by the steri

repulsions and ele trostati intera tions between the surfa tant heads, aptured in

gsteric

and

gelec ,

respe tively. 6

A distin tion is made in the literature between two types of atanioni systems: (1) In the `simple mixtures' of ationi and anioni surfa tants or atanioni surfa tant systems with ex ess salt, both surfa tants still behold their own ounterions. (2) The `true atanioni s' (ion pair amphiphiles) onsist of surfa tant systems where the original ounterions have been removed and repla ed by hydroxide and hydronium ions.

The ombination of the ounterions at equimolarity thus forms

water mole ules. Ea h surfa tant stands as ounterion for the surfa tant of opposite

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

ounterions).

1.1.4 Appli ation of Catanioni Vesi les in Cosmeti Formulation Vesi les are ommonly used in osmeti s and pharma y as vehi les for a tive agents. A tive mole ules an thus be en apsulated in the bilayer membrane if they are lipophili or in the ore of the vesi le if they are hydrophili . En apsulation is useful to prote t a tives in preventing any undesired rea tion. Vesi les an thus be used as ve tors to deliver drugs to a spe i pla e, without being destroyed. The pharma euti al appli ations ontinuously in rease and vesi les are used more and more in the dermatology for prevention, prote tion and therapy. The rst en apsulation experiments on atanioni systems were performed by Hargreaves and Deamer

61

on the etyltrimethylammonium bromide / sodium dode ylsul-

fate system. They su

essfully entrapped glu ose, however, the system was limited to high temperatures (>

47◦ C).

Ten years later, Kaler at al.

24

pro eeded with sim-

ilar experiments using vesi les formed from etyltrimethylammonium tosylate and sodium dode ylbenzenesulfonate (CTAT/SDBS) mixtures. A more omprehensive study of the entrapment ability was made by Tondre et al. and by Kondo et al.

50

62

on the CTAT/SDBS

on the didode yltrimethylammonium bromide / sodium do-

de ylsulfate (DDAB / SDS) system.

The surfa tant on entration as well as the

ratio of the two parti ipating surfa tant have proved to have a big ee t on the entrapment e ien y. 7

Another problem is the release of the entrapped a tive ingredient. In most ases a non-ioni surfa tant is used (i. e. Triton X-100)

50

. A more elegant solution for tar-

geted drug delivery is designing vesi les that be ome unstable at an easily tuned pH value. It is known that, for example, tumors and inamed tissues exhibit a de reased extra ellular pH

6367

. For this reason a large number of groups have fo used their

attention on the preparation of pH-sensitive liposomes

6878

as possible drug arrier

systems. Furthermore, improving the bio ompatibility of produ ts used in osmeti formulation even further is always sought after. For this reason it is important to identify the skin irritating properties of ommer ially used surfa tants. In addition to their entrapment abilities, vesi les serve also as models for membranes of biologi al ells

7981

and as templates

rare earth metal ions

86

8285

for the synthesis of nanoparti les, extra tion of

, and as gene delivery systems

87

.

1.2 Ion Ee ts 1.2.1 Ions in Water It has long been known that the dissolution of ions brings about hanges in solvent stru ture

88

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

as a osphere of the ion

89

. The degree and manner in whi h ospheres overlap in

the lose-range en ounter of two ions depends spe i ally on the nature of both ions and the primary for es between them. Ion hydration has been studied extensively experimentally

88,9093

as well as theoreti ally

9498

.

Terms su h as ` onta t'

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

omplete and partial elimination of solvent mole ules from between two intera ting ions (see Se tion 1.2.3.). The basi features responsible for ion-spe i short-range intera tions are, in the ase of monoatomi ions, their harge, their size, their polarizability, and availability of ele trons and/or orbitals for ovalent ontributions. Additional features of polyatomi ions are the harge density distribution, and, in some ases, the presen e of hydrophobi groups

99

. The ease with whi h hydration

ee ts a

ompanying asso iation-disso iation pro esses an be observed, depends 8

on the number of ion pairs existent at any instant. Thus the ee ts are observable more readily with weak ele trolytes and with pairs of multivalent ions, than with strong 1-1 ele trolytes. Ions have long been lassied as being either kosmotropes (stru ture makers) or

haotropes (stru ture breakers) a

ording to their relative abilities to indu e the stru turing of water. The degree of water stru turing is determined mainly by two quantities:

the in rease or de rease of vis osity in water due to added salt, and

entropies of ion solvation. For example, the vis osity

η

of an aqueous salt solution

typi ally has the following dependen e on ion on entration

c 91 :

η/η◦ = 1 + Ac1/2 + Bc + · · · where

η◦

(1.1)

is the vis osity of pure water at the same temperature.

stant independent of

c;

A

is a on-

its orresponding term an be explained by Debye-Hü kel

theory as being due to ounterion s reening at low ion on entrations. The onstant

B,

whi h is alled the Jones-Dole

degree of water stru turing.

B

B

oe ient, is the quantity that des ribes the

is positive for kosmotropi ions and negative for

haotropi ions. (One issue in interpreting experiments is how to separate the ontributions of the anion from the ation. The standard assumption is that the same

B

oe ient as

ioni ondu tan e

100

Cl− ,

be ause

K+

and

and be ause the value of

B

Cl− for

K+

has

have approximately the same

KCl

is approximately zero.)

Water stru turing is also ree ted in entropies of ion solvation. Again, the effe ts 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. Subtra ting the former,

∆SII

is obtained, whi h des ribes the hange in entropy of hydration water due to

the presen e of an ion

90

. Ions whi h are kosmotropi in vis osity 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

∆SII .

Figure

1.3 plots the entropy of water near monovalent ions as al ulated from the entropy of hydration of the ion (from dissolving the ion in water) versus the ioni radius of the ion

92

. A negative

∆SII

(upper portion of Figure 1.3) indi ates tightly bound 9

15 -

F Li

10

+

Na

0 0.5

-

S

II

5

+

r (Å)

1.0

1.5

Cl

-

2.0

2.5

-5

Br

+

-

K -10

Rb

+

+

Cs

-

I

-15

Figure 1.3:

The entropy of pure water minus the entropy of water near an ion in calK −1 mol−1 .

The rystal radii of the ions in angstroms are plotted along the abs issa. Positive values of ∆SII (lower portion of gure) indi ate water that is more mobile than bulk water. Negative values of ∆SII (upper portion of gure) indi ate water that is less mobile than bulk water. Kosmotropes

are in the upper portion of the gure; haotropes are in the lower portion of the gure (Figure reprodu ed from Collins 101 ).

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

∆SII

(lower portion

of Figure 1.3) indi ates loosely held water that is more mobile than bulk water. In reasing ion size (de reasing ion harge density) is asso iated with in reasing mobility of nearby water mole ules. If this mobile, loosely held water is immediately adja ent to the ion, as suggested by x-ray and neutron dira tion data horizontal line in Figure 1.3 indi ating

∆SII = 0,

102

, then the

separates strongly hydrated ions

(above the line) from weakly hydrated ions (below the line). Sin e this transition from weak to strong hydration o

urs at a larger size for anions than for ations, the anions must be more strongly hydrated than the ations, sin e anions begin to immobilize adja ent water mole ules at a lower harge density than do ations. The experiments show that water is ordered by small or multivalent ions and disordered by large monovalent ions. Therefore, water ordering has generally been interpreted in terms of ion harge densities

90,103

. Charge densities are high on ions that have a

small radius and/or a large harge. The water-ordering ee t of ions is also theoreti ally extensively studied. Contin10

104

uum ele trostati s models su h as that of Debye and Hü kel

utilize a ma ros opi

diele tri onstant and assume that all intera tions involving ions are stri tly ele trostati , implying the existen e of long range ele tri elds strong relative to the strength of water-water intera tions. In these models, ions are often thought of as point harges and water as a dipole whi h orients in the long-range ele tri eld. Su h models are unable to a

urately des ribe su h simple ion-spe i behaviors as their tenden y to form onta t ion pairs, whi h is a major determinate of the solubility of spe i salts and of the role of spe i ions in biologi al systems. For example, models employing a ma ros opi diele tri onstant predi t that all ions are strongly hydrated and will be repelled from nonpolar surfa es by image for es. In fa t, weakly hydrated ions (e.g., ammonium, hloride, potassium, and the positively

harged amino a id side hains) a tually adsorb to nonpolar surfa es terfa es

108,109

105107

and in-

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

weakly bound water to be ome strongly intera ting bulk

110

, a pro ess not in luded

in the al ulations utilizing the ma ros opi diele tri onstant. Sophisti ated mi ros opi al ulations have indi ated a role for the polarizability

111,112

of weakly

hydrated ions (as opposed to their dehydration energy) and dispersion for es

113

in

driving them to neutral interfa es.

1.2.2 Hofmeister Ee t A related property is the Hofmeister ee t

114,115

. In 1888, Franz Hofmeister om-

pleted the rst systemati study on spe i -ion ee ts. He reported that salts ae t the solubility of proteins in water. Certain ions pre ipitate proteins in water (`salting out') while others help solubilize them (`salting in').

This behavior has been

interpreted as a modulation of the hydrophobi ee t by salts due to the hanges in the water stru ture brought about by ions. Su h salt ee ts orrelate with harge densities of the salts.

Small ions tend to ause `salting out', that is, to redu e

hydrophobi solubilities in water, whereas large ions tend to ause `salting-in', in reasing nonpolar solubilities. In Hofmeister's papers an ordering of salts, and later an ordering of ions, usually denoted as `The Hofmeister series' was developed. `The Hofmeister series' orders ions as a monotoni fun tion of their surfa e harge den11

sity and thus water anity, with the strength of water-water intera tions separating strongly hydrated from weakly hydrated spe ies. It is most onvenient to generate a separate series for anions and for ations ( . f. Figure 1.4).

Chaotropes Cosmotropes

Salting-in

Salting-out

“Soft”

“Hard”

Organic

SO42- > OH- > F- > Cl- > Br- > NO3- > I- > SCN- > ClO4NH4+ > K+ > Na+ > Cs+ > Li+ > Rb+ > Mg2+ > Ca2+ > Ba2+

Figure 1.4:

A typi al Hofmeister series.

1.2.3 Ion Pairing in Water Ion pairing des ribes the (partial) asso iation of oppositely harged ions in ele trolyte solutions to form distin t hemi al spe ies alled ion pairs. If the ion asso iation is reasonably strong (the value depends on the harges of the ions and the relative permittivity of the solvent), there is usually little di ulty in separating the properties of the ion pair from the long-range nonspe i ion-ion intera tions that exist in all ele trolyte solutions. However, when the ion asso iation is weak, there is a strong orrelation between these nonspe i ion-ion intera tions ( hara terized in terms of a tivity oe ients) and ion pair formation ( hara terized in terms of an asso iation onstant). Spe ies are generally des ribed as ion pairs if two oppositely harged ions in solution stay together at a separation some spe ied uto distan e

R.

Ions further apart than

r, whi h is smaller than R

Various theories have been proposed for hoosing the value of

are onsidered `free'.

R and for des ribing

the properties of the ion pairs and free ions that together produ e the observed behavior of ele trolyte solutions. It is generally a

epted that ions an not approa h ea h other more losely than some `distan e of losest approa h'

a due to the strong

repulsive for es of the ele tron shells of the ions, even if polarizable. The distan e

a is understood

to bear some relation to the sum of the ( rystal ioni ) radii of the

oppositely harged ions, generally

a ≥ r+ + r− .

In summary, two ions of opposite

sign are onsidered to form an ion pair if their distan e apart is between 12

a and R for

a time longer than the time needed to diuse over su h a distan e

116

. On e ions are

paired, they are thought to have no tenden y to asso iate with other ions in dilute solutions, although, at higher ele trolyte on entrations, ion triplets, quadruplets, or larger aggregates may form. A major role in the asso iation of ions in solution into pairs is thought to be played by long-range ele trostati for es between the ions, usually modeled as a Coulomb's law attra tion, attenuated by the solvent permittivity. Very short-range intera tions (hard or nearly-hard sphere repulsions) involve the mutual ex lusion of ions at

r < a.

However, at distan es

a < r < R, solvation of

the onstituent ions must be onsidered. On this basis an ion pair may be lassied as a (double) solvent-separated ion pair (2SIP), when the primary solvation shells of both ions remain essentially inta t, as a solvent-shared ion pair (SIP), if a single solvent layer exists in the spa e between the ion partners of the pair, or as a onta t ion pair (CIP), if no solvent exists between the partners and the ions are in dire t

onta t (Figure 1.5).

Figure 1.5:

S hemati representation of ion-pair types: (a) solvent separated (2SIP), (b) solvent

shared (SIP), and (c) onta t (CIP). The omplete solvation shell around the ion pair is not displayed. (Reprodu ed from Mar us and Hefter 117 )

1.2.4 Collins' Theory of Mat hing Water Anities Taking into a

ount experimental ndings a simple model for the ion-indu ed stru turing and disordering of water has been proposed

103

. Collins

103

proposed that ion

ee ts on water stru ture ould be explained by a ompetition between ion-water intera tions, whi h are dominated by harge density ee ts, and water-water intera tions, whi h are dominated by hydrogen bonding. For example, lithium is a small ion with high harge density, so it intera ts strongly with the water dipole to strongly 13

orient the water mole ules in the ion's rst solvation shell. Larger ions having lower

harge density have a lower tenden y to orient water in the ion's rst solvation shell ( haotropes). A

ordingly, ions with high harge density have a high propensity to order water (kosmotropes). He suggested that anions are stronger than ations at water ordering be ause of the asymmetry of harge in a water mole ule: the negative end of water's dipole is nearer to the enter of the water mole ule than the positive end. Therefore, anions feel a larger ele trostati potential at the surfa e of a water mole ule than ations.

The al ulations of Kalyuzhnyi et al.

118

indi ate that the

solvation model of Collins yields qualitative agreement with the experimental data.

10 CsI

CHAOTROPE-CHAOTROPE 5

CsCl

-1

q (kcal mol )

KOSMOTROPE-KOSMOTROPE NaCl

0

NaBr

NaF

-5

KF

CHAOTROPE-KOSMOTROPE -10

LiCl

CsF LiBr

KOSMOTROPE-CHAOTROPE -15

-60

LiI

-40

-20

W

Figure 1.6:

0

-

A

- W

20

40

60

-1

+

C

(kcal mol )

Relationship between the standard heat of solution of a rystalline alkali halide (at

innite dilution) in kcal · mol−1 and the dieren e between the absolute heats of hydration of the

orresponding gaseous anion and ation, also in kcal · mol−1 . The ions are assied as haotropes (weakly hydrated) or kosmotropes (strongly hydrated). The enthalpy of solution of haotrope haotrope and kosmotrope-kosmotrope salts is positive (takes up heat), whereas the enthalpy of solution of haotrope-kosmotrope and kosmotrope- haotrope salts is either negative (gives o heat) or positive (takes up heat). Figures reprodu ed from Collins 101 . Collins' law of mat hing water anities states that oppositely harged ions in 14

free solution form inner sphere ion pairs spontaneously only when they have equal water anities

103

. Experimental observations of the systemati dependen e of the

heats of solution of simple alkali halides on the water anity of the individual ions (absolute free energies of hydration) and the dependen e of the solubilities of the alkali halides on ion size ontributed to the realization that a simple law ontrolled the tenden y of ions of opposite harge to form inner sphere ion pairs. In Figure 1.6, the enthalpy of solution is plotted on the verti al axis: those salts learly above the line at at

0

0

produ e old solutions upon dissolution; those salts learly below the line

produ e hot solutions upon dissolution. Plotted on the horizontal axis is the

dieren e in absolute free energies of hydration (water anity) of the onstituent ions of the salt. We see that when the onstituent ions of a salt are mat hed in water anity (kosmotrope-kosmotrope and haotrope- haotrope salts), old solutions are produ ed, suggesting that no strong intera tions with water have o

urred (whi h would release heat) and that the oppositely harged ions of the dissolved salt tend to stay together. This is to be expe ted: the point harge at the 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 mole ule); and, the point harges at the enters of the two harges on the medium size zwitterions

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

harge at the enter of a (large) haotrope. In ontrast, when the onstituent ions are mismat hed in water anity (kosmotrope- haotrope and haotrope-kosmotrope salts), hot solutions are often produ ed, suggesting that a strong intera tion of the small ion with water has o

urred and that the oppositely harged ions of the dissolved salt have separated. This is also to be expe ted, sin e the point harge at the enter of a (small) kosmotropi ion an get loser to the point harge at the

enter of the oppositely harged portion of a medium size zwitterion than to the point

harge at the enter of the oppositely harged (large) haotrope. The requirement of a haotrope-kosmotrope or kosmotrope- haotrope salt for an exothermi heat of solution is a ne essary but not su ient ondition sin e when su h a salt is dissolved the kosmotropi ion will generate heat as it goes from a (large) haotropi partner

15

to a (medium size zwitterioni ) water mole ule, and the haotropi ion will take up heat as it goes from a (small) kosmotropi partner to a medium size zwitterioni ) water mole ule. The net ee t an be exothermi or endothermi .

+ + Small

+ _

_ Small

Small-Small

+ + _ Big

+

Big

+ + _

_

Big-Big

X

Small Big Figure 1.7:

Ion size ontrols the tenden y of oppositely harged ions to form inner sphere ion

pairs. Small ions of opposite sign spontaneously form inner sphere ion pairs in aqueous solution; large ions of opposite harge spontaneously form inner sphere ion pairs in aqueous solution; and mismat hed ions of opposite harge do not spontaneously form inner sphere ion pairs in aqueous solution.

This is s hemati ally represented in Figure 1.7. Small ions of opposite harge will tend to ome together be ause the point harges at their enters an get loser to ea h other than with the point harges at the enters of the medium size water mole ules. Large ions of opposite harge will ome together be ause the released water mole ules an form stronger medium - medium intera tions.

And (small)

kosmotropi ions will not spontaneously dehydrate to form an inner sphere ion pair with an oppositely harged (large) haotropi ion be ause the point harge at the

enter of the kosmotropi ion an get loser to the point harge at the enter of the oppositely harged portion of a medium size zwitterions than to the point harge at the enter of an oppositely harged (large) haotrope. Thus, it an be on luded that oppositely harged ions in free solution spontaneously form inner sphere ion pairs only when they have equal water anities. 16

1.2.5 Ion-Spe i Ee ts in Colloidal Systems Ion-water intera tions are important throughout biology and hemistry. fe t the onformations and a tivities of proteins and nu lei a ids

119123

Ions af-

. Ion om-

plexation in ells is ru ial for the a tivities of biomole ules su h as enzymes and drugs

124,125

. Ions regulate the ele trostati potentials, ondu tan es, and permeabil-

ities of ell membranes

126,127

, the stru tures of mi elles, and the hydrophobi ee t,

whi h drives partitioning, permeation, and folding as well as binding pro esses In hemistry, ions ae t the rates of hemi al rea tions

129,130

107,128

, rates of gelation

(widely used in food appli ations), ion-ex hange me hanisms

132

.

131

(widely used for

hemi al separations), and the expansion and ontra tion of lays, responsible for environmental pro esses su h as mudslides

133,134

.

It has been observed that in asso iation olloids, the hanges in the balan e of for es ontrolling the aggregate stru ture are ree ted in the hanges in interfa ial

on entrations of water and other omponents

135

. In surfa tant systems, this hange

is ree ted in the dierent self-aggregation morphologies. This is espe ially true for

atanioni mixtures, due to their enhan ed sensitivity to outside parameters. For this reason, atanioni surfa tant systems have been hosen to elu idate the ionspe i behavior and role of the surfa tant headgroups in olloidal hemistry.

17

18

Part I Salt-Indu ed Morphologi al Transitions in Non-Equimolar Catanioni Systems

19

Chapter 2 Blastulae Aggregates: Spontaneous Formation of New Catanioni Superstru tures 2.1 Abstra t The transition of ioni mi elles to vesi les upon the addition of salt was explored. The atanioni surfa tant solution was omprised of sodium dode ylsulfate (SDS) and dode yltrimethylammonium bromide (DTAB) with an ex ess of SDS. The hange in aggregate size an be a

ommodated by the in rease of ounterion binding and

onsequent dehydration of the surfa tant headgroups. A new type of intermediate stru ture was found: a symmetri ally shaped spheri al super-stru ture, whi h we named blastulae vesi le. In ontrast to known raspberry-like or egg- arton stru tures, we believe that harge u tuations within the bilayers are responsible for this spontaneous super-aggregation to o

ur in the presen e of only a small amount of sodium hloride.

A possible me hanism for the observed pattern formation is

proposed. 21

2.2 Introdu tion It is known for a long time that double hain surfa tants an spontaneously form vesi les

43

.

A similar phenomenon an be observed with mixtures of ationi and

anioni surfa tants (here alled atanioni s). These systems display a wide variety of phase behavior and stru tures su h as mi elles, vesi les, dis s and folded bilayers an be observed

24,136,137

. Re ently, new self-assembled stru tures, su h as onion

phases and i osahedra were found in `true' atanioni solutions with no other ions than the surfa tant mole ules

138,139

. Spontaneously formed vesi les are of appli a-

tional interest, espe ially in atanioni systems, sin e they an be tailored at will by varying the anioni / ationi surfa tant ratio, the size of the hain length or the nature of the polar heads

26

.

For dilute systems, lo al aggregate urvature determined by geometri al onstraints embodied in a surfa tant pa king parameter

P = v/(lmax a), where v and lmax are the

volume and length of the hydrophobi part, respe tively, and

a the area per mole ule

at the interfa e (headgroup) is a onvenient variable that hara terizes phase diagrams

15

. A ne essary ondition for the formation of vesi les from either single or

mixed surfa tants an be shown to be that the pa king parameter is

1/2 < P < 1.

For a single surfa tant that ondition an be satised by hoosing a double hained surfa tant, e.g. didode yldimethylammonium bromide (DDAB); for mixed surfa tant systems a pseudo-double hain surfa tant is obtained by ion-pair formation between an anioni and a ationi surfa tant However, this ondition is not su ient.

18,45

.

While the type of aggregate that forms

with ioni surfa tants an be broadly understood in terms of a balan e between for es due to the pa king properties of surfa tant tails and those due to double-layer ele trostati intera tions, onditions for the formation of single or few walled vesi les are very subtle

16,140,141

. For ee ts of global pa king onstraints and inter-aggregate

intera tions, see André et al. vature) varies in the range

142

. As the surfa tant parameter (intrinsi lo al ur-

1/2 <

P

<1

by e.g. varying head group area via salt

addition or temperature or mixing hain lengths, then, if the surfa tant hains are exible, the system forms vesi les that grow as 22

P

in reases to form a lamellar phase

at

P = 1.

If the hains are not exible, the system at rst forms multiwalled vesi les, then vesi les, then lamellar phases again. At

P =1

this urious phenomenon is due to

pa king onstraints that emerge be ause the interior and exterior surfa tants of a

urved bilayer experien e very dierent onstraints. Depending on those hain and headgroup onstraints the system forms ubi phases at P

≈ 1.

These are phases

of zero average urvature but varying Gaussian urvature. Again, with in rease in surfa tant on entration equivalent to in reased repulsion between aggregates, the system an form equilibrium states of supra aggregation. In these the interior an be mi elles or ubi phases prote ted by few walled vesi les. The appearan e of so many dierent stru tures is known for a very long time

20,45,143149

But apart from the system of double hained didode yldimethylammonium salts with dierent ounterions studied by Ninham and Evans, they have been little explored. Vesi les from atanioni systems are easily prepared. There is an expe tation that they might also be used as vehi les for ontrolled delivery of drugs templates for the synthesis of hollow parti les

150152

62,150,151

or as

. One advantage of atanioni

vesi les as ompared with more robust genuinely double hained surfa tants is their greater sensitivity to parameters su h as temperature

153

or the presen e of salts

47

used to indu e transitions from vesi les to mi elles or to pre ipitation. Of parti ular interest is the dire t transition from mi elles to vesi les. Su h a phenomenon oers in prin iple an easy way of en apsulating a tive agents by dissolving them in the mi ellar phase prior to vesi le formation. Mi elle-to-vesi le transition was already observed when diluting a mi ellar solution with water / ationi surfa tant ratio organi additives

160

156158

and salt

26,154,155

, in reasing temperature

161163

159

, hanging the anioni

, or upon the addition of

.

The ee t of ioni strength on atanioni systems was previously studied experimentally by Brasher et al.

47

. Their results show that the addition of monovalent

salt hanges the phase behavior and aggregate properties of mixed surfa tant solutions. Theoreti ally the ee t of salt on the atanioni s was des ribed by Yuet et al.

59

, however, due to the onstri tions of the model (smeared surfa e harges and

23

.

point-sized ions), they ould not reveal the spe i ity of dierent ions. In the present hapter, we explore salt-mediated transition of mi elles to vesi les in a well-studied system

47,48,164

. We are on erned with the inuen e of salts on a

atanioni system omposed of sodium dode ylsulfate (SDS) and dode yltrimethylammonium bromide (DTAB) in aqueous solution, with an ex ess of anioni surfa tant. In reasing amounts of sodium hloride was su

essively added to a solution of mixed SDS / DTAB mi elles. The mi ellar system was rst hara terized by rheometri measurements and ryo-TEM. We report on the parti ular morphologies that arise during the salt-indu ed mi elle to vesi le transition. The formation of irregular onvex- on ave patterns and a se ondary self-assembly of vesi le-like stru tures upon the addition of sodium hloride is presented. In order to study this transition in detail, the on entration of salt in the system was in reased in small in rements and the ee t was studied using dynami light s attering, ryo- and freeze-fra ture transmission ele tron mi ros opy. Two dierent mi ros opy te hniques were used in order to ex lude the artifa ts that might arise during the preparation of samples. FF-TEM provides a dire t visualization of the three-dimensional stru ture of parti les. The fra ture follows the path of least resistan e, and in olloidal dispersions, the fra ture surfa e propagates along the interfa e of two phases. This makes FFTEM ideal to study membrane surfa es. To observe whether the vesi les are losed and if the membranes are inta t ryo-TEM was employed. Furthermore, ryo-TEM is very appropriate to study mi ellar solutions.

2.3 Experimental Pro edures Materials

Sodium dode yl sulphate (SDS) (purity:

99%) and sodium hloride

were pur hased from Mer k, Germany. The ationi surfa tant used was 99% dode yltrimethylammonium bromide (DTAB) pur hased from Aldri h, Germany. All

hemi als mentioned above were used as re eived without further puri ation. Millipore water was used as solvent in all ases. 24

Sample Preparation

Surfa tant sto k solutions were prepared by dissolving weighed

amounts of dried substan es in Millipore water. The solutions were then left for 24 hours to equilibrate at

25◦ C.

The atanioni solutions were prepared by mixing the

surfa tant sto k solutions to obtain a xed anioni / ationi surfa tant mass ratio of

70/30

(this orresponds to a molar ratio of about

on entration was kept at 1 wt.% at all times. solution at in reasing on entrations. equilibrate for a week at

25◦ C

2.5/1).

The total surfa tant

Salts were added to the mi ellar

The solutions were then stirred and left to

before making measurements.

Dynami Light S attering (DLS) Measurements

Parti le size analysis was

performed using a Zetasizer 3000 PCS (Malvern Instruments Ltd., England), equipped with a 5 mW helium neon laser with a wavelength output of 633 nm. The s attering angle was

90◦

and the intensity auto orrelation fun tions were analyzed using the

CONTIN software. All measurements were performed at

Rheology

25◦ C.

Rheologi al experiments were performed on a Brookeld DV - III+ rate

ontrolled rheometer. A one-and-plate geometry of deg one angle was used (spindle model CP -

48 mm diameter and with a 0.8

40).

Cryo-Transmission Ele tron Mi ros opy ( ryo-TEM) TEM were prepared by pla ing a small drop ( a.

Spe imens for ryo-

4µl) of the sample on a holey arbon

grid. Immediately after blotting with lter lm to obtain a thin liquid lm over the grid, the sample is plunged into liquid ethane (at its melting temperature). The vitried lm is then transferred under liquid nitrogen to the ele tron mi ros ope. The grid was examined with a Zeiss EM922 EF Transmission Ele tron Mi ros ope (Zeiss NTS mbH, Oberko hen, Germany). Examinations were arried out at temperatures around

90 K. The TEM was operated at an a

eleration voltage of 200 kV. Zero-loss

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

2 e/nm ). Images were registered digitally by a bottom mounted CCD amera system (Ultras an 1000, Gatan, Muni h, Germany) ombined and pro essed with a digital imaging pro essing system (Digital Mi rograph 3.9 for GMS 1.4, Gatan, Muni h, Germany). 25

Freeze-Fra ture Ele tron Mi ros opy

Samples used for ryo-fra ture were

ryoprote ted by 30% gly erol and frozen in liquid N2 . formed in a Balzers (Balzers, Switzerland) apparatus at of

10−6

Freeze-fra ture was per-

−150◦ C

under a va uum

Torrs. Metalli repli as were obtained by Pt and arbon shadowing of fra -

ture surfa es. The repli a were examined and photographed with a Philips CM 12 transmission ele tron mi ros ope.

2.4 Results 2.4.1 Chara terization of SDS / DTAB Mi ellar Solution The system under study is a well-known mixture of ationi and anioni single- hain surfa tants. The total surfa tant on entration (1 wt.%; / ationi molar ratio (2.5 /

1)

≈ 33 mM) and the

anioni

remained onstant throughout all the experiments.

The initial sample was olorless and isotropi , orresponding to the mi ellar region of the phase diagram (Figure 2.1).

Figure 2.1:

S hemati ternary phase diagram of the SDS / DTAB system at 25◦ C (the bla k

ross shows the starting point (referen e sample), to whi h sodium hloride was added) DLS measurements onrm a mi ellar solution indi ating a hydrodynami radius (RH ) of

10

nm and a relatively high polydispersity index (0.27). Figure 2.2 (left)

shows a ryo-TEM image of our referen e solution (without added salt), exhibiting very long rod- or ribbon-like mi elles, in equilibrium with spheri al mi elles (hen e 26

explaining the high polydispersity). Long rod-like mi elles have already been observed by SANS measurements in systems similar to ours

165

. Results from rheometry

experiments show that the vis osity de reases with applied strain rate (Figure 2.2 (right)) therefore exhibiting properties of non-Newtonian shear-thinning uids. This kind of behavior is ommon for solutions ontaining large non-spheri al mole ules in a solvent with smaller mole ules. It is generally supposed that the large mole ular

hains tumble at random and ae t large volumes of uid under low shear, but that they gradually align themselves in the dire tion of in reasing shear and produ e less resistan e.

This behavior onrms the presen e of rod-like mi elles. No enthalpy

hange ould be dete ted by dierential s anning alorimetry so that no information

ould be dedu ed about possible phase transitions o

urring in the system between

10

and

80◦ C 166168 .

Probably, the amount of surfa tant was too low for su h a

dete tion with our equipment.

13 12 11 10 / Pas

9 8 7 6 5 4 3

200

400

600

800

1000

-1

/ s

Figure 2.2:

Left: Cryo-TEM image of a SDS/DTAB aqueous solution at the molar ratio of 2.5/1

and a total surfa tant on entration of 1 wt.% (reprodu ed from ref. 169 ); right: vis osity of the same sample as a fun tion of shear rate.

2.4.2 Salt-Indu ed Mi elle-to-Vesi le Transition Upon the addition of sodium hloride, the solutions exhibited a transition from a

olorless to a blue solution, the blue olor being typi al of the presen e of large obje ts. Samples with dierent salt on entrations were analyzed by dynami light 27

90

80

70

50

40

R

H

/ nm

60

30

20

10

0 0.00

0.01

0.02

c

Figure 2.3:

0.03

0.04

-1

NaCl

/ molL

In rease in the mean hydrodynami radius of atanioni aggregates upon the addition

of sodium hloride.

s attering. The addition of hloride salts auses an in rease in average parti le size and a ertain turbidity of the solution (Figure 2.3).

DLS indi ated a signi ant

in rease in the mean hydrodynami radius (RH ) of the mi elles from

10

to a.

70

nm. It an be expe ted that the salt s reens the ele trostati intera tions, whi h leads to smaller headgroups and therefore a higher pa king parameter. Freeze-fra ture and ryo-TEM onrm the formation of lamellar sheets in the sample (Figure 2.4). However, by arefully observing the ryo-TEM images one an see that at lowest on entration of added salt (10 mM) the long rod-like mi elles present in the starting solution start to break-up and luster together, see Figure 2.5 (left). Other images from the same solution show that these lusters seem to form small pie es of lamellae, whi h eventually lose to form round vesi les. The urving of membranes is represented by the presen e of darker, sti-looking edges, due to the higher ele tron density in these points. Figure 2.5 (right) shows some urved pie es of lamellar sheets as well as some omplete vesi les. The vesi les represented in ryo-TEM appear to be perforated; su h perforated vesi les have previously been 28

Figure 2.4:

Cryo-TEM (left) and FF-TEM (right) photographs representing the formation of

multi-lamellar sheets upon the addition of 10mM NaCl. The arrows show regions where we an observe the unraveling of ribbon-like mi elles into lamellar sheets. The molarity in the ase of FF-TEM experiments orresponds to the on entration of the solutions prior to ryo-prote ting.

observed in various surfa tant systems

170174

. Surprisingly, FF-TEM images do not

onrm su h perforations. The addition of salt produ es dramati ee ts dete ted by freeze-fra ture.

20

At

mM of NaCl large spheri al, highly undulated aggregates are observed. As men-

tioned previously, FF-TEM exploits the property that surfa es fra ture along the area of least resistan e.

In the ase of vesi les this is within the bilayer.

fore, only 3-dimensional obje ts an be observed. is from

150

to

500

There-

The size range of the parti les

nm, see Figure 2.6 (left). This apparent polydispersity is most

likely due to the hara terization te hnique used; the measured size of the obje t depends on the region where the samples are fra tured. Some of the aggregates are fra tured lose to the middle; Figure 2.6 (right) shows a ring of vesi les. Sin e the aggregates pi tured in Figure 2.6 are observed in high amounts, they most likely represent the same obje t, fra tured in dierent pla es ( lose to the `poles' of the blastulae vesi les, as opposed to the middle of the vesi le).

The images suggest

that the inside of these parti les are hollow and lled with the same solvent as the surrounding (water). Due to the similarities in appearan e we named the observed stru ture blastulae, taking the name from biologi al origin; the blastulae are an early 29

Figure 2.5:

Cryo-TEM photographs representing the breaking-up of ribbon-like mi elles and

onsequent lustering of the pie es. Clusters of elongated aggregates are indi ated by bla k (left), whereas individual aggregates are designated by white arrows. The image on the right shows

lusters of smaller aggregates (bla k arrows) and vesi les with perforated surfa es (white arrows).

Figure 2.6:

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

the addition of 20mM NaCl ut near the surfa e (left); ut through the middle (right), learly representing the solvent lled avity. The arrow shows an individual vesi le with its own membrane.

stage of embryoni development onsisting of a spheri al layer of ells surrounding a uid-lled avity. On the basis of the present pi tures we annot say, whether the blastula vesi le is an aggregate onsisting of one individual membrane or a luster of elongated mi elles as observed in ryo-TEM. Both possibilities will be explored 30

further in the text. However, it is interesting to note that some unilamellar individual vesi les are also present. Interestingly, the average diameter of the vesi les ( a. 60 nm) is of the same size as the bulges forming the blastulae stru ture. This might speak for the possibility that the blastula is a tually a luster of individual small vesi les. These, however, are not deformed in a way that it is usually observed in aggregates. A me hanism for this type of lustering will be dis ussed later on, pointing to the similarity with spe i -site (or ligand-re eptor) binding. It should be noted that similar aggregates have been observed in another ontext

175,176

and

will also be dis ussed later.

Figure 2.7:

FF-TEM image of individual unilamellar vesi les upon the addition of 30 mM NaCl.

The bar represents 100 nm. As more salt is added to the system the lusters begin to disaggregate. At sodium

hloride on entrations of

30

mM only individual unilamellar vesi les are observed

as an be seen in Figure 2.7. Finally, at NaCl on entrations of

40 - 45

mM, a loose and unstru tured, randomly

pa ked aggregation of vesi les is observed, see Figure 2.8.

These aggregates are

not spheri ally symmetri al and the individual vesi les forming the aggregates are 31

Figure 2.8:

FF-TEM photograph representing the aggregation of vesi les upon the addition of

45 mM NaCl to the referen e SDS/DTAB mi ellar solution. The bar represents 200 nm.

deformed. In summary, two features are new in this system: i) the appearan e of blastulae stru tures, and ii) the series of dierent stru tures that are indu ed by salt addition only, without any further additives.

2.5 Dis ussion 2.5.1 Models of the Mi elle-to-Vesi le Transition In the following we propose a possible me hanism for the observed pattern formation. The dierent steps are represented in Figure 2.9.

`Lamellar Model'

It is well known that, as an ele trolyte is added to a mixed

mi ellar solution, the ee tive surfa e area of the surfa tant headgroups be omes smaller. This ee t is mainly due to ounterion on entration and s reening as well as onsequent dehydration of the neutralized heads. 32

This ee t favors a lamellar

pa king (step

a → b′ ).

The formation of large multi-lamellar sheets is energeti ally

unfavorable, so these start urving and onsequently forming large spheri al obje ts. A theoreti al model has been suggested by Safran et al.

13

, showing that in ases of

mixed surfa tants vesi les are more stable than lamellar stru tures. This is due to the formation of surfa tant bilayers, where the two monolayers onsist of dierent surfa tant on entrations, whi h results in equal and opposite monolayer spontaneous urvatures.

This model is in a

ordan e with our result, where the large

spheri al aggregates are shown to form from mi elles via lamellar sheets. The main fa tor governing the pa king arrangement in these phases is the degree of hydration of the polar headgroup. As the polar headgroups are dehydrated even more by the addition of salt, the pa king parameter in reases and the formation of individual vesi les is favored. In the present atanioni system the outer layer of the vesi les is omposed of positively and negatively harged groups with the latter being in ex ess. A redistribution of

harges may then take pla e in the highly exible vesi le membranes leading to lo al pat hes of positive and negative harges. The lo ally positive harges of one vesi le

an intera t favorably with the negative harges of another vesi le and vi e versa (Figure 2.9, stage

+, −

f ).

In other words, the vesi les an attra t ea h other due to the

intera tions among the headgroups of surfa tants belonging to dierent vesi-

les. This is somewhat similar to harge u tuations in polarizable obje ts leading to van der Waals intera tions. But the dieren e is that here the ion harge distribution of the headgroups u tuates, and not the ele tron distribution. The lo al Coulomb attra tion may be strong enough to over ome the overall repulsive for e of equally harged vesi les a ting at larger distan es. Sin e vesi les are spontaneously formed from lamellar sheets, they are, at this starting point, in lose enough onta t for this to happen. In this ase only lo alized parts of the membrane (pat hes) are intera ting, similarly to spe i site binding.

As the ion strength in the solution is in reased the blastulae vesi les disaggregate. We suppose this is due to the fa t that the intera tions between the oppositely

harged headgroups of neighboring vesi les are weaker than those within a vesi le (one must onsider the additional strong van der Waals for es between the hydropho33

f

+ NaCl

+ NaCl

+ NaCl

+ NaCl

e

a

b

b’

Figure 2.9:

d

c

e

c’

S hemati al representation of the observed stru tures that form through in reasing

salt on entration: From the starting mi ellar solution, whi h exhibits rod-like mi elles (a) two possibilities are presented: In the ` lustering model', the rod-like mi elles (a) start breaking-up into smaller pie es (b), whi h start lustering together to form spheri al aggregates. In the `lamellar model', multi-lamellar sheets are formed upon addition of salt (b′ ). These start losing to form undulated giant vesi les(c′). At higher ioni strength, the formation of blastulae lusters (c) is observed, where the outer layer is omposed of individual vesi les, whi h en lose a uid-lled

avity. The vesi les intera t through oppositely harged headgroups of surfa tants belonging to dierent vesi les (f ; the open and lled ir les represent headgroups of opposite harges). As salt

on entration is further in reased, the attra tion is s reened and the vesi les segregate (d). Still higher on entrations of salt in the system produ e a non-spe i intera tion, where van der Waals for es dominate, ausing the vesi les to loosely aggregate while deforming (e).

34

bi surfa tant tails). Therefore, as more salt is added and the harges on the vesi les are s reened, the `bonds' between the vesi les are broken. The luster begins to disaggregate due to the net negative harge of the individual vesi les, resulting in the formation of individual unilamellar vesi les. At still higher ioni strength the ele trostati repulsions are s reened and the ee t of van der Waals for es be omes signi ant. A loose and unstru tured, randomly pa ked aggregation of vesi les is observed. A similar behavior (o

ulation of vesi les) was observed upon the addition of NaCl to the mi ellar solution of dode ylbenzene sulfoni a id

177

`Clustering Model'

.

In this model, the ribbon-like mi elles start breaking up into

smaller ylindri al aggregates ( orresponding to step

a → b).

These ould be small

vesi les, whi h grow until retaining a radius at whi h they are most stable (small vesi les are thermodynami ally unfavorable due to high urvature).

Again these

aggregates start lustering together despite an overall net negative harge.

c→e

Steps

remain the same as in the `lamellar model'.

2.5.2 Blastulae Vesi les Mi ros opy images learly show that the blastulae lusters are spheri al and hollow. Figure 2.6 (right) pi tures the uid-lled avity and the astounding symmetry of the aggregate. The interesting phenomenon is that in this luster the vesi les are not deformed in a way that is usually observed in aggregates. Aggregation of vesi les without a

ompanying deformation of membrane had until now been observed only in systems to whi h spe i ligands and re eptors were added.

In su h ase no

deformations o

ur when vesi les are brought together by a spe i site-binding (ligand-re eptor) intera tion. This is be ause not the whole vesi le surfa e is involved in the intera tion, but only a dis rete number of onta t points on ea h surfa e. This kind of self-assembly of vesi les driven by ligand-re eptor oupling was reported by Chiruvolu et al. and Walker et al.

178180

.

It is interesting to translate this on ept to blastulae formation by proposing a u tuation of the ele tron density in the membrane as was des ribed above. Su h 35

a formation of lo alized partitions of opposite harge has been previously reported by Aranda-Espinoza et al.

181

.

A spontaneous partitioning of positively harged

mixed bilayer vesi les in the presen e of negatively harged parti les resulting in an ele trostati repulsion between oppositely harged parti les was observed.

In our

ase the ee t is reversed, ele trostati attra tion of vesi les of equal net harge are found. Re ently, the asymmetry of harge in lipid bilayers indu ed by monovalent salts was reported.

A

ording to Gurtovenko

182

, the dieren e in the headgroup

orientation on both sides of the bilayer, oupled with salt-indu ed orientation of water dipoles, leads to an asymmetry in the harge-density proles and ele trostati potentials of the bilayer. In this report lipid vesi les with only one type of mole ules are present. In our ase, where two oppositely harged surfa tants are present, the salt-indu ed asymmetry may be the reason for the pat hes of the single harged surfa tants of opposite sign. Finally, the overall shape of the blastulae formed by the `atta hed' vesi les an be explained by the preferential lo al aggregate urvature determined by geometri al onstraints, obeying the same rules as the formation of vesi les from surfa tants. It should be noted that the attra tion of overall equally

harged obje ts is dis ussed from time to time in literature

183

. The present ndings

and the model proposed here may help to understand su h phenomena. As far as oexisten e of vesi les and mi elles are on erned, intermediate stru tures have been reported in the literature: open vesi les, mesh phases, or even pat hes and dis s

56,170,184

.

However, to our knowledge su h symmetri ally shaped hollow

stru tures as the blastulae vesi les have never been observed before in atanioni systems. One of the possible explanations is that blastulae are the diluted ounterpart of the `oyster phase' whi h has been observed for other harged bilayers in the absen e of salt

185

.

The rationale in the sequen e of observed shapes (as s hemati ally represented in Figure 2.9), when spontaneous urvature is varied via omposition, is given by a general me hanism: a o- rystallization o

urs, followed by a segregation of ex ess material.

The amount of non-stoi hiometri omponent distributes between the

latti e and the edges and thus ontrols the shape of the rystallized olloids

36

186

.

2.5.3 The O

urren e of Convex-Con ave Patterns in Biologi al Systems

Experimentally, the o

urren e of onvex- on ave deformations of high regularity, similar to our blastulae, was rst observed in a study of lipid extra ts from ba terial membranes

187,188

. The egg arton pattern is mostly found in omplex lipid mixtures

of biologi al origin su h as Streptomy es hygros opi us and brain sphingomyelin

189

.

Later on, this kind of urved patterns has also been observed in a few examples with a more simple omposition su h as in vesi les of DMPC mixed with a polymerized amphiphile with butadiene groups

190

, N-nervonoyl sphingosylphosphoryl holine

191

both in their gel state and in systems ontaining the lipid omplex soybean le ithin and poly(diallyldimethylammonium hloride)

192

.

However, the repeating onvex-

on ave patterns in these so- alled `egg arton' stru tures are found on at sheets and the obje ts are multilamellar vesi les with diameters largely ex eeding the diameters of the bulges. By ontrast, FF-TEM images of the present atanioni system show that the building blo ks forming the blastulae aggregate are of the same average diameter as the individual vesi les present ( a.

60

nm) in solution.

The

origin of onvex- on ave bilayer deformation is believed to result from onstraints imposed by limiting hydration of the headgroup and a frustration arising from the spontaneous urvature of the bilayers Gebhardt et al.

194

193

. This ee t had already been dis ussed by

. Undulation and the formation of so- alled egg arton stru ture

had been extensively studied theoreti ally. A `hat and saddle' model was proposed by Helfri h

195

to explain the existen e of orrugated membranes of biologi al origin.

Fournier has shown that anisotropi in lusions an indu e spontaneous bending in at lipid membranes, whi h attra t ea h other to form an egg arton stru ture

196,197

.

A model al ulation based on bilayer bending elasti ity yielding disordered egg arton textures was proposed by Goetz and Helfri h

198

. However, all these al ulations

are not appropriate to explain the pe uliar vesi le patterns experimentally observed in present work. 37

2.5.4 Raspberry Vesi les For the same reason the blastulae are dierent from the so- alled raspberry vesi les. The term `raspberry vesi les' was used previously to des ribe onvex- on ave vesi les obtained after the indu tion of osmoti sho k on giant unilamellar vesi les in dierent phospholipid systems

175

.

The `raspberry ee t' is related to the deating of the

liposome due to the volume redu tion, the onsequen e being the existen e of an ex ess membrane. This ex ess membrane indu es the formation of inverted `daughter vesi les'. Similar observations have been reported by Ménager and Cabuil

176

for the

osmoti shrinkage of liposomes lled with a ferrouid and in the ase of liposomes subje ted to a gradient of glu ose

194

.

However, the evolution of these vesi les is

dierent from the one observed in our ase.

The initial membrane undulation of

giant vesi les seems to be a ommon step in all ases. In the aforementioned ases, the osmoti shrinkage of vesi les was shown to be reversible whi h is proof of a persistent membrane ne k onne tion between the mother and daughter vesi les. At high enough osmoti strength the membrane ruptured. No vesi les with individual membranes were observed. This is not the ase in our system, where the in rease of ioni strength promotes the formation of individual membranes, resulting in a spheri ally symmetri al luster of unilamellar vesi les, the blastulae. Higher ioni strength results in a separation of the vesi les.

2.5.5 Blastulae Vesi les: A General Trend in Catanioni Systems? Finally, we would like to report that similar images have been found also in samples

ontaining esium hloride and in a system omposed of sodium dode yl arboxylate and DTAB in the presen e of sodium hloride ( . f. Figure 2.10). Further experiments and theoreti al insights are ne essary in order to larify the exa t me hanism of formation of su h stru tures and the reason for this intermediate stru ture to be found only in atanioni systems ontaining salt. 38

Figure 2.10:

Blastulae vesi les in an SDS / DTAB system with the addition of 20 mM CsCl.

2.6 Con lusions We have presented a way to make hollow regularly shaped stru tures by spontaneous se ondary self-assembly of vesi les without additives ex ept salt. These spheri al, symmetri al aggregates of individual vesi les were never observed previously, even in systems with ligand-re eptor binding. A me hanism of formation for this type of super-stru tures was proposed, showing the importan e of harge u tuation. Finally, su h blastulae aggregates ould be onsidered as an intermediate step in the formation of unilamellar vesi les from bilayers in non-equimolar atanioni systems.

39

40

Chapter 3 Spe i Alkali Cation Ee ts in the Transition from Mi elles to Vesi les Through Salt Addition 3.1 Abstra t A transition from mi elles to vesi les is reported when salts are added to atanioni mi ellar solutions omposed of sodium dode anoate (SL) / dode yltrimethylammonium bromide (DTAB) and sodium dode ylsulfate (SDS) / DTAB, with an ex ess of the anioni omponent. The ounterion binding and in rease in aggregate size was monitored by mass spe trometry, rheology and dynami light s attering measurements, whereas the vesi les were hara terized by freeze-fra tion and ryotransmission mi ros opy experiments. The ee t of ounterions on the formation of vesi les in both systems was studied and ompared to evaluate the role of the surfa tant headgroups on the ounterion spe i ity. The hange in aggregate size an be a

ommodated by the in rease of ounterion binding and onsequent dehydration of the surfa tant headgroups. A lassi ation of the ations ould be made a

ording to their ability to in rease the measured hydrodynami radii. It was observed that, if the sulfate headgroup of the anioni surfa tant is repla ed by a arboxyli group, the order of the ions was reversed. 41

3.2 Introdu tion The investigation of spe i ion ee ts has engaged resear hers for de ades. Despite that, ion properties and their intera tions with other mole ules are still not understood in detail, and we are far away from being able to predi t their behavior. A major di ulty in the study of salts presents the fa t that many phenomena involve the a tion of ations and anions of the ele trolyte. Mole ular self-assembly in surfa tant systems is largely dependent on the number of water mole ules surrounding the headgroups. When ions are added to a solution, they dehydrate the surfa tant headgroups

a

3,59

. This auses a de rease in the value

(ee tive area per mole ule at the interfa e) and onsequently an in rease of the

stru tural pa king parameter

P,

whi h may result in the formation of vesi les. The

ee t of salts on a harged system an dier mu h depending on their kosmotropi or haotropi hara ter. It is well known that small ions of high harge density (e.g. sulfate, arboxylate, sodium) are strongly hydrated (kosmotropes), whereas large monovalent ions of low harge density (e.g. iodide, potassium) are weakly hydrated ( haotropes)

101,199

. Ea h salt is therefore expe ted to have an individual inuen e

on vesi le formation, whether it tends to adsorb at the interfa e between mi elle and water or remains strongly hydrated in the bulk. Collins' on ept of `mat hing water anities'

101

provides us with a simple model

of spe i ion-ion intera tions. From various experimental results (for an extensive review see ref.

200

) Collins on luded that the dominant spe i for es on ions of

the same valen y in water are short-range for es of hemi al nature and that the long range ele trostati for es generated by simple ions in water are weak relative to the strength of water-water intera tions

200

. Therefore, onta t ion-pair forma-

tion is a tually dominated by hydration-dehydration. A good agreement was also found with re ent al ulations of water pairing, where expli it water mole ules were modeled

201

.

It is hallenging to see if this simple on ept holds also for spe i

ion-headgroup intera tions. A salt indu ed mi elle-to-vesi le transition was studied in two atanioni systems: sodium dode anoate (SL) / dode yltrimethylammonium bromide (DTAB) and sodium 42

dode ylsulfate (SDS) / DTAB. An ex ess of the anioni surfa tant was present in both systems. The two systems diered only in the headgroup of the anioni surfa tant in order to elu idate the fa t that not the types of the ion alone, but rather the spe i ation-surfa tant intera tions shape the surfa e behavior. Large ation spe i ity was found, however the series experien ed a reversal when the arboxylate ion was ex hanged for a sulphate one. SDS / DTAB is a well-known throughly studied system

47,48

, whereas, by using a fatty a id based surfa tant, the system re-

sembled more to a biologi al membrane than a system ontaining SDS; it had been determined that ellular membranes onsist of roughly 2 to 5 % of free long- hain

arboxyli a ids. The atanioni surfa tant trimethylammonium headgroup, in omparison, resembles the holine group that is often present in membranes

202,203

. The

ee t of dierent anions and ations on the mi ellar solutions was studied by phase diagrams, rheology, dynami light s attering and mass spe trometry, whereas the vesi les were hara terized by freeze-fra ture and ryo-TEM imaging. The me hanism of mi elle-to-vesi le transition was investigated and ompared.

3.3 Experimental Pro edures Materials >

The surfa tants, sodium dode yl sulfate (SDS; Mer k, Germany; assay

99%), sodium dode anoate (SL; Sigma, Germany; grade: 99-100%) and dode-

yltrimethylammonium bromide (DTAB; Mer k, Germany; assay

> 99%) were used

as re eived. All sodium and hloride salts used in the experiments were supplied by Mer k, Germany. Millipore water was used as solvent in all ases.

Sample Preparation

Surfa tant sto k solutions were prepared by dissolving weighed

amounts of dried substan es in Millipore water. The solutions were then left for 24 hours to equilibrate at

25◦ C.

The anioni -ri h region of the phase diagrams was

used in both ases. The atanioni solutions were prepared by mixing the surfa tant sto k solutions to obtain a xed anioni / ationi surfa tant mass ratio: (this orresponds to a molar ratio of about

2/1)

and

70/30

(molar ratio

60/40

2.5/1)

was

used for SL / DTAB and SDS / DTAB mixtures, respe tively. The starting ratio 43

was determined from phase diagrams (see Figures 2.1 and 3.1). The total surfa tant

on entration was kept at 1 wt.% at all times. solution at in reasing on entrations up to and left to equilibrate for a day at

25◦ C

50

Salts were added to the mi ellar

mM. The solutions were then stirred

before making measurements.

Ele trospray Mass Spe trometry (ES-MS)

Cation anities for the vesi ular

interfa e/ arboxylate group were determined by ele trospray mass spe trometry. ES-MS was arried out using a Thermoquest Finnigan TSQ 7000 (San Jose, CA, USA) with a triple stage quadrupole mass spe trometer. The solutions were sprayed through a stainless steel apillary held at 4 kV, generating multiple harged ions. Data were olle ted using the X alibur software.

Other Methods

Cryo- and freeze-fra ture transmission ele tron mi ros opy, dy-

nami light s attering and rheology were performed as des ribed in Chapter 2.

3.4 Results 3.4.1 Phase Diagrams The addition of salts to the atanioni mixtures at a ertain ratio of the surfa tants indu ed an easily observable aggregation, due to a formation of a bluish olor or turbidity. Salts were added to samples from all parts of the phase diagrams, however homogeneous vesi ular systems upon salt addition were observed only at a ertain anioni / ationi surfa tant ratio (see Phase Diagrams, Figures 2.1 and 3.1). For this reason, the starting solution was taken from the mi ellar region of the phase diagram, a little way from the equimolarity line, with an ex ess of the anioni spe ies (with a mass ratio of

60/40

and

70/30

for SL / DTAB and SDS / DTAB mixtures,

respe tively).

44

4% SL 0.00 1.00

0.25

0.75

I_

equimolarity

0.50

0.50

0.75

1.00 water 0.00

Figure 3.1:

0.25

I+ 0.25

0.50

0.75

0.00 1.00 4% DTAB

S hemati ternary phase diagram of the SL / DTAB system (right) at 25◦ C (the bla k

arrow shows the starting point (referen e sample), to whi h dierent salts were added). Figure reprodu ed with permission from the do toral dissertation of A. Renon ourt.

3.4.2 Counterion Ee ts The ee t of various ations was studied by varying the on entration of dierent alkali hloride salts (LiCl, NaCl, KCl, CsCl).

As the on entration of salt in the

system was in reased, the solutions exhibited a transition from a olorless to a blue solution, the blue olor being typi al of the presen e of large obje ts. This transition

ould be observed for all salts, however, the on entration at whi h vesi les appeared was strongly dependent on the nature of the ation. Further in rease of added salt

on entration nally led to a phase separation, with one of the two phases being blue and isotropi and the other turbid. Samples with dierent salt on entrations were rst analyzed by dynami light s attering. The addition of hloride salts auses an in rease in average parti le size and a ertain turbidity of the solution (Figure 3.2). DLS indi ated a signi ant in rease in the mean hydrodynami radius (RH ) of the mi elles from

10

up to a.170 nm. In

agreement with our visual observations the rate of in rease of the measured hydrodynami radius was dierent for ea h hloride salt. This highlights strong ation spe i ity. 45

For instan e, in the SL / DTAB system (Figure 3.2, left) a small amount of LiCl (<

10

mM) was su ient to dramati ally in rease the hydrodynami radius (over

nm), whereas with CsCl, on entrations higher than

60

100

mM were needed to obtain

parti les of that size. A lassi ation of the ations an be made a

ording to their ability to in rease the measured hydrodynami radius of the aggregates forming in the SL / DTAB surfa tant mixture: Cs

+

<

+

K

<

+

Na

<

+ Li .

200 60

R (nm)

100

20

50

0

40

H

R

H

(nm)

150

0

10

20 c

SALT

Figure 3.2:

30

40

0

50

0

10

20

(mmol/L)

C

30

SALT

40

50

(mmol/L)

The ee t of various ations on the growth of the hydrodynami radii RH of the

atanioni aggregates in SL / DTAB (left) and SDS / DTAB (right) systems with an ex ess of anioni surfa tant in both systems: LiCl (△), NaCl (N), KCl (), CsCl (). However, if the arboxyli headgroup of the anioni surfa tant was repla ed by a sulfate group, the order in whi h the ions assist the formation of vesi les was reversed ( ompare with Figure 3.2, right).

Salts ontaining big ations having a

smaller harge density (for instan e CsCl) more e iently indu ed vesi le/aggregate formation than small highly harged and highly hydrated ations (su h as LiCl). A

+ reversed Hofmeister series was therefore observed: Cs

>

+

K

>

Na

+

>

+ Li .

Cation anities for the vesi ular interfa e / sulphate (or arboxylate) group were determined by ele trospray ionization mass spe trometry.

Although in this

method the ionization pro ess takes pla e in the gas phase, the hemi al nature of surfa tant monomers and simple ions is the same in the liquid and in the gas phase. Therefore, the preferential ion-surfa tant intera tions an easily be noted 46

204,205

. Re-

sults show that most of the mass signals (peaks) remain un hanged independently of the nature of the added salts. A typi al ES-MS spe trum of the omplete gion (m and

m/z

re-

z are the mass and harge of an ion, respe tively) is shown in Figure 3.3.

100

A

-

Relative Abundance

80

60

-

+

(2A + H )

40

-

20 -

(2A +Na+) -

-

+

-

0

200

300

400

+

(2A +K )

- -

(A + K + Cl ) 500

-

600

700

m/z Figure 3.3:

ES-MS spe trum of 1 wt.% SL / DTAB mi ellar solution with 15mM NaCl; A− :

dode yl arboxylate anion (199 Da); C+ : dode yltrimethylammonium ation (228 Da).

Figure 3.4 represents the anion fragmentation patterns of the SL / DTAB atanioni mixture upon the addition of various salts: for better visibility only a part of the

m/z

region is presented. A loser look reveals that the position of the peak

representing the binding of sodium ions to the arboxylate (2A un hanged.

−

+

+ − Na ) remained

Also the size of the peak was omparable in all spe tra with the ex-

eption of spe trum (A). When LiCl was added, two same-sized peaks appeared, one representing the binding of sodium, the other of lithium ions to the arboxy-

+ late anion. This suggests that Li was able to ome loser to the vesi ular surfa e, repla ing a part of the sodium ions at the interfa e. The exa tly opposite was observed in the ase of CsCl (Figure 3.4D). The latter suggests a very small o

urren e of esium ions at the vesi le interfa e (very small (2A

−

+

+ − Cs ) peak). A general

ordering of the ations ould be determined from the ES-MS spe tra, with lithium showing the greatest anity for the anioni group and the other ations following: Li

+

>Na+ >K+ >Cs+ .

As observed previously, this order exhibited a reversal if the 47

surfa tant mixture ontained a sulphate headgroup (SDS).

-

-

+

(2A + H )

100

+

(2A + H )

100

-

-

80

Relative Abundance

Relative Abundance

80

60

40

60

40

-

-

20

(2A + Na+) -

(2A + Li+)

-

-

(2A + C )

- -

+

+

-

400

500

-

(2A + H+)

+

-

(A + C + Cl )

600

700

400

500

+

(2A + H )

100

600

m/z

-

-

-

80

Relative Abundance

80

Relative Abundance

-

(2A + C ) - -

+

B

0

m/z

100

-

-

(A + C + Cl )

A

0

(2A + Na+)

20

-

-

60

40

60

40

-

20

-

+

-

+

-(A

(2A + Na ) -

C

(2A + K )

-

+

(2A + C ) -

-

- -

-

+ C+ + Cl )

0 400

500

600

0

700

-

-

+

(2A + C ) +

-

- -

(A + C + Cl ) + (2A + Cs )

D 400

m/z

Figure 3.4:

+

(2A + Na )

20

500

600

m/z

Ion binding as determined by ES-MS: addition of 15mM of (A) LiCl, (B) NaCl, (C)

KCl and (D) CsCl to a SL / DTAB mi ellar solution; A− : dode yl arboxylate anion (199 Da); C+ : dode yltrimethylammonium ation (228 Da). Results from rheology experiments performed on atanioni solutions with various salts showed that the vis osity de reases with applied strain rate ( . f. Figure 3.5). This behavior is ommon for solutions ontaining large non-spheri al mole ules, whi h tumble at random under low shear, but align themselves in the dire tion of

3

in reasing shear and produ e less resistan e as the shear rate is in reased .

This

behavior pointed to the presen e of rod-like mi elles in the solutions, whi h was

onrmed also by mi ros opy (see ahead). No hange in the rheologi al behavior of the samples was observed however as the nature of the salt is varied. Probably, the

hange in the overall on entration of elongated mi elles present in the solution was 48

too low for su h dete tion to be possible with our equipment.

5

/ Pas

4 3 2 1 0

0

50

100

150 / s

Figure 3.5:

200

250

300

-1

Vis osity of 1 wt.% SL / DTAB mi ellar solution upon the addition of 15 mM LiCl

(△), NaCl (N), KCl (), CsCl () as a fun tion of shear rate.

Homogeneous samples exhibiting a bluish olor typi al for solutions ontaining vesi les were further investigated by ryo- and FF-TEM. The images onrmed an in rease in the size of the aggregates and high polydispersity ( ommon to nonequimolar atanioni systems). Figure 3.6(A) shows a pi ture similar to what we have reported in the previous hapter. Not only the SDS / DTAB system, but also the SL / DTAB system onrmed the presen e of long ribbon-like mi elles unraveling and forming sheets. It seems that the addition of salts produ ed a similar ee t in both systems, hinting at a general me hanism of mi elle-to-vesi le transitions in

atanioni systems. Figure 3.6(B) shows many half- losed and some already fully losed vesi les.

Cryo-TEM images of the SDS / DTAB system onrm the high polydispersity in su h mixtures.

Vesi les of various sizes were seen to exist in equilibrium with

ribbon-like mi elles.The in rease in measured RH was therefore most likely due to 49

B

A

Figure 3.6:

Cryo-TEM photographs of a SL/DTAB aqueous solution at the molar ratio of

approximately 2/1 and a total surfa tant on entration of 1wt.% upon the addition of 20 mM of NaCl. The arrows in (A) show the presen e of long ribbon-like mi elles unravelling and forming sheets. These sheets then urve and form spheri al aggregates (B).

the ombined ee ts: formation of vesi les as well as the elongation of rods. The latter ee t is well known in literature

206,207

.

FF-TEM, be ause of the advantageous fra ture ourse within hydrophobi zones, provides us with the information regarding the membrane surfa e of the vesi les. A pe uliar dieren e ould be observed.

The addition of sodium ions to the two

systems assisted in the formation of very dierent vesi les. While the sulfate group

ontaining surfa tant mixture formed polyhedral (fa eted) vesi les with very sti looking membranes, the arbooxylate system exhibited the presen e of individual unilamellar vesi les with ompletely smooth membranes (Figure 3.7). Fa eted vesi les are present when the tails of the surfa tants are rather sti due to rystallization. This is ommonly seen in atanioni systems. This stiness may also des ribe the hydration of the surfa tant mole ule.

Similar behavior ould be found within one system, when the ee t of various

ations on the membrane stiness was ompared. The stronger preferential binding of ions ould also be observed by the distortion of the SDS / DTAB vesi le membrane

+ + as it be ame dehydrated when Cs and Li ions were ompared (see Figure 3.8). 50

200nm

Figure 3.7:

FF-TEM photographs representing the ee t of 30 mM NaCl on the SL / DTAB (left)

and the SDS / DTAB (right) mi ellar solution. The left image presents vesi les with ompletely smooth membranes. When the arbooxylate group is ex hanged for a sulphate group, the vesi les be ome sti-looking and fa eted.

3.4.3 Co-ion Ee ts The binding of ounterions to a mixed atanioni mi elle/solution interfa e depends

1

on the surfa tant molar ratio , i.e.

the harge of the aggregates.

Zeta potential

measurements of the atanioni solutions with and without salt onrmed the presen e of negatively harged aggregates.

Therefore, no anion spe i ity was to be

expe ted. Samples were nevertheless he ked also in the presen e of various sodium salts (NaCl, NaBr, Na2 CO3 , NaSCN, NaOA , NaNO3 ). All salts proved to have a signi ant inuen e on the growth of the mi elles, however, no signi ant spe i ity

ould be found for the dierent anions (the urves overlap - . f. Figure 3.9).

3.4.4 Nonioni Ee ts Two non ioni additives, glu ose and urea, having respe tively salting-out and salting-in ee ts, were also added to the same mixed mi elles solutions. No ee t whatsoever on the growth of the measured hydrodynami radius ould be noti ed. Even though glu ose and urea were added at a on entration as high as 1 mol/L, no in rease in the hydrodynami radius of the aggregates was found. 51

Figure 3.8:

FF-TEM images the ee t of 45 mM CsCl (left) and LiCl (right) on the referen e

SDS / DTAB mi ellar solution. The bars represent 100 nm in both ases.

3.4.5 Ee ts of `Hydrophobi Ions' Above observations on alkali salts have shown that all alkali ations aused an in rease in the measured hydrodynami radii. At high ele trolyte on entrations the ele trostati repulsions are s reened and the vesi les aggregate. This ee t has not been observed when so alled `hydrophobi ' ions are present.

Tetramethylammo-

nium hloride (TMAC) and holine hloride (ChCl) were added to mixed mi ellar solutions of SDS / DTAB and SL / DTAB. Results obtained by DLS show only a slight in rease of the hydrodynami radius upon the addition of TMAC to a mi ellar solutions (from 20 to 30 nm).

A larger dieren e in the ation anity for the aggregate surfa e ould be determined by ES-MS. Figure 3.10 shows anion fragmentation patterns upon the addition of salt, tetramethylammonium hloride (for better visibility only a part of the region is presented).

m/z

+ ES-MS spe tra hint that TMA was able to ome loser to

the vesi ular surfa e, repla ing a part of the sodium ions at the interfa e (Figure 3.10(A)). The exa t opposite was observed in the ase of arboxylates (Figure 52

175 80

150 125 R [nm]

H

[nm] H

R

60

100 75

40

50 20

25 0

0

10

20

30

40

50

0.00

0.01

Figure 3.9:

0.02

0.03

c [mM]

c [mM]

The ee t of various anions / sodium salts on the growth of the hydrodynami radii

RH of the atanioni aggregates in SL / DTAB (left) and SDS / DTAB (right) systems: NaCl ((•), NaSCN (), NaBr (), Na2 CO3 (△), NaOA (◦) and NaNO3 (N)

3.10(B)). The results obtained for holine hloride salts were omparable.

FF-TEM images of the SDS / DTAB solution showed the presen e of vesi les at low TMAC on entrations ( .f. Figure 3.11, left), similarly to the ase of alkali salts. However, the membranes of the vesi les were more exible and are therefore more likely to fuse. Surprisingly, at higher on entration only small dis s and mi-

-

(2DS- + K+)-

(2DS- + TMA+)-

100

+ -

(2L + H )

100

80 -

(2DS- + Na+)-

60

40

Relative Abundance

Relative Abundance

80

(2DS- + H+)-

60

(2L- + K+)-

40

+ (L- + C+ + Cl - )- (2L + TMA )

20

20

A

0 500

Figure 3.10:

600

m/z

700

0

800

+ -

(2L + Na )

B 400

500

600

700

m/z

Ion binding as determined by ES-MS: addition of 40 mM of TMAC and to a (A)

SDS / DTAB and (B) SL / DTAB mi ellar solution. DS- : dode ylsulfate anion (265 Da); L- : dode yl arboxylate anion (199 Da); K+ : dode yltrimethylammonium ation (228 Da). 53

100 nm Figure 3.11:

FF-TEM photographs representing the ee t of 20 mM (left) and 40 mM (right)

TMAC on the referen e SDS/DTAB mi ellar solution.

elles ould be observed (Figure 3.11, right). In the ase of SL / DTAB, however, no vesi les were observed.

Small dis s and mi elles were present over the whole

on entration region. The same pi ture ould be found when 50 mM of ChCl was added to SDS/DTAB and SL/DTAB.

3.5 Dis ussion 3.5.1 Aggregation Behavior of Catanioni Systems The main thermodynami driving for e for the asso iation of a ationi and an anioni surfa tant mixture is the release of ounterions from the aggregate surfa es. This results in a large entropy in rease. For these systems a non-monotoni hange in

P

is observed, with a pronoun ed maximum as the mixing ratio is varied

1,3

. In

the system presented here vesi les ould only be formed when the anioni surfa tant is in molar ex ess ompared to the ationi surfa tant. Therefore, only this ratio was onsidered here. The geometry of aggregates in olloidal systems is attributed to the pa king of the amphiphili mole ules. The pa king parameter is dependent on the length and vol54

ume of the hydrophobi tail and the size of the hydrophobi head of the surfa tant mole ule. These fa tors are often expressed in a pa king parameter where and

a

v

P = v/(lmax a),

and lmax are the volume and length of the hydrophobi part, respe tively,

the area per mole ule at the interfa e. If the hydro arbon part of the surfa -

tant is kept onstant and only the headgroup is varied, then the dieren e in the aggregation behavior an be attributed solely to the properties of the polar headgroup. Be ause

a

des ribes the ee tive headgroup size, whi h in the ase of ioni

surfa tants is largely determined by repulsive ele trostati for es, and not the ioni radius, the hange of morphology in surfa tant systems is largely dependent on the number of water mole ules surrounding the headgroups. It has been observed by

hemi al trapping method

135

that in asso iation olloids, the hanges in the balan e

of for es ontrolling the aggregate stru ture are ree ted in the hanges in interfa ial on entrations of water and other omponents. The ions, when added to a mi ellar solution, an dehydrate the surfa tant headgroups. This auses a de rease in the value

P.

a

and onsequently an in rease of the stru tural pa king parameter

As a onsequen e, the riti al mi ellar on entration is redu ed

aggregate morphology hanged

209

60,208

and the

. The ee ts are aused by destru tion of the hy-

dration layer of the surfa tant, de reased ele trostati repulsions, and an in reased

ounterion binding

60

. Consequently, the surfa tant monomers an be pa ked loser

together leading to a bilayer formation.

3.5.2 Counterion Properties The ee t of salts on a harged system an dier mu h depending on the salting-in or salting-out properties of their ions

107,115,210

. Salting-out ions are usually small,

with relatively small polarizabilities. They have high ele tri elds at short distan es and have tightly bound hydration water. NMR eviden e

211

suggests that small ions

do not show spe i binding to mi elles and retain their mobility and their water of hydration right up to the mi ellar `surfa e'

212

although this result depend on the

type of surfa tants. Salting-in ions are usually large, with signi ant polarizabilities. They have weak ele tri elds and a loose hydration sheath, whi h an be easily removed. NMR proves that they perturb phospholipid (surfa tant) headgroups in a 55

signi ant way, probably through ion pairing

213

. Ea h salt is therefore expe ted to

have an individual inuen e on vesi le formation, whether it tends to adsorb at the interfa e between mi elle and water or remains strongly hydrated in the solution.

3.5.3 Collins' `Law of Mat hing Water Anities' Re ently, a simple `law of mat hing water anities' has been proposed

101

.

It is

based on the tenden y of oppositely harged ions to spontaneously asso iate in aqueous solution. Only oppositely harged ions with mat hing absolute free energies of hydration should spontaneously form inner sphere ion pairs.

This is supposed

to be due to the fa t that the strength of intera tion between the ions and the water mole ules is orrelated to the strength with whi h the ion intera ts with other ions

200

. Small osmotropi ions an ome lose together forming inner sphere ion

pairs without intermediate water mole ules. The same is supposed to be true for big

haotropi ions, whereas when a osmotropi ion approa hes a haotropi ounterion, the ions should remain separated by at least one water mole ule. With this model an impressive number of phenomena and properties an be des ribed.

3.5.4 Counterion Sele tivity of Alkyl Sulfates In the present ase the headgroups in ex ess are alkylsulfates. In ontrast to double harged sulfate ions, su h headgroups have a more haotropi behavior. With this assumption and a

ording to Collins' on ept, alkylsulfates should ome in lose

onta t with haotropi ions like esium, whereas lithium ions remain further away.

+ Therefore it is expe ted that Cs s reen more e iently the negative harge ex ess + on the aggregates than Li , and this is pre isely what is observed, .f. Figure 3.2 (right). The same ordering of ations was found in systems ontaining pure SDS mi elles

214

and in the ase of another anioni surfa tant with a sulfate group, sodium

dode ylbenzenesulfonate

177

. To make a more quantitative analysis we ompare the

salt on entrations to the ex ess on entration of SDS ( ompared to DTAB), whi h is approximately

0.015

mol/L. From Figure 3.2 (right) it an be inferred that this

on entration roughly orresponds to the asymptoti limit for mi elle to vesi le 56

+ + transition in the ase of K and Cs

hlorides.

Therefore it may be on luded

that these ations have a strong propensity to the sulfate groups and that sulfate headgroup- ation pairs are formed; in this ase the ex ess negative harge of the vesi les is neutralized. This nding is in agreement with the asso iation onstants found for a similar surfa tant n-o tylbenzenesulfonate

154

.

3.5.5 Counterion Sele tivity of Alkyl Carboxylates We have seen that with the sulfate group the binding in reases with the de reasing size of the hydrated alkali metal ions. The order is reversed when the headgroup is ex hanged for a arboxylate. The arboxylate headgroup exhibits water-ordering properties and the addition of an alkyl hain should not hange the kosmotropi behavior of the headgroup. A

ording to Collins' on ept, alkyl arboxylates should then ome in lose onta t with kosmotropi ions like lithium, making it more possible to form inner-sphere ion pairs, whereas esium ions remain further away. Therefore, it is expe ted that Li

+

s reens more e iently the negative harge ex ess on

+ the aggregates than Cs , and this is pre isely what is observed in Figure 3.2 (left). The same order of ounterion-binding to arboxylate headgroups that was found in our atanioni surfa tant system are found in polyele trolyte solutions measuring the ation anity to ion-ex hange resins potentials ena

222

220

, of ele trophoreti mobility of olloids

219,220

221

215218

, when

, in studies of membrane

, and of ion-transport phenom-

. Our results are also in agreement with measurements of ounterion binding

to soap mi elles

81,212,223,224

and long- hain fatty a ids

222,223,225

.

Re ent MD simu-

lations have shown that the arboxylate groups dominate the ounterion behavior even in omplex systems su h as proteins

210

.

3.5.6 Alkyl Sulfates vs. Alkyl Carboxylates Similar ounterion spe i ity was observed in the ase of polyele trolytes.

As in

the ase of surfa tants, the spe i -ion ee ts do not depend only on the individual properties of the parti ipating ioni group and its ounterion ( harge, size, harge distribution and polarizability), but also on the overall harge of the polyion, as well 57

as on the possible ooperating binding sites whi h might inuen e the binding site. Furthermore, even at high dilution a substantial number of ounterions is for ed into lose proximity to the polyion by the long-range ele trostati for es

226,227

, so

that there always exist a large number of ion pairs for whi h solvation ee ts should be observable. Strauss

217,218

used the dilatometri method to measure the volume

hanges that o

urr when polyele trolyte solutions are mixed with solutions ontaining dierent spe i ally intera ting ounterions. Polyele trolytes ontaining sulfate and arboxylate headgroups exhibit opposite series of ounterion binding, similar to the series observed in our experiments on surfa tant mi elles. Dilatometri results

learly show spe i ities depending on both the polyion and on the metal ion whi h would not be expe ted on the basis of long-range ele trostati density of the polyion. The ee ts of the latter are governed predominantly by the linear harge density of the polyion and the linear harge densities of the studied sulfonates and a rylates are the same. It follows that the intera tions giving rise to the observed volume hanges involves spe i sites on the polyions. Similar results of alkali ations to polya rylates and polysulfonates have been dis ussed also by other authors

215,216,228230

. The

reversal of the ation series in the presen e of polya rylates was attributed to a

ompetition between water of hydration and the anion for a given ation, whi h is related to the harge distributions, polarizabilities, and the ee tive eld strengths of the ions

221,222

. The on ept was however not studied thoroughly.

The reversion of series was observed also when measuring the ation anity to ionex hange resins

219,220,231

, ion-transport phenomena

ounter-ion binding to long- hain fatty a ids

222

, swelling of hydrogels

212,215,223,224,233,234

232

and

.

3.5.7 Mole ular Dynami s Simulations Also MD simulations show similar results.

Chialvo and Simonson

short- hain polystyrenesulfonate and found the intera tion with Li

ause desolvation. Similarly, Lipar et al.

230

235

+

modeled a

too weak to

reported the binding to polyanethole-

+ sulfoni a id to be strongest for Cs . Re ently, an extended omputational study of pairing of sodium and potassium with a broad set of biologi ally relevant anions was des ribed

201,236

. The monovalent sulfate and the arboxylate groups and their 58

pairing with sodium vs. potassium were examined. The stru tures of the four investigated onta t ion pairs, together with the ation - anion oxygen distan es are shown in Figure 3.12.

Figure 3.12:

Geometries of the onta t ion pairs of a) sodium-a etate, b) potassium-a etate, )

sodium-methylsulfate, and d) potassium-methylsulfate. The ations bind bidentally to two anioni oxygens. Reprodu ed from Vla hy et al. 236 . A ombination of

ab initio

al ulations with a polarizable ontinuum solvent

model and mole ular dynami s simulations were used to quantify the relative ationanion asso iation free energies, i.e., the values of

∆∆G

onne ted with repla ing

potassium with sodium in a onta t ion pair with a etate or methylsulfate anion

− − These two free energy dieren es for CH3 COO and CH3 SO4 are

−2.50

and

236

.

+0.37

k al/mol, respe tively. We see that while a etate strongly prefers sodium over potassium (by about 2.5 k al/mol), methylsulfate weakly (by roughly 0.4 k al/mol) prefers potassium over sodium. These ndings are onsistent with our experimental results 59

and also with the empiri al Law of mat hing water anities

101

.

3.5.8 Generalization of the Con ept: Hofmeister Series of Headgroups In the pre eding paragraphs a omparison of two headgroups is given. However, more headgroups are of interest in olloidal hemistry and biology, espe ially sulfonates and phosphates. Con erning hydrogen phosphate, both the harge density as well as several experimental results

201,214,237,238

suggest that it an be lassied between

the arboxylate and the sulfonate headgroup. Following omputational as well as experimental results, we propose a way to lassify headgroups in a Hofmeister like series, ordering them from osmotropi to haotropi headgroups: - arboxylate, hydrogen phosphate, -sulfonate, -sulfate.

(presented in Figure 3.13).

With this

series many spe i -ion phenomena in olloidal systems an now be explained.

3.5.9 The Anioni Ee t The addition of sodium salts proved to have a signi ant ee t on the growth of the mi elles (Figure 3.9). This in rease of parti le size is mainly due to a de rease of the head group repulsions be ause of ele trostati s reening. Sin e the mi elles are very probably negatively harged, the anions will not ome into lose onta t with the mi ellar surfa es and the onset of vesi le formation o

urs at approximately the same on entration for all sodium salts. The small dieren es in radius size observed are likely to be due to the dieren es in the hydration of the anions; in the ase of strongly hydrated Cl

−

larger vesi les were observed than in the presen e of other

less hydrated ions. A similar ee t was previously observed in studies of negatively

harged phospholipid vesi les

239

.

3.5.10 The Non-Ioni Ee t The fa t that the addition of glu ose and urea had no ee t on the growth of the mi elles (no mi elle to vesi le transition was observed) suggests that ele trostati intera tion is ne essary and is the rst order ee t. Hydration numbers show that 60

Figure 3.13:

Ordering of anioni surfa tant headgroups and the respe tive ounterions regarding

their apabilities to form lose pairs. The green arrows represent strong intera tions ( lose ion pairs).

glu ose dehydrates the headgroups as well as the salts (the hydration numbers for Cs

+

and glu ose are 4.3

116

and 3.5

tions of glu ose and urea (up to aggregates.

240

1M)

respe tively.

Even mu h higher on entra-

had no ee t on the growth of the surfa tant

This onrms that dehydration of the headgroups alone is not su-

ient to trigger the mi elle to vesi le transition. The reason must be sought in the ele trostati s. Even if these un harged mole ules modied the surrounding water stru ture, this ee t is not strong enough to signi antly hange the urvature via adsorption to the headgroups.

If the mi elles were onsisted of just one type of

surfa tant (one harge), then the addition of salt would s reen the repulsion between the headgroups, ausing the surfa tant mole ules to arrange in a bilayer. The opposite ase would be if the surfa tants were mixed in equimolar amounts. Then 61

the salts would s reen the attra tion between them, most likely ausing the distan e between the headgroups to in rease. Sin e our mixed mi elles ontain an ex ess of the anioni surfa tant, we an assume an asymmetri distribution of harges (as was also proposed in the pre eding hapter). Therefore the addition of salt should have a similar ee t as in the ase of `single- harged' mi elles.

3.5.11 Ee ts of `Hydrophobi Ions' Ammonium ations have a salting-in hara ter and enhan e the solubility of otherwise poorly water-soluble ompounds. They are well-known as phase transfer atalysts. In water the ioni hara ter of these ions is dominant, whereas in hydrophobi environments the organi hara ter prevails

241

. However, there is a signi ant dif-

feren e between the properties of ammonium and tetramethylammonium ions. The ammonium ation behaves mu h like a big alkali ation; it is weakly hydrated, its

+ hydration sphere being similar to the hydration sphere of the Cs ion. By ontrast, the tetramethylammonium ation is an intermediate ase between `simple' ions and hydrotropes su h as xylene sulfate

242

.

It seems plausible to assume

that this `hydrophobi ' ation an penetrate into the mi ellar surfa e mu h as anions like tosylate and sali ylate

243246

in the ase of ationi surfa tants. If this is

true, the tetramethylammonium ion an a t as a o-surfa tant, perhaps even partially repla ing the ationi surfa tant and perturbing lamellar stru tures - a feature that is well known from hydrotropes

247

. Interestingly, ES-MS suggest that a strong

binding of the hydrophobi ions to the mixed mi elles is present only in the ase of sulfates, but not also when a arboxylate surfa tant is present. The initial formation of the vesi les, observed in the SDS / DTAB system at low salt on entrations ould be solely due to the in reased ioni strength.

However, this does not explain the

observed hanges in membrane exibility. The destru tion of vesi les at higher salt

on entrations suggests the in orporation of the hydrophobi ion into the membrane and a hange in the pa king of the surfa tants. Although the information obtained from ele troni mi ros opy is more or less the same for both systems at high salt on entrations, the reason for the presen e of only mi elles and small dis s are most likely dierent. 62

In the ase of SDS, the

strong binding of the `hydrophobi ions' should ause a disruption of the membrane, destroying the large vesi ular stru tures. TMA

+

On the other hand, it is likely that the

ion does not ome into lose onta t to the SL / DTAB mi ellar surfa e.

Therefore, it assists aggregate growth only by hanging the properties in the bulk. As ammonium ions have salting-in properties, they do not indu e the dehydration of the ioni surfa tant headgroups and therefore also not assist in vesi le formation. Further dis ussion on alkyl trimethylammonium groups an be found in Chapter 4.

3.6 Con lusions Through the addition of salts a transition from rod-like mi elles to vesi les was observed in aqueous solutions omposed of DTAB and an ex ess of either SL or SDS. Whereas no anion spe i ity for the added salts appeared in the formation of vesi les, the nature of the ation was found to inuen e strongly the riti al salt

on entrations around whi h mi elles turn to vesi les. In the present ase of negatively harged vesi les, ion-spe i ee ts were expe ted for ations, sin e they a

umulate in high on entration in the vi inity of the vesi le. The observed ation spe i ity followed the lassi al Hofmeister series for ation adsorption to sulfate headgroups. When alkyl arboxylates were present in solution, the ations followed a reversed Hofmeister series.

The ion spe i ity ould be qualitatively explained

a

ording to Collins' on ept of mat hing water anities.

To this purpose, the

headgroup of an alkylsulfate had to be regarded as a haotrope and the alkyl arboxylate as a kosmotrope. The morphologies observed during the mi elle-to-vesi le transition are analogous to those presented in the pre eding hapter, hinting at a general me hanism ommon to atanioni systems.

63

64

Chapter 4 Anion Spe i ity Inuen ing Morphology in Catanioni Surfa tant Mixtures with an Ex ess of Cationi Surfa tant 4.1 Abstra t In the previous hapter we reported on an ion spe i mi elle-to-vesi le transition, when salts were added to a atanioni mi ellar solution omposed of sodium dode yl arboxylate (sodium laureate, SL) and dode yltrimethylammonium bromide (DTAB), with an ex ess of SL. In the present hapter, we illustrate the ion spe i ity, when DTAB was in ex ess in the same system. In this ase, no transition to vesi les was observed, but an elongation of mi elles upon salt addition. The ounterion binding and in rease in aggregate size was monitored by mass spe trometry, dynami light s attering measurements, and ryo-transmission mi ros opy.

The

me hanism was argued by employing the ability of the ounterion to dehydrate the surfa tant headgroup. This theory was onrmed in phase with Collins' on ept of mat hing water anities. 65

4.2 Introdu tion Mixtures of surfa tants show enhan ed performan e in te hni al pro esses (e.

g.

detergen y, tertiary oil re overy, drug arrier systems, otation), when ompared to pure surfa tant systems.

Surfa tant mixtures for spe i appli ations are of-

ten hosen based on empiri al eviden e and experien e. However, to optimize the appli ations it is important to have a general understanding of the interplay of intera tions between the surfa tants in a mixed system and of the fa tors inuen ing the phase diagram.

Therefore, it is of interest to study the self-aggregation and

mi ellization of su h mixtures. When oppositely harged surfa tants are mixed, new properties appear. Aqueous

atanioni mixtures exhibit a wide range of unique properties that arise from the strong ele trostati intera tions between the oppositely harged heads. They exhibit low riti al mi elle on entration (CMC) values and a non-monotoni hange in the

2

surfa tant pa king parameter (P ) as the mixing ratio is varied . For this reason a large number of aggregate stru tures su h as spheri al and rod-like mi elles, vesi les, lamellar phases, and pre ipitate have all been observed depending on the on entration, the size of the hain length or nature of the polar heads, and the ratios of the surfa tants in solutions

24,26,48,5357

. One advantage of atanioni systems as

ompared with more robust genuinely double hained surfa tants are their greater sensitivity to parameters su h as temperature

153

or the presen e of salts

47

.

The ee t of salt type on various physi o- hemi al properties of a system was rst observed over one hundred years ago by Franz Hofmeister who dis overed the dependen e of protein solubility on the type of added inorgani ele trolyte

114

. Sin e

then, a wide range of ion-spe i phenomena in biology, pharma y, and hemistry was observed. They have been re ently reviewed in a spe ial issue of `Current Opinion in Colloids and Interfa e S ien e'

115

.

Extensive studies have shown that the

ounterion has a strong ee t on the thermodynami s and aggregation properties of surfa tants

248250

.

Salt ee ts on the m , mi ellar size, and degree of disso i-

ation for a given surfa tant may follow a lyotropi (or Hofmeister) series

251

.

An

ion's position in the lyotropi series an be orrelated with its harge and hydrated 66

radius. Depending on the harge density of the anion, it an intera t more or less strongly with the ationi headgroups of mi ellar surfa e. Su h a `binding' de reases the ele trostati repulsion between the surfa tant headgroups and hen e favors aggregation.

This lowers for instan e the CMC and the degree of ionization of the

2− mi elles. A typi al Hofmeister series for anions is as follows: SO4 −

Cl

<

− NO3

<

− − Br < ClO4

<

I

−

<

CNS

−

<

− C2 H3 O2

<

− − (the positions of the NO3 and Br ions

are often swit hed in the lytropi series). Single-tailed surfa tants usually form globular mi elles in aqueous solution above

4

their CMC .

An in rease in surfa tant on entration may indu e the formation

of worm-like mi elles ons

206,252254

252,253

. Similarly, addition of organi and inorgani ounteri-

, un harged ompounds like aromati hydro arbons

harged surfa tant

26,158

255

, or an oppositely

an transform spheri al mi elles into worm-like mi elles.

In the previous hapter, we have reported a way to lassify headgroups in a Hofmeister like series.

We also were able to explain how salt-indu ed mi elle-to-vesi le

transitions in the anioni -ri h regions of the phase diagrams in atanioni systems depend on both ion and headgroup spe i ities. In the present hapter, this study is extended to the ationi -ri h region. We fo us on the inuen e of the ounterion identity on the aggregation behavior of non-equimolar mixed surfa tant solutions,

omposed of sodium dode anoate (SL) and dode yltrimethylammonium bromide (DTAB) with an ex ess of DTAB. The ee t of dierent anions and ations on the mi ellar solutions will be shown by means of phase diagrams, ryo-TEM, mass spe trometry and dynami light s attering. Collins' on ept of mat hing water anities

101

will be shown to be also very valuable for the omprehension of the found

series of salt sensitivity. And nally, the aggregation behavior will be ompared to the one found in the anioni -ri h region of the orresponding phase diagram.

4.3 Experimental Pro edures Materials

The surfa tants, sodium dode anoate (SL; Sigma, Germany; grade: 99-

100%) and dode yltrimethylammonium bromide (DTAB; Mer k, Germany; assay

>

99%) were used as re eived. All sodium and hloride salts used in the experiments 67

were supplied by Mer k, Germany. They were also used as re eived without further puri ation. Millipore water was used as solvent in all ases.

Phase Diagrams

Surfa tant sto k solutions were prepared by dissolving weighed

amounts of dried substan es in Millipore water. 24 hours to equilibrate at

25◦ C.

The solutions were then left for

The atanioni solutions were prepared by mixing

the surfa tant sto k solutions to obtain a xed anioni / ationi surfa tant mass ratio of 30 / 70. The starting ratio was determined from phase diagrams. The total surfa tant on entration was kept at 1 wt.% at all times. Salts were added to the mi ellar solution at in reasing on entrations. The solutions were then stirred and left to equilibrate for a day at

Other Methods

25◦ C

before making measurements.

Ele trospray mass spe trometry, ryo-transmission ele tron mi-

ros opy and dynami light s attering were performed as des ribed in the previous

hapters.

4.4 Results and Dis ussion 4.4.1 Ion Binding in Catanioni Surfa tant Mixtures In the studied atanioni system, the ationi surfa tant DTAB was in ex ess. Therefore, it was expe ted that the variation of the anions will produ es a larger ee t as the variation of the type of ations. Figure 4.1 shows ele trospray ionization mass spe tra of the SL / DTAB atanioni solutions (mass ratio 30 / 70) upon the addition of dierent salts. Although in this method the ionization pro ess takes pla e in the gas phase, the hemi al nature of surfa tant monomers and of simple ions is the same as in the liquid phase. Therefore, ounterion anities for the surfa tant

an easily be noted

204,205

. Be ause our surfa tant mixtures already ontained oun-

terions, we added the same on entration of salts. In this way, we ould observe the

ompetition of the ounterions for the surfa e of the surfa tant aggregate.

+ Figure 4.1 represents the DTA fragmentation patterns upon the addition of 68

100

K

100

+

+

80

Relative Abundance

80

Relative Abundance

K

60

+

- +

(2K + SCN )

40

+

- +

(2K + Br )

20

60

40

+

- +

(2K + Br )

20

+

+

- +

(2K + A )

- +

(2K + A )

B

A 0

0 100

300

200

400

500

200

100

700

600

300

400

m/z

100

700

+

Relative Abundance

K

600

500

m/z

80

60

60

+

Relative Abundance

80

- +

(2K + Br )

40

+

- +

(2K + NO3 )

20

+

- +

(2K + A )

+

- +

(2K + Br ) 40

20

+

- +

(2K + A )

D

C 0 100

200

K

Figure 4.1:

300

400

500

600

0

700

100

200

300

400

500

600

700

m/z

m/z

+

Ion binding as determined by ES-MS: addition of 15mM of (A) NaSCN, (B) NaBr,

(C) NaNO3 and (D) Na2 SO4 to a SL / DTAB mi ellar solution; A− : dode yl arboxylate anion (199 Da); K+ : dode yltrimethylammonium ation (228 Da); the peaks between 250 and 300 (m/z) are solvent related.

various salts: for better visibility only a part of the

m/z

region is presented.

It

turns out that most of the mass signals (peaks) remain un hanged, independently of the nature of the added salts. A loser look revealed that the position of the peak representing formation of ion

+ − + pairs between the oppositely harged surfa tant ions (2K + A ) remained un hanged.

Also the size of the peak was omparable in all spe tra.

Similarly, the

peak orresponding to the binding of bromide ions to the dode yl trimethylammo-

+ − + was present in all spe tra, regardless of the nature of nium ation (2K + Br ) the added salt. The height of this peak, however, was less pronoun ed in spe trum (A). When omparing the binding of anions to the dode yl trimethylammonium

−

ation, we observed that only the SCN ex hange the bromide anions at the mi− −

ellar surfa e. NO3 and Cl showed approximately the same anity for the alkyl trimethylammonium group, whereas in spe trum (D), when Na2 SO4 was added to 69

the atanioni mixture, no peak representing the binding of sulfate ions was visible. These results suggest that the strongly hydrated sulfate anion did not ome lose to the mi ellar surfa e, whereas the loosely hydrated thio yanate anion was able to ome loser to the mi ellar surfa e, repla ing a part of the bromide ions at the interfa e. A general ordering of the ations ould be determined from the ES-MS spe tra, with thio yanate showing the greatest anity for the anioni group and the

− other ations following: SCN

>

−

Br

>

− NO3

>

Cl

−

>

− CH3 COO

>

2− SO4 . The

variation of the ation of the salt produ ed no ee t on the ation fragmentation patterns, as an be expe ted in a system with an ex ess of ationi surfa tant. The

m/z

peaks remained un hanged for all added hloride salts.

4.4.2 Phase Behavior upon Salt Addition 4% SL 0.00 1.00

0.25

0.75

I_

equimolarity

0.50

0.50

0.75

1.00 water 0.00

Figure 4.2:

0.25

I+ 0.25

0.50

0.75

0.00 1.00 4% DTAB

S hemati ternary phase diagram of the SL / DTAB system at 25◦ C (the arrow

represents our starting solution (referen e sample; mass ratio 30 / 70). The inuen e of salts on the SL / DTAB atanioni mixture was further followed by observing the phase behavior; the starting phase point in the phase diagram being a mi ellar solution, . f. diagram shown in Figure 4.2. Dierent salts ae ted the system in various ways. While the addition of some salts produ ed no visible ee t, others indu ed an easily observable aggregation (formation of a bluish olor 70

or turbidity). As the salt on entration was in reased the aggregation be ame more pronoun ed. The ee ts are reported in Table 4.1.

Salt

25 mM

50 mM

NaSCN

bluish/turbid

turbid

NaBr

bluish/turbid

turbid

NaNO3

bluish

turbid

NaOA

lear

lear

Na2 SO4

lear

lear

NaCl

lear

lear/bluish

LiCl

lear

lear/bluish

KCl

lear

lear/bluish

CsCl

lear

lear/bluish

Choline Cl

lear

lear

Visual observations of the ee t of various salts on the SL / DTAB (mass ratio: 30 /

Table 4.1:

70) atanioni solution. The referen e sample was a lear homogeneous solution.

The aggregation was he ked by light s attering; however problems were reported by the apparatus due to the high s attering intensity and the high polydispersity of the solutions. Despite that we are able to onrm an in rease in the hydrodynami radius of the parti les in a

ordan e with our visual observations ( . f. Figure 4.3). We emphasize that the absolute values of the hydrodynami radii should be taken with a grain of salt, be ause the aggregates formed in solutions are rods (as will be shown with ryo-TEM further on), and the CONTIN software used by the Zetasizer 3000 al ulates the radius of spheri al aggregates. However, the trend in the in rease of RH is important. The degree of mi ellar growth varied in exa tly the

2− inverse order of the hydrated radius of the ounterion: SO4 − NO3

<

−

Br

<

≈

− C2 H3 O2

<

−

Cl

<

− CNS .

− Salts ontaining big anions having a weakly distributed harge (CNS ) indu ed 2− more e iently the mi ellar growth than the divalent sulphate (SO4 ). Apparently 71

130 120 110 100 90

R

H

[nm]

80 70 60 50 40 30 20 10 0

0

20

40

60

80 c

Figure 4.3:

salt

100

120

140

160

[mM]

The ee t of various ations / hloride salts on the growth of the hydrodynami

radii RH of the atanioni aggregates in SL / DTAB systems: NaOA (N), Na2 SO4 (), NaCl (◦), NaNO3 (), NaBr (•) and NaSCN (△).

the large hydrated radius of the sulfate ion hinders its ability to bind to the mi elle

ontaining an ex ess of DTAB.

4.4.3 Anion Spe i ity in Physi o-Chemi al Properties of Alkyltrimethylammonium Systems The ompetition of various anions for the binding sites on the surfa e of CTAB mi elles was he ked by Larsen and Magid

256

. They found a strong binding of nitrate

to the mi elle, displa ing bromide. The positions of bromide and nitrate ions are often ex hanged in the lyotropi series. As an example, Cohen and Vassiliades

257

− − found that NO3 was less ee tive than Br at lowering the CMC of CTAB. The lyotropi series was observed also when mi ellization of alkyltrimethylammonium halides, heats of ounterion binding, surfa e tension, and the thermodynami and 72

mi roenvironmental properties of the mi elles were ompared

256,258261

.

The on-

trolling fa tor in this ion-spe i ity seems to be the distan e of losest approa h of the ion to the mi elle.

4.4.4 Inuen e of Salt on the Aggregation Behavior of Surfa tants The geometry of aggregates in olloidal systems is attributed to the pa king of the amphiphili mole ules. The fa tors governing the shape of the aggregates are expressed in its simplest form by a pa king parameter

P = v/(lmax a),

whi h is

dependent on the length (lmax ) and volume (v ) of the hydrophobi tail and the effe tive size of the hydrophili headgroup (a). The area per mole ule at the interfa e depends on the hydration of the surfa tant headgroup, whi h in turn depends on the ion harge density and the distan e to the small ounterions. Furthermore, it

an be inuen ed by the ioni strength of the solution. When ions ome lose to the headgroups, the harges are more or less neutralized the value

a

(area per mole ule

at the interfa e) de reases, and onsequently the stru tural pa king parameter

reases

60

P

in-

. Therefore, a de rease of the riti al mi ellar on entration and a hange

in the aggregate morphology an be observed

60,208,209

.

As was mentioned above, the system under onsideration has an ex ess of DTAB. Previous studies have shown that the self-aggregation behavior of alkyl tetramethylammonium surfa tants is relatively independent of the hydro arbon hain length

256

.

The phase behavior of DTAB (C12 ) and CTAB (C16 ) is therefore omparable. The formation of rodlike mi elles in CTAB systems at high on entrations is widely known

256,262266

. Alkylammonium halides have also been reported to exhibit a tran-

sition from spheri al to rod-like mi elle shape with in reasing on entration of added salt. At lower ioni strength only spheri al mi elles are formed, while at salt on entrations higher than a ertain threshold, larger rod-like mi elles are formed in equilibrium with the spheri al mi elles. The length of the rod-like mi elle in reases strongly with in reasing ioni strength on entration

253,267278

. Ozeki, et al.

277

re-

ported that the sphere-rod transitions of mi elles took pla e in aqueous NaBr solu73

tions of DTAB when the salt on entration ex eeded 1.8 M, whereas measurements of Sudan red B in the same system indi ated that the minimum NaBr on entration required to indu e the sphere-rod transition was 1.0-1.5 M. This may be due to a de rease in intrami ellar repulsion by ele tri al shielding due to ioni atmosphere. At higher salt on entrations, there is a dehydration of the spheri al mi elles due to the salting-out ee t of NaBr. By adding an anioni surfa tant, we lower the harge density of the previously

Figure 4.4:

Left: spheri al and small rod-like mi elles in the SL / DTAB referen e solution

(before the salt was added); right: signi ant growth and networking of rods upon the addition of salt (25 mM NaBr).

ationi mi elles.

In this way, we also lower the on entration of salts ne essary

to produ e ylindri al mi elles.

Similar ee ts are observed with the addition of

anioni hydrotropes to ationi surfa tants

279281

. Hydrotropes bind strongly to op-

positely harged surfa tant ions and redu e the headgroup area of the surfa tant by redu ing the headgroup repulsions.

Thus, they are ee tive at promoting the

elongated mi elle formation. The elongation of ylindri al mi elles was studied by ryo-transmission ele tron mi ros opy. Figure 4.4 shows that our initial mi ellar solution (referen e sample) exhibits the presen e of spheri al mi elles, as well as some short rods. An elongation of the ylindri al mi elles was observed as salts were added to the solution. These 74

Figure 4.5:

Cryo-TEM images representing the ylindri al growth as a fun tion of anion type;

the ee t of 25 mM NaNO3 (left) and NaBr (right).

mi elles were quite exible; small loops were observed, and the long mi elles formed networks. This aused an in rease in the vis osity (observed at the preparation of the solutions) and an in rease of the turbidity of the solutions. However, the growth and the on entration in rease of the rods was salt spe i (Figure 4.5). The addition of NaBr produ ed a higher on entration of longer rods than the addition of NaNO3 . These then started networking together and forming a net-like stru ture. Surprisingly, we ould not see any formation of vesi les. Upon the addition of salts, the system transformed from spheri al mi elles and small rods to long ylindri al mi elles, and eventually to pre ipitation as the salt on entration was in reased further. On the other hand, we have observed the transition from rod-like mi elles to vesi les in the anioni -ri h region of the phase diagram of this sample (when SL was in ex ess).

A similar system to ours was studied by Sierra et al.

282

.

They inves-

tigated the phase behavior of atanioni mixtures omposed of DTAB and sodium unde anoate. They found the presen e of spheri al mi elles on the anioni ri h side of the phase diagrams and short rods on the ationi ri h side. The rods elongated and aggregated as the omposition neared equimolarity. No hexagonal mesophase was dete ted and the phase separation at equimolarity was pre eded by the formation of bundles of rod-like mi elles. The entanglement of long thread-like mi elles 75

in CTAB mi elles upon salt addition was observed also by Aswal et al.

283

.

4.4.5 Explaining Counterion Spe i ity in Surfa tant Systems Sin e the ationi surfa tant was in ex ess in the atanioni system, and also in the mixed mi elles, it is reasonable to suppose that anions were in average loser to the mi ellar surfa e than ations. Consequently, the ee t of anion variation was more pronoun ed than the ee t of ation variation. But what makes the dieren e between anions of the same harge? It is their harge density and polarisability, in line with the ordering from hard (high harge density) ions, su h as a etate, to soft (low harge density and often highly polarizable) ions, su h as thio yanate. Again Collins' on ept of so- alled `mat hing water anities' will be used to explain ounterion spe i ity.

A

ording to Collins' idea, soft ions should ome in

lose onta t with soft ions and hard ions in lose onta t with hard ions

101

.

By

ontrast, when hard and soft ions ome together, they do not approa h so far that they loose their hydration sphere.

Therefore, the intera tion between a hard and

a soft ion should be weak in water.

an be explained by this on ept.

Numerous phenomena in physi al hemistry

For quaternary ammonium ions, it is not sur-

prising that they behave like soft, low-density ions. A

ording to Collins' on ept it

+ is therefore lear that DTA intera t more with softer ions than with harder ones, − and this is pre isely what was found in the present study. For example, SCN ions

ome in lose onta t to the ationi headgroup and they share a ommon hydration shell, whereas a etate anions will stay away from the headgroup and keeps its own hydration shell. Consequently, the pa king parameter will in rease more, when NaSCN is added to the mi ellar solution than when NaOA is added, and therefore the tenden y to form rod-like mi elles is more pronoun ed in the ase of NaSCN. This ee t was already dis ussed in detail in Chapter 3, where mole ular dynami s (MD) simulation was used to lassify surfa tant headgroups from hard to soft, in the same spirit as the ions

236

. A similar ordering as was done in the previous hapter

for anioni headgroups an be done here to represent the ounterion binding to the 76

alkyl ammonium headgroup surfa tant ( . f. Figure 4.6).

Figure 4.6:

Ordering of anioni ounterions regarding their anity for the alkali ammonium

headgroup.

4.4.6 Dierent self-aggregation behavior of atanioni systems in the atanioni - and anioni -ri h regions Why we are able to get vesi les in the anioni -ri h region, but only thread-like mi elles in the atanioni -ri h region is a di ult questions. This phenomenon was already observed by other authors, but never really explained.

If the hanges in

the pa king parameter are onsidered, the values of the hydrophobi part remain the same, only the

a

referen e Sierra et al.

value diers.

282

However,

a

(as reported in literature, e.

g.

) does not dier enough to explain this signi ant dieren e

in the end pa king. One of the best explanations is the dierent hydration behavior of the headgroups ompared to the hydration of the ounterions. of diele tri relaxation spe tros opy Bu hner et al.

284

With the help

were able to observe that

the mi elle surfa e of the SDS surfa tant is strongly hydrophili and the adsorbed

ounterions are generally separated by a layer of water mole ules.

On the other

hand, is the surfa e of the alkyltrimethylammonium bromides hydrophobi , with the bound halide ions dire tly atta hed

284

.

77

4.5 Con lusions A study of the inuen e of added salt on the aggregation behavior of non-equimolar mixed surfa tant solutions was ondu ted.

The system was omposed of sodium

dode anoate (SL) and dode yltrimethylammonium bromide (DTAB) with an ex ess of ationi surfa tant.

The addition of salts produ ed a sphero ylindri al growth

of the mi elles, markedly dependent on the anion identity.

The e ien y of the

anions to elongate the mi elles ould be explained by Collins' on ept of mat hing water anities and the lassi ation of the ationi surfa tant headgroup as a soft, polarizable entity.

78

Part II In reasing the Stability of Catanioni Systems

79

Chapter 5 Inuen e of Additives and Cation Chain Length on the Kineti Stability of Supersaturated Catanioni Systems 5.1 Abstra t The stability of mixed surfa tant solutions of sodium dode ylsulfate (SDS) with

etyltrimethylammonium bromide (CTAB) and with dode yltrimethylammonium bromide (DTAB) was studied as a fun tion of time. These spe i mixtures were shown to have a solubility temperature below that of pure surfa tant solutions in the anioni -ri h region. The stability of su h supersaturated solutions was studied with and without dierent additives. Surfa tant mixtures without additives were shown to destabilize with time varying between

3 and 28 days, depending on the surfa tant

ratio. Generally, the stability of solutions in reased by in reasing the per entage of the anioni surfa tant. The variation of the hain length of the ationi surfa tant produ ed a large ee t on the stability of su h mixed surfa tant systems. The presen e of simple ele trolytes de reased, while the addition of middle- hain al ohols in reased its stability.

Bluish solutions orresponding to a vesi ular region were 81

observed at ratios lose to equimolarity in samples without salt, and in the anioni ri h region upon the addition of middle- hain al ohols. Fluores en e and dynami light s attering measurements showed that the destabilization of the solutions is not due to the formation of bigger aggregates, but rather due to a shift of the equilibrium between mi elles and monomers, leading to the liberation of monomers, whi h pre ipitate.

The lifetime of vesi les and mi elles ould therefore be ontrolled by

varying the omposition of the surfa tant solutions and by additives. Controlling the pre ipitation phenomena is of importan e for a large number of industrial pro esses, su h as oil / solute re overy pro esses after extra tions or hemi al rea tions.

5.2 Introdu tion Mixtures of ationi and anioni surfa tants ( atanioni s) are onstituted of ve types of spe ies, in luding two ounterions. In these systems a ompetition between various mole ular intera tions due to van der Waals, hydrophobi , ele trostati and / or hydration for es may result in a wide variety of phase behaviors and mi rostru tures

24

. Among others, atanioni mi elles, vesi les and bilayer stru tures, an be

more parti ularly found.

In mixtures of anioni and ationi surfa tants a pre-

ipitation zone is observed around equimolarity

24,134

.

The pre ipitation zone an

be ontrolled by hanging the temperature or/and the ioni strength of the solution

47,153

.

In reasing temperature results in a general redu tion in the tenden y

of pre ipitation, whereas the in rease of ioni strength generally shows the opposite ee t.

The formation of nite stru tures in equimolar atanioni mixtures

is thus limited to high temperatures.

It is therefore of obvious interest to study

the parameters whi h ould extend the temperature range in whi h atanioni surfa tant self-aggregation an take pla e. Furthermore, ontrolling the pre ipitation and mi ellization phenomena of atanioni mixtures are of pra ti al importan e in many industrial pro esses, su h as oil / solute re overy pro esses after extra tions or

hemi al rea tions

47,153,285287

, foaming pro esses and wetting time of textiles

288,289

.

Mi elles and surfa tant monomers are in dynami equilibrium and individual surfa tant mole ules are onstantly being ex hanged between the bulk and the mi elles. 82

Furthermore, the mi elles themselves disintegrate and reassemble ontinuously

290

.

While the pro ess of surfa tant mi ellization is primarily entropi ally driven, mi elle formation depends on the balan e of for es between the fa tors favoring mi ellization (van der Waals and hydrophobi for es) and those opposing it (ele trostati repulsions and kineti energy of the mole ules)

60,291,292

. The relaxation times of mi-

elles are dire tly related to the mi ellar stability and are mu h longer for nonioni surfa tants than for ioni surfa tants, be ause of the absen e of ioni repulsions between the headgroups

290

. For the same reason mi elle kineti s in atanioni systems

were also found to be very slow

293,294

. Thus, mixed surfa tant solutions an remain

supersaturated for long periods of time before pre ipitation is omplete equilibrium states of su h systems are questionable

296

295

. As the

, the time dependent stabil-

ity of ommon atanioni mixtures was studied in this paper.

The ee t of ioni

strength and the inuen e of the variation of ounterions were investigated, and also the inuen e of added al ohols as well as the importan e of a dierent hain length.

5.3 Experimental Pro edures Materials

The surfa tants, sodium dode yl sulfate (SDS: Mer k, Germany, grade:

99%), dode yltrimethylammonium bromide (DTAB: Mer k, Germany; assay > 99%) and etyltrimethylammonium bromide (CTAB; Sigma, Germany; grade:

99%) were

used as re eived. All sodium and hloride salts used in the experiments were supplied by Mer k, Germany. The al ohols: methanol, 1-propanol, 1-butanol, 1-de anol

> 99%),

(Mer k, Germany, (Aldri h, Germany, Aesar, Germany;

99.5%),

99%),

(Mer k, Germany,

98%)

ethanol (J. T. Baker, Holland, 1-hexanol (Fluka, Germany,

99%),

1-o tanol (A ros Organi s, U.S.A., were also used as re eived.

99.9%),

1-pentanol

1-heptanol (Alfa

98%),

and itronellol

Millipore water was used as

solvent in all ases.

Determination of the solubility temperature

(This measurement was per-

formed by A. Arteaga during his graduate studies. Results are represented with his permission.)The solubility temperature at dierent time intervals was determined 83

by visual observation. All systems were observed at 1 wt.% total surfa tant on entration. The ationi / anioni mixtures at dierent mass ratios were prepared from 5 wt.% surfa tant sto k solutions. The samples were rst heated to

ooled down and left to equilibrate for 5 hours at was in reased by approximately peratures (between

0◦ C

and

0.5◦ C

99◦ C)

0◦ C,

per minute.

39◦ C,

then

and then the temperature

The measured solubility tem-

orrespond to the transition from pre ipitate to

isotropi phase; i. e. to a mi ellar or vesi ular solution. The pro edure was repeated after a ertain time to analyze the kineti s of the system.

Phase Diagrams

Phase diagrams of the systems were onstru ted a

ording to

the pro edure des ribed above. The samples had to be heated after mixing in order to obtain a homogeneous lear solution. Dierent surfa tant ratios exhibited dierent initial solubilization temperatures,

TI

(more throughly explained in the Results

se tion). The ee t of additives was studied by adding an appropriate amount of salt or al ohol to the SDS / CTAB mixture. Again, in some ases the sample had to be heated in order to obtain a lear solution. The maximum temperature to whi h the samples were heated was at

20◦ C.

39◦ C.

The samples were then ooled down and kept

Phase diagrams were onstru ted by visual observation and by measuring

transmission using a Hita hi U-1000 spe trophotometer. The samples were also observed at

0◦ C in order to shorten the destabilization times and to he k

whether the

order of destabilization is the same at both temperatures.

Counterion Binding

Cation anities for the surfa tant aggregate interfa e were

determined by ele trospray mass spe trometry. ES-MS was arried out using a Thermoquest Finnigan TSQ 7000 (San Jose, CA, USA) with a triple stage quadrupole mass spe trometer. The solutions were sprayed through a stainless steel apillary held at 4 kV, generating multiple harged ions. Data were olle ted using the X alibur software. The surfa tant on entration was kept 1wt.% and the on entration of the various hloride salts was

System Kineti s

0.1

M in all ases.

The evolution of the surfa tant aggregates was further followed

using uores en e spe tros opy and dynami light s attering (DLS) measurements. 84

Pyrene (Aldri h, opti al grade) was used as the external uores en e probe to monitor the hange in surfa tant self-assembly. The preparation of water saturated with pyrene was as reported previously

297,298

. The uores en e emission spe tra of pyrene

were re orded on a Cary E lipse uores en e spe trophotometer (Varian, In .) at

20◦ C.

From the spe tra, the ratio of intensities of the rst and the third vibra-

tional peak of pyrene,

I1 /I3 ,

was al ulated. Parti le size analysis was performed

using a Zetasizer 3000 PCS (Malvern Instruments Ltd., England), equipped with a 5 mW helium neon laser with a wavelength output of was

90◦

633

nm. The s attering angle

and the intensity auto- orrelation fun tions were analyzed using the Contin

software.

Ee t of Al ohols on the System Morphology

Spe imens for ryo-transmission

ele tron mi ros opy ( ryo-TEM) were prepared as des ribed before

169

; frozen sam-

ples were examined with a Zeiss EM922 EF Transmission Ele tron Mi ros ope (Zeiss NTS mbH, Oberko hen, Germany), whereas the repli a were examined and photographed with a Philips CM

12

transmission ele tron mi ros ope.

5.4 Results and Dis ussion 5.4.1 Shift of Solubility Temperature with Time The inuen e of the ationi surfa tant hain length on the solubility temperatures of atanioni mixtures was investigated by omparing mixtures of sodium dode ylsulfate (SDS) with etyltrimethylammonium bromide (CTAB) and with dode yltrimethylammonium bromide (DTAB). CTAB and DTAB have the same quaternary ammonium polar headgroups, but CTAB has a longer hain (16 arbon atoms) than DTAB (12 arbon atoms).

Owing to stronger hydrophobi repulsions of its

hydro arbon hain, CTAB is therefore less soluble in water than DTAB. The Krat point of pure CTAB in water at 1 wt.% is onsequently higher (≈ that of DTAB (<

0◦ C) 300 .

25◦ C) 299

than

The solubility temperatures (TS ) of the orresponding

atanoni mixtures, i. e. DTAB / SDS and CTAB / SDS with a total surfa tant

on entration of 1 wt.% and dierent anioni / ationi surfa tant (molar) ratios 85

were followed as a fun tion of time ( . f. Figures 5.1 and 5.2). Both systems displayed the same marked dieren es in their solubility temperatures over the whole anioni / ationi ratios in the initial days after mixing. The regions were generally higher than the

TK

TS

in the ationi -ri h

of the pure ationi surfa tant. The same

trend was observed for only a part of the phase diagram in the anioni -ri h region. Around equimolarity, a maximum of solubility temperatures (TS ) was rea hed. This maximum lay within the zone of pre ipitation usually observed at room temperature. A bluish zone, orresponding to a vesi ular region, was lo ated in both sides of the entral area of insolubility

301,302

region (approximately between

0.35).

0.6

. It was mu h more extended in the anioni -ri h -

0.7)

than in the ationi -ri h region (around

The vesi ular regions ould be extended by raising the temperature. Harg-

reaves and Deamer SDS above

61

≈ 47◦ C.

noted the formation of liposomes in mixtures of CTAB and The sequen e of dierent phases resembled those observed in

similar atanioni systems

24,49,303

. Obviously, the behaviour of two single- hain sur-

fa tants is mu h dierent in a atanioni system. The pa king between the ationi and anioni surfa tants is quite dierent from the single ioni one and ontributes to in rease the stability of the atanioni rystal, leading thus to higher solubility (and Krat) temperatures. Interestingly, however, at a ertain ratio of anioni / ationi surfa tants a `solubility temperature depression' exists. This depression was lo ated in a range of a molar fra tion of the anioni surfa tant between

0.65 − 0.85

and

ould be observed immediately after mixing. With time, the mixtures undergo a

ontinuous evolution of

TS

toward higher temperatures. Surprisingly, the solubility

temperature was almost onstant over time in the ationi ri h region, whereas a large time dependen y ould be observed in the anioni ri h region, . f. Figures 5.1 and 5.2. Furthermore, the shift in the solubility temperature was more pronoun ed in systems ontaining a longer hain ationi surfa tant (Figure 5.2). For this reason we fo used our attention on the anioni part of the phase diagram of the CTAB / SDS system.

86

100 90

Temperature (°C)

80 70 60 50 40 30 20 10 0 0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0

Molar fraction SDS

Figure 5.1:

Shift of solubility temperature in DTAB / SDS mixtures at dierent surfa tant ratios

as a fun tion of time (ctot = 1wt.%): observations after (N) 1 day, () 4 weeks and (•) 6 weeks.

100 90 Tempertarure (°C)

80 70 60 50 40 30 20 10 0 0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0

Molar fraction SDS

Figure 5.2:

Shift of solubility temperature in CTAB / SDS mixtures at dierent surfa tant ratios

as a fun tion of time (ctot = 1wt.%): observations after (N) 1 day, () 4 weeks and (•) 6 weeks. Both Figures reprodu ed with permission from the do toral dissertation of A. Arteaga. 87

5.4.2 Behavior of the Anioni -Ri h Region of the Phase Diagrams Without Additives The samples (with an ex ess of SDS) were prepared as des ribed in the Experimental Se tion and heated in order to obtain a lear homogeneous solution. The temperature needed an be des ribed by the equation where

TI (◦ C)

TI (◦ C) = −0.43 · X + 64.9,

is the initial solubility temperature expressed in Celsius and X is the

per entage of SDS in the solution ( . f. Figure 5.3 (left)). Then the samples were left

20◦ C.

of the SDS / CTAB system at

20◦ C

The results from spe tros opy measurements are presented in Figure 5.3 (right).

40

100

38

80

Transmission (%)

T (oC)

to ool down and observed at

36 34

60 40

32

20 30 60

65

70

75

0

80

XSDS

Figure 5.3:

0

5

10

15

20

25

time (day)

Left: The temperature needed to obtain lear homogeneous solutions in the anioni -

ri h region an be des ribed by a straight line with a slope of k = −0.43(±0.03) and n = 64.9(±2.6); right: Transmission of the SDS / CTAB atanioni system as a fun tion of time for the following ratios: 65/35 (), 70/30 (•), 75/25 (N), 80/20 (), and 85/15 (◦) We see that the higher the ratio of the anioni surfa tant, the more stable the system is. Whereas the solution with

10

days, solutions with

80%

and

65%

85%

and

70%

of SDS were pre ipitated after

were still lear after

30

days.

The ele tro-

stati intera tions and onsequent harge neutralization between the SDS mole ules and the oppositely harged CTAB promote long relaxation times of atanioni mi elles. These, in turn, are a

ountable for the supersaturation of our system and the temporary solubility temperature depression observed. The anioni and ationi 88

surfa tant mole ules an be present in solution either as monomers, in orporated into surfa tant aggregates (mi elles, vesi les, et .) or as pre ipitate (DSCTA(s) - a

DS − CT A+

salt formed from

ions). Thus, there are two equilibria present in su h

mixtures: monomer-mi elle and monomer-pre ipitate. Two dierent types of phase separation in su h systems an be observed. Phase separation in atanioni surfa tant mixtures may o

ur due to the entanglement of rod-like mi elles, the formation of lamellar phase, or the formation of densely pa ked vesi les. In some ases, however, there seems to be a ompetition between the kineti s of vesi le formation and the kineti s of pre ipitation of ationi -anioni pairs

134,304

. Earlier measurements on

atanioni systems have shown that pre ipitation is primarily enthalpi ally driven, while mi ellar formation is primarily entropi ally driven. This is due to the ordering of the surfa tant headgroups in the rystalline pre ipitate as a onsequen e of harge neutralization

153

.

In order to understand whether phase separation is a onsequen e of pre ipitation of solid atanioni salt or the onsequen e of mi ellar (vesi ular) aggregation, dynami light s attering and uores en e te hniques were employed. Parti le size measurements showed a de rease in the size of the parti les with time, as an be observed in Figure 5.4.

After

3

days the solution was pre ipitated and the laser

beam passing through the upper ( lear) part of the solution dete ted only very small parti les

(RH ≈ 2.5nm).

This behavior is quite dierent from that observed in a previously studied SDS / DTAB system

169

. There, an in rease in turbidity was observed in a

ordan e with

the in rease of the hydrodynami radius of the orresponding parti les. Although the solutions were turbid, no pre ipitation took pla e, even after months of observation. The behavior of the SDS / CTAB was dierent. A pre ipitation was observed without any dete tion of an intermediate turbidity. The reasons for the dierent behaviors of the two systems are most likely due to the dierent Krat temperatures of the two ationi surfa tants. Pre ipitation is a ommon phenomenon in atanioni surfa tant mixtures, whi h is regulated by two ompeting for es: the free energy of the solid rystalline state and that of the mi ellar solution

305

. The strong intera -

tions make pa king into a rystalline latti e energeti ally favorable for surfa tant 89

12

10

% of particles

8

6

4

2

0

0

2

4

6

8

10

12

14

16

18

20

RH [nm]

Figure 5.4:

Parti le size followed by DLS: SDS / CTAB atanioni system with a (molar) ratio

65/35 after 1 day () and after 3 days ().

monomers, parti ularly when the hydrophobi mat h is high.

It has been shown

that by ex hanging the ounterion, we are able to inuen e the energeti state of the mi ellar solution. A mu h bigger variation between systems is observed in the free energies of the rystalline state. As we an see from the phase diagrams, the solubility of the atanioni salt is dire tly orrelated to the solubility of the surfa tant monomers. The solubility (and Krat point) de reases with in reasing hain length, and is therefore lower for the CTAB mixture in omparison to the DTAB surfa tant mixture. We an not ex lude at this point the possibility that the pre ipitate was formed from an entanglement of rod-like mi elles or similar big parti les as was already reported by other authors

282,283

. However, su h transitions take a

longer time, suggesting the formation of larger parti les would be dete ted before the a tual pre ipitation takes pla e. For this reason, we believe our system yields 90

a similarity to the SDS / DPLC (dode ylpyridinium hloride) mixture studied by Stellner et al.

134

. In this ase, the destabilization of solutions was not due to parti le

growth (and formation of big aggregates), rather was the observed pre ipitation due to the liberation of monomers.

Pyrene uores en e emission spe tros opy was used to further investigate the

hanges in phase behavior. The low uores en e ratio,

I1 /I3 ,

of pyrene on the rst

day after preparation of the solutions showed the presen e of highly hydrophobi parti les in the solution (I1 /I3

= 1.1; .

f. Figure 5.5). The

I1 /I3

ratio in

1 week old

solutions (ma ros opi ally the solutions were two-phased: the lower part ontaining the pre ipitant, whereas the upper part onsisted of a lear supernatant) showed an in rease from a value of

1.1

to a value around

the polarity found for SDS mi elles

306

.

1.3.

This value orresponds to

This suggests that as the atanioni salt

pre ipitated, the remaining pyrene was solubilized inside the mi elles formed by the ex ess anioni surfa tant. In this way, a large amount of the surfa tant was removed from the solution (as pre ipitate); only the ex ess amount remained in solution. Had all of the SDS surfa tant pre ipitated as well, a mu h higher pyrene ratio would have been observed (I1 /I3 (water)

≈ 1.8 307,308 ), whereas,

if bigger aggregates had formed,

one would expe t the pyrene intensity ratio to remain un hanged.

In the latter

ase, the pyrene probe would be solubilized either inside rod-like mi elles or the hydrophobi interior of vesi ular membranes, whose environment mi ro-polarity is the same as that in spheri al mi elles.

A higher stability of systems when the fra tion of one of the omponents was in reased in omparison to the other omponent was already observed by Stellner et al.

134

. When the SDS on entration is in reased (and the CTAB on entration

held onstant), pre ipitate an not form due to a hange in the mi ellar (and onsequently monomer) omposition. The monomer on entration then does not ex eed the monomer-pre ipitate boundary (for a more detailed explanation the reader is referred to Stellner et al.

134

). 91

Figure 5.5:

Pyrene uores en e emission spe trum of a SDS / CTAB atanioni system with a

(molar) ratio 70/30 after (left) 1 day, (right) 7 days.

5.4.3 Ee t of Additives on the Stability and the `Solubility Temperature Depression' Ee t of Ioni Strength

Addition of ele trolytes to ioni surfa tant solutions

may modify both the inter- and intrami ellar intera tions.

Therefore, the phase

behavior of surfa tant solutions is expe ted to be ae ted signi antly upon the addition of salts. Sodium bromide was added to the atanioni mixture at dierent ratios (in the anioni ri h region) to study the ee t of ioni strength on the phase stability. The observed ee t was similar for all ratios. Generally, the in rease of ioni strength de reased the destabilization time of the sample ( . f. Figure 5.6). The higher the on entration of the added NaBr was, the shorter were the destabilization times. Whereas the solution with only

2

after

10

days, the solution with

0.1

0.5

M of NaBr was pre ipitated after

M NaBr started showing rst signs of turbidity

days. The destabilization times are shortened down to two hours when the

solutions are kept in an i e bath. A general in rease in the tenden y of pre ipitation of atanioni mixtures upon the in rease of ioni strength was already observed previously

47,153

. When surfa tant monomers are salted out by the presen e of an 92

2

ele trolyte, mi ellization is favored .

Catanioni systems exhibit dierent behav-

iors upon salt addition. This is due to the ability of mixed surfa tant systems to adjust the aggregate omposition to attain the lowest possible free energy state for given onditions. In atanioni systems two salt-indu ed phenomena are observed, depending on whether the equimolar

47

(true) or non-equimolar

169

atanioni sys-

tems are studied. To qui kly review, if the surfa tant mi elles onsist of just one type of surfa tant (one harge) or one of the omponents is in ex ess, then the addition of salt s reens the repulsion between the headgroups, ausing the surfa tant mole ules to arrange in a bilayer. The opposite ase is true, if the surfa tants are present in equimolar amounts. The salts then s reen the attra tion between them, most likely ausing the distan e between the headgroups to in rease. In these ases a vesi le-to-mi elle transition was observed. Sin e we are dealing with non-equimolar

atanioni mixtures, where SDS is in ex ess, the addition of salt to our surfa tant mixture is expe ted to have the same ee t as on pure SDS mi elles. Addition of NaCl to SDS, for instan e, is known to de rease the CMC and in rease the mi ellar

6

size . Furthermore, by raising the ioni strength of the solution, the harges on the surfa tant headgroups are s reened due to a stronger ounterion binding. This, in ee t, de reases the solubility of the surfa tant monomers and therefore assist in pre ipitation.

Ee t of Cation / Anion Variations

A good orrelation has been observed

between the temperature to whi h it is required to heat the solution for omplete solubilization to o

ur (TI ) and the stability of the then isotropi solution. For this reason, the heating temperatures of solutions ontaining various salts and al ohols are reported in Table 5.1. The salt on entration was kept onstant in all ases at M. The solubility temperatures for all sodium salts are

29(±0.5)◦ C.

0.1

The variation of

the anion apparently has no ee t on the solubility of the atanioni mixture. This was expe ted as the overall net harge was negative (we had an ex ess of the anioni surfa tant SDS). A big variation of the temperature was observed when dierent

hloride salts were added to the mi ellar solution. Mass spe tros opy results (Figure 5.7) showed that at this relatively high on entration of added salt (0.05 M `new' 93

Transmission (%)

100 80 60 40 20 0

0

5

10

15

20

25

time (day)

Figure 5.6:

Transmission of the SDS / CTAB atanioni system: the ee t of ioni strength on

the stability of a SDS / CTAB solution (molar ratio 80/20) without salt (), with 0.1 (•) and 0.5 M (N) NaBr.

ounterions vs.

≈ 0.016

M of `original' sodium ounterions) all ounterions were in

the lose proximity of the mixed mi elle surfa e, regardless of the fa t whether they normally exhibit salting-in or salting-out behavior. Despite this, we saw a large dieren e in the stability of the solutions when the solutions ontaining LiCl or NaCl were ompared with solutions ontaining KCl or CsCl. After one week at

20◦ C,

the rst two solutions were still lear and isotropi ,

while the later two were already pre ipitated (see Figure 5.8). This dieren e in

TI

an be attributed to the stronger binding of less hydrated potassium and esium ions to the ex ess dode yl sulfate ions is ommonly used in biology and

T MA+ )

309

310,311

. The use of KCl pre ipitation to remove SDS

. The ee t of `hydrophobi ' ounterions ( holine

was found to be in the middle of both.

A stronger binding of organi ounterions in omparison to sodium to dode yl sulfate mi elles was already observed using uores en e te hniques 94

312

showing that

TI (0.1

Salt

M)

Al ohol

TI (0.1

M)

TI (0.2

M)

TI (0.05

M)

No salt

35.5

Methanol

35.5

LiCl

29

Ethanol

36

36

NaCl

29

n-Propanol

36

36

KCl

37

n-Butanol

35

34.5

CsCl

31.5

n-Pentanol

30.5

27

ChCl

37

n-Hexanol

21∗

21∗

TMACl

37.5

n-Heptanol

21∗

21∗

NaBr

29.5

n-O tanol

21∗

21∗

NaSCN

29

n-De anol

> 39∗∗

> 39∗∗

Na2 SO4

28.5

Citronellol

> 39∗∗

> 39∗∗

Table 5.1:

Ee t of additives on the initial solubility temperature (TI ); the initial sample is a

70/30 mixture (with an ex ess of SDS);

room temperature (21 C); ◦

∗∗

∗

the samples were isotropi and slightly bluish already at

the samples were not lear even at 39◦ C.

the binding of alkylammonium ions is dominated by their hydrophobi ity. As was pointed out previously, we are able to inuen e the energeti state of the mi ellar solution by ex hanging the ounterion.

This an explain the small dieren es in

the stability of the systems with dierent ounterions.

Mukerjee et al.

313

have

investigated the binding of ounterions to dode yl sulphate anions by ondu tivity. They have reported that the Li

+

ion is less rmly atta hed to the mi elle than the

+ + Na ion and this one in turn less than Cs . The lower binding therefore promoted the formation of ionized spe ies.

The gain in free energy was however not large

enough to over ome the free energy of the rystallization, and thus all of the systems eventually pre ipitated. The anity of spe i ion- ounterion pairing has been previously extensively studied on the vesi ular

169

and two-phase regions

31

of a similar system. The ordering

of ations was found to follow the Hofmeister series in all ases. The ee ts of the ions were found to be strongly dependent on the ability of the ounterions to form

lose ion pairs with the surfa tant headgroups and the behavior was explained using Collins' Law of Mat hing Water Anities

101

95

. As was shown in Chapters 3 and 4,

-

+ +

(2A +Na ) -

100

+ +

(2A +Li )

100

80 80

60 60

40

(2A-+M+)+

40

(2A-+H+)+

(2A-+Na+)+

20

(2A-+M+)+

20 -

(A-+M++Cl-)+

- +

(A +M +Cl )

A 0

+

B 0 700

600

500

800

600

m/z

m/z

(2A-+K+)+

100

500

900

700

800

900

(2A-+ Cs+)+

100

80 80

60

60

-

+ +

(2A +M ) -

+ +

(2A +M ) 40

-

+ - + + + (2A +H ) (A +M +Cl )

+ +

-

+

- +

(A +M +Cl ) (2A-+H+)+

20

20

D

C 0

-

(2A +Na )

40

+ +

(2A +Na )

500

700

600

800

0

900

600

m/z

m/z

Figure 5.7:

700

800

Ion binding as determined by ES-MS: addition of 0.1 M of (A) LiCl, (B) NaCl, (C)

KCl and (D) CsCl to an SDS / CTAB mi ellar solution. A− : dode ylsulphate anion (265 Da); M + : etyltrimethylammonium ation (284 Da).

the alkyl sulfate headgroup behaves like a soft, haotropi ion. The anions showed a destabilization time omparable to sodium hloride. The variations of the anion produ ed no hange in the stabilization times and therefore no spe i ity ( . f. Figure 5.9). This is most likely due to the fa t that the anions did not a

umulate near the same- harged mi ellar surfa e, but assisted purely by in reasing the ioni strength of the medium.

Ee t of al ohols of mi elles

314

It is known that al ohol penetrates in the palisade layer

and depresses the surfa tant CMC

Kaneshina et al.

319

315317

.

Nakayama et al.

318

and

have observed a depression of the CMC and the Krat point of

ioni surfa tant in the presen e of longer hain al ohols. The Krat point depression was explained quantitatively by a model in whi h it is proposed that the melting 96

Figure 5.8:

Ee t of additives on the stability of an SDS / CTAB atanioni system with a

(molar) ratio 70/30: (from left to right) LiCl, NaCl, KCl and CsCl (all salts 0.1 M) after 1 week at 20◦ C

Figure 5.9:

Ee t of additives on the stability of an SDS / CTAB atanioni system with a

(molar) ratio 70/30: (from left to right): without salt, with LiCl, NaCl, ChCl (Choline Chloride), NaSCN, Na2 SO4 (all 0.1 M) after 3 hours on i e.

97

point of the hydrated solid agent is depressed owing to the formation of a mixed mi elle of surfa tant and al ohol

Figure 5.10:

318

.

Ee t of additives on the stability of an SDS / CTAB atanioni system with

a (molar) ratio 70/30: (from left to right) methanol, ethanol, n-propanol, n-butanol n-pentanol, n-hexanol, n-heptanol, n-o tanol, n-de anol, itronellol (all 0.1 M) after 1 week. Solutions from methanol to butanol are slightly turbid / pre ipitated, pentanol is lear; hexanol to o tanol are bluish and homogeneous, de anol and itronellol are turbid (due to the low al ohol solubility).

The heating temperatures of solutions ontaining al ohols of various hain length and on entrations are reported Table 5.1. A

ording to the results reported, the al ohols an be lassied into four groups: (i) short- hain al ohols (< no ee t on the

TI

TI

C4 ), whi h have

or on the stability of the system; (ii) pentanol, whi h de reases

and the solubility somewhat; (iii) middle- hain al ohols (between

whi h lower (>

C10 ),

TI

C6

and

C8 )

and in rease the stability substantially; and (iv) long- hain al ohols

whi h are inappropriate for use due to their low solubility ( . f. Figure

5.10). Variation of on entration generally produ ed little ee t. Middle- hain al ohols from hexanol to o tanol have provided best results. temperature was redu ed to below room temperature (<

The initial solubility

21◦ C)

and the stability of

the solutions was in reased drasti ally. The samples were homogeneous and bluish, hinting at the presen e of larger parti les. Best ee ts were observed when the al ohol on entration range was kept between

0.02

and

0.2

M. Lower on entrations

produ ed no ee t and larger amounts of al ohol resulted in an al ohol-surfa tant gel. 98

Figure 5.11:

Pyrene uores en e emission spe trum of a SDS / CTAB atanioni system with a

(molar) ratio 70/30 upon the addition of 0.05 M hexanol after (left) 1 day, (right) 1 week.

Fluores en e measurements reported a presen e of hydrophobi domains in the solution ( .

f.

Figure 5.11).

In fa t, the polarity of the aggregates sensed by

the pyrene probe was even lower when hexanol was added to the atanioni mixture (I1 /I3

= 1.04);

propanol (I1 /I3

lose to that found in toluene (I1 /I3

= 1.07) 307 .

= 1.03) 307,308

or in 2-

This hints at the fa t that hexanol forms mixed mi-

elles/aggregates with the two surfa tants, thus redu ing the on entration of surfa tant monomers.

Solutions with dierent amounts of hexanol were observed at

room temperature for up to Also the

I1 /I3

6

months and still showed no sign of destabilization.

ratio in su h samples remained stable over time.

Furthermore, solutions ontaining small amounts of hexanol (0.05 M) were kept in an i e bath for a period of up to After

2

2

weeks and again no visible hanges appeared.

weeks, the sample was warmed to

mi ros opy.

20◦ C

and he ked by ryo-transmission

Solutions exhibited a high polydispersity.

mi elles, as well as vesi les were observed ( .f. 99

Spheri al and ribbon-like

Figure 5.12).

This proves that it

Figure 5.12:

Cryo-TEM photographs of a SDS / DTAB aqueous solution at a molar ratio of

70/30 and a total surfa tant on entration of 1 wt.% upon the addition of 0.05 M hexanol: spheri al

(bla k dots) and ribbon-like mi elles (top), as well as vesi les (bottom) are seen.

100

is possible to obtain vesi les stable for a long time in mixed surfa tant systems at temperatures mu h lower than the Krat temperatures of the two orresponding surfa tants.

5.5 Con lusions The o

urren e of supersaturation has been investigated in mixed surfa tant solutions omposed of SDS / CTAB and SDS / DTAB. A temporary `solubility temperature depression' has been observed in the anioni -ri h part of the phase diagram. We have shown that the stability of su h supersaturated solutions an be tuned by in reasing the ioni strength, by variation of ounterions, and by the addition of al ohols. The presen e of simple ele trolytes generally de reased, while the addition of middle- hained al ohols in reased its stability. Destabilization and on urrent pre ipitation of the systems was shown not to o

ur due to the formation of bigger aggregates, but rather due to a shift of the equilibrium between mi elles and monomers. The addition of low on entrations of middle- hain al ohols resulted in the formation of vesi les in the atanioni systems at temperatures signi antly lower than the Krat temperatures of the ioni surfa tants. The lifetime of vesi les and mi elles ould therefore be ontrolled by varying the

omposition of the surfa tant solutions and by additives. Controlling the pre ipitation phenomena is of importan e for a large number of industrial pro esses, where formulations need to be tuned.

101

102

Part III Toward Appli ation

103

Chapter 6 Use of Surfa tants in Cosmeti Appli ation: Determining the Cytotoxi ity of Catanioni Surfa tant Mixtures on HeLa Cells 6.1 Abstra t The ytotoxi ity of ommonly used syntheti surfa tants and atanioni mixtures of those was evaluated using MTT on HeLa ells. The 50% inhibition on entration (IC50 ) for MTT redu tion was al ulated. The ee t on hain length in rease and in lusion of polyoxyethylene groups on the toxi ity was tested on single surfa tant systems.

A general trend of in reasing toxi ity with in reasing hain length and

the presen e of polyoxyethylene groups was observed.

The measured IC50 values

of atanioni systems lie between those of parti ipating surfa tants. The in rease in toxi ity as the ationi surfa tant was added to the anioni one was however not linear. A steep de rease of the IC50 values (and therefore in rease in the toxi properties) was observed immediately already at low on entrations of the ationi surfa tants.

This behavior is analogous to the enzyme a tivity in atanoni mi-

roemulsions. 105

6.2 Introdu tion Vesi les are ommonly used in osmeti s and pharma y as vehi les for a tive agents. A tive mole ules an thus be en apsulated in the bilayer membrane, if they are lipophili , or in the ore of the vesi le, if they are hydrophili .

En apsulation is

useful to prote t a tives in preventing any undesired rea tion. Vesi les an thus be used as ve tors to deliver drugs to a spe i pla e, without being destroyed. Improving the bio ompatibility of produ ts used in osmeti formulation further is sought after. For this reason it is important to identify the irritating properties of

ommer ially used surfa tants.

Lately, atanioni systems have been investigated

as potential delivery systems due to their ability to spontaneously form vesi ular phases. Furthermore, new surfa tants are being synthesized ontinuously. For this reason, it is important to establish an easy way to estimate the potential toxi ity of su h substan es. Performing ytotoxi ity tests on established ell lines is a good alternative to expensive and morally questionable animal tests.

In the present study, ytotoxi ity

of ommer ially used surfa tants and ommon atanioni surfa tant mixtures were measured on HeLa ells in order to identify their toxi ity for use in pharma euti al and osmeti appli ations.

HeLa ells are a well-known ell line that is used

to asses the ytotoxi ity of hemi al ompounds

320323

reprodu ibility and a signi ant orrelation with

and reportedly show good

in vivo

results

324

.

Cell viability

was evaluated by the tetrazolium MTT redu tion assay, based on the uptake and the redu tion of the soluble yellow MTT tetrazolium salt by mito hondrial dehydrogenase to a blue insoluble MTT formazan produ t. The IC50 value ( on entration of test substan e that lowers MTT redu tion by 50% ompared with the untreated

ontrol) was al ulated from absorban e data. Previous tests have shown that the mito hondrion-based MTT test is a very sensitive indi ator of surfa tant ytotoxi ity

325

. Intra ellular organelles (su h as mito hondria) are ae ted already by low

surfa tant on entrations due to their high sensitivity to surfa tant membrane a tions.

Furthermore, high orrelation between MTT redu tion assay tests and y-

tosoli la tate dehydrogenase leakage (LDH) 106

in vitro

and the Draize eye irritan y

data

in vivo were

reported

325

. In fa t, ytotoxi ity

sitive and quantitative than the Draize test

326

in vitro proved

to be more sen-

.

A few reports on erning the toxi ity of surfa tants using dierent assays should be noted

325,327332

. The toxi ity of SDS is ommonly investigated as an example of an

ioni surfa tant. It has been established that ioni surfa tants show higher toxi ity values than the non-ioni ones, and that ationi surfa tants are more potent than their anioni ounterparts

328

. However, reports on the toxi properties of atanioni

mixtures are very s ar e. The ytotoxi ity of a SDS/CTAB/ holesterol mixture was tested on murine ma rophage-like ells by Kuo et al.

333

. A dose-dependent apoptosis

was observed. The study did however not in lude the ee t aused by the variation of the ratio of the surfa tants and their hain lengths.

6.3 Experimental Pro edures 6.3.1 Materials HeLa Cell Line

HeLa ells were distributed by the Ameri an Type Culture Col-

le tion (ATCC). Cells were ultured in Earle's minimum essential medium (MEM)

ontaining

0.85

g/L NaHCO3 supplemented with FCS (fetal alf serum)(10%), L-

Glutamin (2 mM), NEA (non-essential amino a ids)(1%), Amphoter in B (0.4µg/ml) and Peni ilin G / Streptomy in sulfate (100 u/mL).

Surfa tant Solutions

The surfa tants, sodium o tanoate (SO; Sigma, Germany;

grade: 99%), sodium dode anoate (SL; Sigma, Germany; grade: 99-100%), sodium dode yl sulfate (SDS; Mer k, Germany; assay > 99%), sodium laurelth sulfate

− + (Texapon 70; Na C12 (EO)2.2 OSO3 , a tive assay: 70%, gift from Cognis, Germany) + − and sodium dode ylether arboxylate (Akypo Soft 45NV; Na C11 (EO)4.5 OCH2 COO , a tive assay: 21%, gift from Kao Chemi als, Germany), o tyltrimethylammonium bromide (OTAB; Mer k, Germany; assay > 99%), de yltrimethylammonium bromide (DeTAB; Mer k, Germany; assay > 99%), dode yltrimethylammonium bromide (DTAB; Mer k, Germany; assay > 99%), etyltrimethylammonium bromide 107

(CTAB; Sigma, Germany; grade: 99%) were used as re eived. Surfa tant sto k solutions were prepared by dissolving weighed amounts of dried substan es in Millipore water. The solutions were then left for 24 hours to equilibrate at

25◦ C.

The atan-

ioni solutions were prepared by mixing the surfa tant sto k solutions to obtain dierent anioni / ationi surfa tant mass ratios.

6.3.2 Growing HeLa Cell Cultures Substan es: MEM - EARLE Medium, PBS - DUBECCO Buer, Trypsin / EDTA, Flask ontaining HeLa ells 1. The old medium was removed from the ask ontaining HeLa ells that we wished to transplant. 2. The ells were rinsed with approximately

4

ml of PBS buer solution. With this

we have removed all the free-swimming ells; only the ells stu k to the bottom of the ask remained. 3.

5

3

ml of Trypsin / EDTA was added and the ells were in ubated at

37◦ C

for

minutes. Trypsin disables the ell's ability to bind to surfa es ( onsequently, the

ells are swimming freely in the suspension). 4. The ell suspension was transferred into a new ask,

3

ml of new MEM medium

was added to stop the rea tion of Trypsin and the ells were entrifuged at for 5 minutes (20

◦

800/min

C).

5. On e entrifuged, the supernatant was arefully removed. 6.

4

ml of new MEM medium was added and the solution was homogenized using a

pipette until ell- lusters disappeared. 7.

A desired amount of ell suspension (depending on when we needed the ells

ready for the next experiments) was transferred into a new ask and MEM medium was added to a olle tive volume of

20

8. The ells were left to in ubate at

37◦ C

ml. for minimum

108

3

days.

6.3.3 HeLa Toxi ity Test 1. The viability of the ells was he ked with Trypanblue (solution: blue in

10

ml PBS buer);

10 µl ell

suspension

+10 µl Trypanblue

0.04 g Tryptan=> we ounted

the living ells under the mi ros ope (the defe ted ells were olored blue, the inta t one's white). 2. Number of ells in the

330 · 2 = 660

squares: f.i.

330

dilution with Trypanblue

660 · 10 = 6.600

ells in

1µl

6.600 · 1.000 = 6.600.000 3. We need

16

150.000

ells in

1

ml

ells per experiment:

6.600.000 : 150.000 = 44 That means we need to dilute 1 ml of the ell suspension with For 1 plate (60 wells with

0.5

ml); we prepare

75 µl) 4.5

44

ml of medium.

ml of ell suspension is needed (2 plates =

ml ell suspension +

22

9

ml of medium.

4. Con entration of substan es of whi h we wish to measure toxi ity: 1 mg/ml (1 wt.%);

0.1

mg/ml if the substan es are very potent ( ationi surfa tants).

5. Filling the plates: o The wells at the borders were lled with

150µl

of pure medium (36

· 150 µl

=

5.4

ml) o The middle o

60 µl

60

wells were lled with

75 µl

medium

medium was added to wells 2 / B-G (the olumns are des ribed using num-

bers, and rows using letters) o Wells 2B/2C were additionally lled with sample 2; 2F/2G with 6.

15 µl

15 µl

of sample 1; 2D/2E with

15 µl

of

of sample 3.

With a 6- anal pipette the solutions were thoroughly mixed in wells 2B - 2G

and then

75 µl

of the solutions was transfered from olumn 2 to olumn 3 (wells

3B - 3G). This pro edure was repeated until row solutions from row 7. Finally,

75 µl

10

was dis arded. Row

11

10 (10B - 10G).

mixing)(in the end the whole plate should ontain

68

60

of

middle wells with (without

150 µl

hours (±1 hour). 109

75 µl

was reserved for our blind probe.

of ell suspension was added to all

8. The ells were left to in ubate for

The last

/ well).

Figure 6.1:

96-well plates used for toxi ity studies immediately after preparation (top) and after

in ubation with MTT (bottom).

110

6.3.4 Dete tion 1.

15 µl

of MTT solution (4 mg/ml of MTT in PBS buer) was added to all wells

ontaining ells (0.9 ml MTT solution per plate). 2. The plates were left to in ubate for 4 hours. 3. The ex ess MTT ontaining medium was arefully remove. Well 1A was emptied as well. 4. The empty wells were lled with

150 µl 10

wt.% SDS solution (in water;

18.3

ml

for two plates). 5. The mi ro-plates were pla ed in laminar ow overnight, then the opti al density of ea h plate was measured with a mi roplate reader at 560 nm. A sample plate immediately after the preparation pro ess and after the MTT in ubation is presented in Figure 6.1. The wells at the rims are lled only with medium (pink olor), the blue olor is a sign of inta t ells and the transparent solution of

ell apoptosis.

6.3.5 Evaluation of spe tros opi al data The IC50 value (in

µg /ml), whi h represents the on entration of test substan e that

lowers MTT redu tion by 50% ompared with the untreated ontrol, was al ulated for ea h substan e from the on entration-response urve. sheet is presented in Figure 6.2.

A sample al ulation

Experiments were repeated four times (n = 5),

and the average IC50 is reported. Maximal observed (absolute) standard deviation was about 15%. Positive ontrol measurements were performed with xanthohumol (HeLa ells: IC50

≈

7

µg/mL).

6.4 Results and Dis ussion 6.4.1 Single-Chain Surfa tants Cell viability was determined by the tetrazolium MTT dye redu tion in the ell

ulture system.

Evaluation of the results revealed a dose-dependent ytotoxi ity

after 68 hours of exposure. The IC50 values are reported in Table 6.1. 111

HeLa -Cells Testsubstance:SDS/DTAB Konz[µg/ml] OD

90/10 Average

corr. ave. Restakt. [%]

StAbw [%]

0.811 0.0098 0.006 0.002 0.031 0.343 0.485 0.665 0.789 0.863

0.1 0.3 0.2 1.4 0.5 7.7 0.4 1.3 0.6

Empty value 0.0428 Control

0.8590 0.8141 0.0519 0.0511 0.0457 0.0661 0.3885 0.4840 0.7098 0.8245 0.9018

1000 500 250 122.5 62.5 31.25 15.63 7.8 3.9

0.043 0.8325 0.8739 0.9065 0.8372 0.854 0.0532 0.053 0.0472 0.049 0.0433 0.045 0.0821 0.074 0.3824 0.385 0.5725 0.528 0.7054 0.708 0.8394 0.832 0.9091 0.905

1.2 0.8 0.2 3.9 42.2 59.9 82.0 97.3 106.4

Conc. >50%: 31.25Value: 59.9µg/ml Conc.<50%: 62.5 Value: 42.2µg/ml IC 50 =48.73µg/ml

Figure 6.2:

Cal ulation of the IC50 value from the spe tros opi al data.

Comparison of the IC50 values of the surfa tants revealed the following ytotoxi properties: 1) Anioni surfa tants generally exhibited a lower ytotoxi ity than atanioni surfa tants. 112

Anioni surfa tants

Cationi surfa tants

name

IC50 [µg/mL℄

name

IC50 [µg/mL℄

SO

530.5 ± 41.7

OTAB

23.3 ± 1.8

SL

115.8 ± 8.2

DeTAB

4.8 ± 0.6

SDS

177.1 ± 7.8

DTAB

5.2 ± 0.3

Texapon 70

69.2 ± 1.7

CTAB

1.6 ± 0.2

Akypo Soft 45NV

76.3 ± 4.8

Table 6.1:

The IC50 values ( on entration of test substan e that lowers MTT redu tion by 50%

ompared with the untreated ontrol) of singe- hain ioni surfa tants.

2) Surfa tants ontaining sulphate headgroups showed higher IC50 values than those bearing a arboxylate (having the same hain-length). 3) An in rease of the toxi ity of the surfa tant was observed as the length of the hydro arbon hain was in reased ( . f. Figure 6.3). An exponential de rease in the IC50 values was observed, whi h leveled o at a ertain hain length. 4) The presen e of the oxyethylene groups in reases the toxi properties of the surfa tants. Two surfa tants ontaining 12 arbon atoms, and polyoxyethylene groups, one with a sulphate (Texapon 70) and one with a arboxylate headgroup (Akypo Soft 45 NV) were ompared to those without the EO groups. An in rease in the toxi properties is observed for both surfa tants, however Akypo Soft 45 NV exhibits a higher IC50 value, than its sulfate ontaining ounterpart, despite the fa t that SDS shows higher IC50 values than SL.

6.4.2 Catanioni Surfa tant Systems The toxi ity of atanioni surfa tant mixtures omposed of SDS / DTAB and SL / DTAB were analyzed for dierent surfa tant ratios. The results are presented in Table 6.2 and Figure 6.4. We an see that the presen e of only a small amount of a ationi surfa tant in reased the toxi ity of the mixture signi antly. This was somewhat surprising, as a linear in rease of the toxi ity was expe ted. 113

600 500 400 300 200

30

IC

50

[ g/mL]

100

20 10 0

8

10

12

14

16

n (C)

Figure 6.3:

Ee t of hydro arbon hain length on the ytotoxi ity of ioni surfa tants: n(C)

represents the number of arbons in ationi CnTAB () and anioni SCn () surfa tants.

SDS / DTAB

SL / DTAB

xSDS

IC50 [µg/mL℄

xSL

IC50 [µg/mL℄

0

4.2 ± 0.2

0

4.2 ± 0.2

0.1

3.5 ± 0.1

0.1

5.1 ± 0.4

0.2

3.8 ± 0.3

0.2

4.6 ± 0.6

0.3

3.7 ± 0.2

0.3

6.3 ± 0.5

0.4

6.7 ± 0.5

0.4

7.1 ± 0.2

0.5

8.2 ± 0.8

0.5

7.2 ± 0.4

0.6

7.9 ± 0.6

0.6

13.0 ± 1.2

0.7

11.6 ± 1.0

0.7

16.4 ± 0.9

0.8

20.1 ± 1.6

0.8

28.6 ± 2.1

0.9

43.1 ± 3.2

0.9

49.7 ± 3.4

1.0

177.1 ± 7.8

1.0

115.8 ± 8.2

Table 6.2:

The IC50 values of atanioni surfa tant mixtures. 114

200

175

150

100

75

IC

50

[ g/mL]

125

50

25

0

0.0

0.2

0.4

0.6

0.8

1.0

X

SDS/SL

Figure 6.4:

The mito hondrial redu tion of MTT after a 68-hour in ubation of SDS / DTAB

() and SL / DTAB () mixture at dierent surfa tant ratios with HeLa ells.

This behavior is similar to the one found when the enzymati a tivity in atanioni emulsions was studied

334

. It was observed that the presen e of DTAB exerts

an inhibiting ee t on the enzyme. At weight fra tions higher than

0.22, the enzyme

was ompletely inhibited. This is approximately the weight fra tion at whi h the toxi ity of the mixtures equals that of a pure DTAB solution. It is highly probable that at weight fra tion lower than

0.22

DTAB is ompletely in orporated in the

atanioni mi elles and its toxi ity is redu ed by the strong ele trostati intera tions with the oppositely harged anioni surfa tant. Results suggest that in order to keep the toxi ity of potential drug delivery systems low, a high fra tion of the anioni omponent is ne essary. So far, vesi les have been shown to exist in the anioni -ri h region of the atanioni phase diagrams, however at ratios lose to equimolarity. The vesi ular region was shifted to higher anioni fra tions when salt was added to the system

169,335

. A system of lower toxi ity om-

posed of a mixture of anioni surfa tants will be reported in the following hapter 115

(Chapter 7).

6.5 Con lusions The ytotoxi ity of single- hain ioni surfa tants and atanioni mixtures was evaluated using MTT on HeLa ells in order to examine their potential use in osmeti and pharma euti al formulation. It was onrmed that anioni surfa tants generally exhibit higher IC50 values than atanioni ones. The toxi ity an further be inuen ed by the hydro arbon hain length and the presen e of polyoxyethylene groups. A general trend of in reasing toxi ity with in reasing hain length and the presen e of polyoxyethylene groups was observed. A non-linear in rease was observed as ationi surfa tants were added to anioni ones.

A steep de rease of the IC50

values was observed already at low fra tions of the ationi surfa tants, suggesting that in potential drug delivery systems a high fra tion of the anioni omponent is ne essary.

116

Chapter 7 Spontaneous Formation of Bilayers and Vesi les in Mixtures of Single-Chain Alkyl Carboxylates: Ee t of pH and Aging 7.1 Abstra t We report the observation of bilayer fragments, some of whi h lose to form vesi les, over a large range of pH at room temperature from mixtures of single- hain bio ompatible ommer ially available non-toxi alkyl arboxyli surfa tants after neutralization with HCl. The pH at whi h the morphologi al transitions o

ur, was varied only by hanging the ratio between two surfa tants: the alkyl-oligoethyleneoxide arboxylate and sodium laureate. The ee t of aging of the mixed surfa tant systems in the pH region desired for dermatologi appli ation (4.5

<

pH

< 7)

was also

studied.

7.2 Introdu tion There is a growing interest in the eld of syntheti surfa tants used for dermatologi purposes. These surfa tants exhibit a wide range of stru tures; parti ularly useful is 117

the formation of vesi les, whi h an be used as delivery systems. For osmeti reasons, the surfa tants forming vesi les must be skin ompatible (non-irritant), easy to manufa ture, and the vesi le region must be stable within the range of physiologi al pH at room temperature. A simple geometri al hara terization of hain pa king an be used to analyze trends in surfa tant phase behavior

15,60

. The geometri properties of surfa tants depend

on the ratio between the ross-se tional area of the hydro arbon part and that of the headgroup.

Low pa king parameters (around

1/3)

are found for single hain

surfa tants with a strongly polar headgroup. These systems tend to form spheri al mi elles, whereas a pa king parameter value around one favors the formation of lamellar stru tures.

An in rease in the pa king parameter an be obtained by

adding a se ond hain, whi h doubles the hydro arbon volume. Double- hain surfa tants

18,21

, two surfa tants of opposite harge

tant and a o-surfa tant

2733

2326

, or the asso iation of a surfa -

, an be used. In the latter two ases a pseudo-double

hain surfa tant is obtained by either an ion-pair formation between the anioni and

ationi surfa tant, or due to asso iation of the two dierent mole ules via hydrogen bonds. As ationi surfa tants and short al ohols are undesirable in osmeti formulations due to their toxi ity, and be ause long- hain al ohols (>

C12 )

exhibit high melting

points, monoalkyl arboxylates were hosen for our study. Fatty a ids form a range of aggregates depending on the a id on entration and the ionization degree of the terminal arboxyli group

336,337

a ids has long been known.

.

The formation of vesi les from mono arboxyli

Gebi ki and Hi ks

338

vesi les from unsaturated, long- hain fatty a ids.

rst observed the formation of Later, Hargreaves and Deamer

showed that also saturated fatty a ids an form vesi les taneously formed, when short or middle hain (< with HCl

61,81

C12 )

61

. A vesi le phase is spon-

fatty a ids are neutralized

; two types of amphiphiles are then present in the solution, the proto-

nated and the ionized form. The ratio between the two determines the aggregation morphology. Despite the simpli ity of the me hanism of vesi le formation, possible appli ations of fatty a id vesi les in osmeti s remain largely unexplored

118

339,340

. This may be a

onsequen e of two obsta les: (i) the high solubility temperature of the long- hain

arboxylates and, (ii) the generally too basi pH ne essary for the solubilization of the arboxylate. The problem of the solubility temperature of the alkyl arboxylates has been dis ussed by Hargreaves et al.

61

Below

25◦ C

only alkyl arboxylates with

short alkyl hains are water-soluble. However, these are inappropriate due to their skin irritating properties. The solubility temperature of sodium laureate is reported to be equal to or above room temperature, depending on the on entration longer alkyl hains (n

> 12)

336

. With

the solubility temperature be omes even higher.

It has previously been reported that the formation of vesi les o

urs at pH values at or near the pKa, where approximately half of the arboxyli groups are ionized

61,336,339

. For this reason, fatty a id vesi les are present only over a narrow pH

range. Designing vesi les that be ome unstable at an easily tuned pH value is of great interest for targeted drug delivery. It is known that, for example, tumors and inamed tissues exhibit a de reased extra ellular pH

6367

. For this reason a large

number of groups have fo used their attention on the preparation of pH-sensitive liposomes

6878

as possible drug arrier systems.

A range of sugar-based gemini surfa tants has been re ently studied, be ause they exhibit pH-dependent aggregation behavior. However, these surfa tants are ationi and therefore vesi les are observed only at neutral and high pH values same problem o

urs also in solutions of bola-amphiphiles

343

341,342

. The

. Systems omposed of

mixed single- and double-short-tailed PEO ether phosphate esters might be promising for the formation of pH-sensitive vesi les. observed at higher on entrations.

In that ase a vesi le phase was

The pH ee t, however, was only studied on

vesi ular solutions, so one an not onrm a mi elle-to-vesi le transition as a onsequen e of protonation

344

.

For arboxylates with longer alkyl hains, desirable for osmeti formulations (i.e. lauri a id; pKa

≈ 8 − 8.5 24,61 ,

depending on the on entration), the pH of vesi le

formation is also generally too basi . The addition of medium- and long- hain al ohols has been proven to expand the pH region of vesi le formation even higher pH values, whi h is undesirable.

61,339

, but toward

One way to lower the pH range of

vesi le formation is to introdu e another arboxyli group to the fatty a id hain,

119

thus lowering the pKa of the a id. This has been previously done by de Groot et al. by the formation of 2-(4-butylo tyl) maloni a id

345

. However, to the best of our

knowledge, no one has tried to use a mixture of single- hain fatty a id soaps yet. Re ently, spontaneous formation of vesi les below room temperature, at a idi pH (between

2 and 4), by neutralization of a parti ular industrial single- hain alkyl ar-

boxylate surfa tant was reported

346

. Our intent was to obtain spontaneously-formed

vesi les over a wide range of physiologi al pH at room temperature, by hanging the ratio of two surfa tants: Akypo Soft 45NV (an alkyl-oligoethyleneoxide- arboxylate already used in osmeti formulations; AS) and sodium laureate (SL). The ee t of pH and aging on vesi le formation was studied by visual observation, dynami light s attering and transmission ele tron mi ros opy. The bio ompatibility of the surfa tant mixture was he ked by measuring the ell viability. Based on our results, we propose a general method to obtain vesi les on a large range of physiologi al pH using only non-toxi alkyl arboxylates.

7.3 Experimental Pro edures Chemi als

Sodium laureate (SL) was pur hased from Sigma-Aldri h at a purity of

+ − 99%. Sodium dode ylether arboxylate (Akypo Soft 45NV; Na C12 (EO)4.5 OCH2 COO ) was a gift from Kao Chemi als (Germany). A

ording to the information given by Kao Chemi als, AKYPO Soft 45NV ontains mole ules with roughly a Gaussian distribution of the number of EO groups with an average value of 45NV is supplied as an approximately

21

4.5.

AKYPO Soft

wt.% aqueous solution. Sto k solutions of

1wt.% were prepared by dissolving weighed amounts of sodium laurate in deionized water and by dilution of the original Akypo Soft 45NV solution. Dierent solutions were then prepared by mixing dierent ratios of the two sto k solutions.

Phase Diagrams

The phase behavior as a fun tion of temperature was deter-

mined by visual observations. The samples were ooled down and left to equilibrate at

0◦ C,

then the temperature was raised by approximately

0.5◦ C

per minute. The

measured solubility temperatures orrespond to the transition from pre ipitate to 120

isotropi phase, i.e., to a mi ellar or vesi ular solution. The samples were titrated with

0.1 M HCl at 25◦ C.

A Con ort, type C831 pH-meter, with a Bioblo k S ienti

glass ele trode (Consort ref. nr. SP02N), was used.

Dynami Light S attering

Parti le size analysis was performed by a Zetasizer

3000 PCS (Malvern Instruments Ltd., England), equipped with a 5 mW helium-neon laser with a wavelength output of 633 nm. The s attering angle was

90◦

and the

intensity auto orrelation fun tions were analyzed using the CONTIN software. All measurements were performed at

Cytotoxi ity Tests Culture Colle tion). varied from

25

to

(MEM) ontaining

25◦ C.

HeLa ells were provided by the ATCC (Ameri an Type The passage numbers of the HeLa ells used in the proje t

30.

Cells were ultured in Earle's minimum essential medium

0.85

g/L NaHCO3 supplemented with FCS (10%), L-Glutamine

(2 mM), NEA (1%), Amphoter in B (0.4

µg/ml)

and Peni illin G / Streptomy in

sulfate (100 u/mL). Keratyno ytes (SK-Mel-28) were provided by the ATCC. The passage numbers varied from

ontaining

3.7

15

to

20.

Cells were ultured in Dulbe

o's MEM

g/L NaHCO3 supplemented with FCS (10%), L-Glutamine (2 mM),

NEA (1%), Amphoter in B (0.4

µg/ml) and Peni illin G / Streptomy in sulfate (100

u/mL). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay pro edure was prepared a

ording to Mosmann

347

in

96-well mi ro-plates using

the

doubling dilution method (as des ribed in the previous hapter). First, the wells of the rst olumn were lled with were lled with

75 µl

135µl

of medium.

of MEM medium. All subsequent olumns

Then,

15µl

of 1wt.% test solutions was added

to ea h well in the rst olumn and the solutions were thoroughly mixed with a 6- hannel pipette.

75µl

of the test substan e / medium mixture was transferred

from the wells in the rst olumn to those in the se ond. Again the solutions were mixed and the pro edure was repeated for the whole plate. were seeded (2.5

· 103

ells / well).

After a

68-hour

MTT (5 mg /mL) were added to ea h well. After a

ontaining MTT was removed and

150µl 121

of

10%

Then,

in ubation at

4-hour

75µl

of ells

37◦ C, 15µl

of

in ubation, the medium

SDS solution was added.

The

mi ro-plates were pla ed in laminar ow overnight, then the opti al density of ea h plate was measured with a mi roplate reader at

560

nm. The IC50 value (µg /ml),

whi h represents the on entration of test substan e that lowers MTT redu tion by

50%

ompared with the untreated ontrol, was al ulated for ea h substan e from

the on entration-response urve. Experiments were repeated four times (n = 5), and the average IC50 is reported. Maximal observed (absolute) standard deviation was about

15%.

Positive ontrol measurements were performed with xanthohumol

(HeLa ells: IC50

Cryo-TEM

≈

7

µg/mL).

We prepared vitried ryo-TEM spe imens in a ontrolled environ-

ment vitri ation system (CEVS), at a ontrolled temperature of

25◦ C

and xed

100% relative humidity, followed by quen hing into liquid ethane at its freezing point

348

.

We examined the spe imens, kept below

transmission ele tron mi ros ope, operated at

120

−178◦ C,

by an FEI T12 G2

kV, using a Gatan 626 ryo-

holder system. Images were re orded digitally in the minimal ele tron-dose mode by a Gatan US1000 high-resolution ooled-CCD amera with the DigitalMi rograph software pa kage.

7.4 Results and Dis ussion 7.4.1 Lowering of the Solubility Temperature of Fatty A ids To form vesi les at room temperature, the solubility temperature

TSol

of the sur-

fa tant mixture has to be equal to or below this temperature. The use of sodium laureate is therefore limited, be ause it is insoluble in water at temperatures below

24◦ C

(for solutions of 1 wt.%). We were able to lower the solubility temperature by

mixing sodium laurate with a surfa tant that is soluble at lower temperatures. AS was hosen be ause its

TSol = 5◦ C.

Figure 7.1 shows that the solubility temperature

de reased linearly with in reasing amounts of AS for a xed total surfa tant on entration of 1wt.%, and ould be des ribed by the equation: where

TSol

TSol (◦ C) = −19.9·A+23.9,

represents the solubility temperature of the mixture, and

of AS, expressed in wt.%. 122

A

the amount

25

T

solubility

o

[ C]

20

15

10

5

0 0.0

0.2

0.4

0.6

0.8

1.0

amount of Akypo Soft 45NV (wt.%)

Figure 7.1:

Solubility temperature of mixed surfa tant solutions (AS and sodium laureate; ctot S =

1 wt.%) as a fun tion of the amount of AS in the solution. A linear t des ribes the dependen e

with the equation: TSol (◦ C) = (−19.9 ± 0.1) · A + (23.9 ± 0.1), where TSol represents the solubility temperature of the mixture and A the amount of Akypo expressed in wt.% (the total surfa tant

on entration was kept at 1 wt.%)

7.4.2 The Ee t of pH on Vesi le Formation The titration of alkaline soaps with HCl has been des ribed by Rosano et al.

225

,

when investigating the ee ts of surfa e harge on lipid-water interfa es. In these experiments, the appearan e of a plateau (buering apa ity) at a pre ise pH during titration was oin ided with the formation of lipid liquid- rystals.We observed this plateau in solutions of dominating quantities of sodium laureate. As more SL was repla ed by AS, the plateau region be ame smaller, disappearing below 0.5 wt.% SL (mass ratio

1 : 1).

By redu ing the pH of the samples we observed a su

es-

sion of two (or three) phases, depending on the mass ratio of the two surfa tants (Figure 7.2).

At high pH, all solutions were isotropi and olorless, orrespond-

ing to the mi ellar region of the phase diagram.

By de reasing the pH, a bluish

olor appeared, attributed to a formation of vesi ular stru tures. In the presen e 123

of pure AS, only two phases appeared onse utively, while in SL / AS mixtures, a se ond phase transition to pre ipitation was observed at low pH values. In 1 wt.% SL solutions pre ipitation began at pH values below 7, whereas the addition of AS

= 3.5

lowered the pre ipitation boundary to pH

25/75).

(in solutions with SL / AS ratio

Pre ipitation when the surfa tant ratio nears equimolarity, had been ob-

served in previous studies and was attributed to the in reased on entration of the

onjugated a id

169

. The titrations were performed one week after the preparation

of the samples. Ma ros opi ally, the pH values at whi h the phase transitions took pla e remained the same with respe t to time. This is not true mi ros opi ally, as des ribed below. The formation of fatty a id vesi les is restri ted to a narrow pH region lose to the pKa of the a idi omponents anions (from the pKa

61,336,339

. The apparent shift in the pKa of fatty a id

= 4.67 of arboxyli group 349

to the pKa

= 8 of lauri a id 61 )

has been attributed to the lo al de rease in pH at highly harged surfa es

61

. Sin e

pure fatty a id/soap vesi les (without additional amphiphiles) ontain an amphiphile that is not harged (the neutral form of fatty a id), the two bilayer-forming omponents are asso iated by hydrogen bonds instead of ele trostati intera tions

350

.

The formation of vesi les near the pKa of the a id an then be explained by the formation of stable hydrogen bond networks between the ionized and neutral a id forms. It has been observed that the addition of al ohols to fatty a ids auses an in rease in the pH of the region of vesi le formation

339

. In that ase, vesi les are formed due

to stable hydrogen bonds between the al ohol headgroup and the ionized a id headgroup. The addition of the al ohol means larger presen e of the non-ionized spe ies in the solution (due to the high pKa of the al ohols); a larger amount of arboxyli a id will be in the ionized form to a hieve the same protonated/ionized headgroup ratio needed for vesi le formation (1

: 1).

An opposite ee t an then be expe ted

in the ase when the added omponent has a mu h lower pKa than the fatty a id, as is the ase of AS. This will be ompletely ionized at a neutral pH, therefore more protonated a id groups will be required for the formation of vesi les, resulting in a de rease of the pH of vesi le formation. This was onrmed by our titrations, where

124

the pKa (and orresponding bluish region) de reased with in reasing amount of AS in the mixture.

9 8 7 6

pH 5 4 3 2 1 0.00

0.25

0.50

V

0.75

HCl

Figure 7.2:

1.00

1.25

[mL]

Dierent phase transitions observed by titration of mixed surfa tant solutions

(sodium dode anoate and AS; ctot = 1 wt.%; Vo = 15 mL) ontaining (from bottom to top) S 1; 0.75; 0.5; 0.25; and 0 wt.% of AS. The dashed lines represent a bluish solution (possibly vesi les);

the solid lines represent the lear (mi ellar) solution (to the left) and the turbid phase (to the right of the vesi ular phase). The hydrodynami radius of parti les in solutions at dierent surfa tant ratios was measured by dynami light s attering. The results onrmed the presen e of large aggregates (possibly vesi les) over a large pH region (Figure 7.3) in mixed surfa tant solutions. Be ause solutions of industrial surfa tants are highly polydisperse (the polydispersity of AS has been previously reported radius ould be measured.

169

), only the average

The regions where RH was found to be

80 − 120

nm

(the average radius of vesi les) is plotted in Figure 7.3 for dierent surfa tant ratios. In reasing the amount of AS lead to a de rease of the pH, at whi h vesi les were formed. The width of the pH range depended strongly on the surfa tant ratio. This range is the largest for the ratio AS / SL :

0.75 / 0.25, where

125

it extends from pH 3.5

to 7.5. At this ratio the omplete range of skin pH or physiologi al pH is overed. A redu tion of this range was observed when the amount of Akypo Soft 45NV was either in reased or de reased.

10

Micelles

8

pH

6

4

Precipitation

2

0 0.0

0.2

0.4

0.6

0.8

1.0

AS [wt.%]

Figure 7.3:

pH range where (after 3 weeks) the average hydrodynami radius RH was approx-

imately 100 nm (possible region of vesi le formation) in mixed surfa tant solutions (SL and AS) as a fun tion of AS on entration (ctot S = 1 wt.%); the open squares () represent the solutions examined by ryo-TEM.

The (average) hydrodynami radius (RH ) of the obje ts present in the vesi le

ontaining solutions is shown in Figure 7.4 for dierent surfa tant ratios as a fun tion of the pH. The obtained values suggest the presen e of vesi les, but large variations exist. In presen e of

0.75

wt.% of AS only small vesi les are measured with a small

polydispersity index (around

0.2).

At smaller ratios of AS, or in presen e of pure

sodium laurate, bigger obje ts of higher polydispersity are observed. Be ause light s attering is insu ient to hara terize systems of high polydispersity, ryo-TEM was used to analyze the system further. 126

200 175

R

H

[nm]

150 125 100 75 50 25

2

3

4

5

6

7

8

9

pH

Figure 7.4:

Average hydrodynami radius, RH , of the aggregates present in bluish solutions as

a fun tion of pH for dierent surfa tant ratios: (△) 0; (◦) 0.25; (•) 0.5; (N) 0.75; () 0.85; () 1 (all expressed in wt.% of AS; ctot S = 1 wt.%).

7.4.3 Cryo-TEM Study of Time-Dependent Vesi le Formation The AS / SL ratio (75/25), at whi h the largest pH interval of vesi le formation was measured, was further investigated by ryo-transmission ele tron mi ros opy. The evolution of self-assembly aggregates was followed over a period of two months at three dierent pH values. The sequen e of observed morphologies is summarized in Table 7.1. We see from Table 7.1 that in the rst weeks after preparation mi elles and dis s are the observed stru tures; these are presented in Figure 7.5.

With time,

membranes started forming and eventually vesi les appeared (Figure 7.6).

The

higher the pH of the solution, the faster this transition took pla e; in samples with pH

= 6.7

vesi les were observed already after 2 weeks, whereas in solution with

pH

= 5.5,

these were visible just after 4 weeks. After 2 months vesi les were still 127

pH

No. of days after preparation 5

4.5 5.5

10

17

28

60

D, MP

M

D

MP

M, D

M, V

M, MP

MP, V

M, D, V (C)

M, MP, V (I)

M, D

6.7 Table 7.1:

Development of various surfa tant self-assembled aggregates with time; M - mi elles,

D - dis s, MP - membrane pie es, V - vesi les, V (C/I) - vesi les with either usps, or in omplete vesi les.

not observed in solution with the lowest pH ( .

f.

Figure 7.7 a).

However, the

two samples at higher pH values already showed in omplete vesi les and membrane pie es ( .

f.

Figure 7.7 b).

It seems that after a ertain time, the vesi les in

these systems begin to destabilize. We noted that despite the ri h morphologi al development that ould be observed by mi ros opy, ma ros opi ally the solutions appeared about the same during the period of observation. Furthermore, we an see that not all regions where big obje ts were dete ted by DLS ontain vesi les. These were only found at higher pH values (above

5.5),

whereas at low pH, dis s

and membrane pie es were the dominating form. The results suggest it is possible to obtain vesi les in the region dened by the two pKa; at ertain surfa tant ratios an even broader pH range was observed. This is in a

ordan e with results obtained by de Groot et al.

345

for a bran hed monoalkyl

surfa tant with a malonate headgroup. A bluish zone was observed between the two pKa values (the pKas of the two arboxylate groups on maloni a id are

5.70,

respe tively)

345

2.85

and

. In that region o-existen e of small unilamellar (SUV) and

multilamellar (MLV) vesi les was found. Above pH = 5.8 the solutions were lear and only SUVs were observed by TEM

345

.

Su h a high polydispersity of morphologies, where at dis s oexist in equilibrium with spheri al open and omplete unilamellar vesi les, was previously observed in

atanioni systems. A possible explanation for the oexisten e of these stru tures was proposed by Jung et al. spontaneous urvature

351

.

by evaluating the parameters ontributing to the

In some systems however (namely mixtures of anioni 128

Figure 7.5:

Cryo-TEM images of a SL / AS mixed surfa tant solution at (left) pH = 4.5, 10

days after mixing, and (right) pH = 5.5, 5 days after mixing. The bla k arrows point to dis s, the double arrows to a partially folded dis , whereas the white arrowheads to globular stru tures (possibly mi elles). The white arrow points to an elongated stru ture.

Figure 7.6:

The formation of small vesi les after 2 weeks in samples with pH = 6.7 and large

multilamellar vesi les after 4 weeks (pH = 5.5); arrowheads point to very small perfe t vesi les, while the arrow points to an in omplete two-lamellar vesi le (`F' indi ates a frost parti le).

129

Figure 7.7:

Phase behavior after 2 months: (a) small membrane fragments at low pH (= 4.5);

(b) destabilization of vesi les at high pH (= 6.7).

and zwitterioni surfa tants), dis oidal stru tures are only short-lived intermediates in the mi elle-to-vesi le transitions

352

.

The vesi ulation depends on the balan e

between the unfavorable edge energy of the disks and the bending energy required to form spheri al stru tures. In the ase of atanioni surfa tant mixtures, the edges of the dis s are stabilized by the ex ess ationi surfa tant. It is possible that the relatively bulky EO groups stabilize the dis s observed in our system.

7.4.4 Cytotoxi ity Potential of Surfa tant Mixtures Akypo Soft 45 NV is ommonly used in osmeti formulations, su h as hair onditioners and hair dyes

353357

. Improving the bio ompatibility of produ ts used in

osmeti formulation even further is always sought after. Cytotoxi ity of our mixtures on HeLa ells and Keratyno ytes was measured in order to identify the toxi properties of the two surfa tants for use in pharma euti al and osmeti al appli ations. Cell viability was evaluated by the tetrazolium MTT redu tion assay, based on the uptake and the redu tion of the soluble yellow MTT tetrazolium salt by mito hondrial dehydrogenase to a blue insoluble MTT formazan produ t. The IC50 value was al ulated from absorban e data.

HeLa ells were used, be ause they

reportedly show good reprodu ibility and a signi ant orrelation with 130

in vivo

re-

sults

324

, whereas Keratyno ytes were hosen to he k the skin ompatibility of su h

surfa tant mixtures.

120

80

IC

50

[ g/mL]

100

60

40 0.0

0.2

0.4

0.6

0.8

1.0

Akypo / SL

Figure 7.8:

The mito hondrial redu tion of MTT after a 3-day in ubation of SL / AS mixture

at dierent surfa tant ratios with HeLa ells () and Keratyno ytes (). IC50 values reported in Figure 7.8 show a dose-dependent de rease in toxi ity when AS is ex hanged for SL. Therefore any surfa tant mixture formed with SL is bene ial. The order of the IC50 values was the same for both ell lines; however the absolute values were slightly lower in the ase of the Keratyno ytes. The lowering of the pH of the surfa tant test solutions showed no pronoun ed ee t. The volume of the added surfa tant solution was probably too small to hange the pH value of the ell medium signi antly and the buering apa ity of the medium too large for any ee ts to be observed.

7.5 Con lusions Two obsta les mentioned in the introdu tion, namely, the high solubility temperature of alkyl arboxylates and limited pH region of vesi le formation have been 131

over ome by using a mixture of two alkyl arboxylates with two very dierent pKa values. The presented results show that it is then possible to spontaneously form bilayer stru tures, su h as vesi les, in a pH range between the two onsidered pKa. Sodium laureate (with a pKa around

8.5) and Akypo Soft 45 NV (with a pKa = 4.67)

ould be thus used at dierent mixing ratios to obtain vesi ular solutions over the entire range of pH omparable to skin or physiologi al pH. Furthermore, we have used ommer ially available and bio ompatible surfa tants that are already used for

osmeti purposes and are inexpensive, whi h is of signi ant importan e for appli ation purposes

358

. However, more resear h is required to improve their appli ation

potential by in reasing the olloidal stability of fatty a id vesi les and assure the vesi les are ompletely losed. Both are ommon problems in su h systems

132

81,359

.

Con lusion In this thesis the formation and stabilization of atanioni vesi les was studied. Catanioni surfa tant mixtures were hosen, be ause they were shown to form vesi le spontaneously under ertain onditions and exhibit an enhan ed sensitivity to outside parameters, su h as temperature or the presen e of salts. Due to this sensitivity, atanioni mixtures are a good system to study spe i -ion ee ts.

The

interest in vesi les was further motivated by their potential as models for biologi al membranes and as en apsulation systems. To optimize the appli ations it is important to have a general understanding of the interplay of intera tions between the surfa tants and of the fa tors inuen ing the phase diagram of a mixed system. First, the morphologi al transitions o

urring in mixed surfa tant systems upon the in rease of the ioni strength were explored. The transition of mixed ioni mi elles to vesi les in a atanioni surfa tant solution, omprised of sodium dode ylsulfate and dode yltrimethylammonium bromide, with an ex ess of the anioni omponent, upon the addition of salt is des ribed in Chapter 2.

A new type of intermediate

stru ture was found: a symmetri ally shaped spheri al super-stru ture, whi h we named blastulae vesi le. A me hanism of formation for this type of super-stru tures was proposed, suggesting the importan e of harge u tuation in the vesi le membrane. The spe i -ion ee ts on this system was further studied and ompared to one where the sulphate surfa tant was repla ed by a arboxylate surfa tant (sodium dode anoate). No anion spe i ity was found in the anioni -ri h region of the phase diagram for the added salts, whereas the nature of the ation was found to strongly inuen e the riti al salt on entrations around whi h mi elles turn to vesi les. This was explained by taking into a

ount that in the present ase of negatively harged 133

vesi les, the ations a

umulate in high on entration in the vi inity of the vesi le. The observed ation spe i ity followed the lassi al Hofmeister series for ation adsorption to sulfate headgroups. When alkyl arboxylates were present in solution, the ations followed a reversed Hofmeister series. The ion spe i ity was qualitatively explained a

ording to Collins' on ept of mat hing water anities. To this purpose, the headgroup of an alkylsulfate had to be regarded as a haotrope and the alkyl arboxylate as a kosmotrope. Based on MD simulations performed on the aforementioned headgroups, a general `Hofmeister series of surfa tant headgroups' is established in Chapter 3. In Chapter 4, the study of ion-spe i ity was extended to the ationi -ri h region of the phase diagram of the atanioni system.

No vesi les were found, however,

the addition of salts produ ed a sphero ylindri al growth of the mi elles, markedly dependent on the anion identity. The e ien y of the anions to elongate the mi elles ould again be explained by the Collins' on ept and the lassi ation of the trimethylammonium surfa tant headgroup as a soft, polarizable entity. The ee t of various additives on the stability of mixed surfa tant solutions of sodium dode ylsulfate with etyltrimethylammonium bromide and with dode yltrimethylammonium bromide was studied as a fun tion of time and is reported in Chapter 5. The lifetime of vesi les and mi elles in these systems ould be ontrolled by varying the omposition of the surfa tant solutions and by additives. Controlling the pre ipitation phenomena is of importan e for a large number of industrial pro esses, where formulations need to be tuned. These spe i mixtures were shown to have a solubility temperature below that of pure surfa tant solutions in the anioni -ri h region. The stability of su h supersaturated solutions was in reased by in reasing the per entage of the anioni surfa tant and shortening the hain of the ationi surfa tant.

The presen e of simple ele trolytes de reased, while the addition of

middle- hain al ohols in reased their stability. Experimental results suggested that the destabilization and on urrent pre ipitation of the systems was not due to the formation of bigger aggregates, but rather to a shift of the equilibrium between mi elles and monomers. The addition of low on entrations of middle- hain al ohols resulted in the formation of vesi les in the atanioni systems at temperatures sig-

134

ni antly lower than the Krat temperatures of the parti ipating ioni surfa tants. In Chapter 6 we evaluated the ytotoxi ity of single- hain ioni surfa tants and

atanioni mixtures using MTT on HeLa ells. It is important to onsider the toxi ity of parti ipating surfa tants when formulating new en apsulating systems for either osmeti or medi al use.

It was onrmed that anioni surfa tants gener-

ally exhibit higher IC50 values (lower toxi ity) than atanioni ones. The toxi ity

ould further be inuen ed by the hydro arbon hain length and the presen e of polyoxyethylene groups. A general trend of in reasing toxi ity with in reasing hain length and in lusion of polyoxyethylene groups was observed. A non-linear in rease was observed as ationi surfa tants are added to anioni ones.

A steep de rease

of the IC50 values is observed already at low fra tions of the ationi surfa tants, suggesting that in potential drug delivery systems a high fra tion of the anioni

omponent is ne essary.

Seeing that the presen e of only a small amount of a ationi surfa tant in the mixture resulted in a large in rease in its toxi ity, we fo used on a mixture of two anioni and in osmeti formulation already ommonly used surfa tants. In hapter 7 we present a way to form vesi les in a mixture of two alkyl arboxylates with two very dierent pKa values. We have shown that it is then possible to spontaneously form vesi les in a pH range between the two onsidered pKa. Sodium laureate and Akypo Soft 45 NV ould be thus used at dierent mixing ratios to obtain vesi ular solutions over the entire range of pH omparable to skin or physiologi al pH.

135

136

Bibliography [1℄ K. Holmberg, B. Joensson, B. Kronberg, and B. Lindman.

Polymers in aqueous solutions, 2nd ed.

Surfa tants and

John Wiley & Sons, 2003.

Surfa tants and Interfa ial Phenomena (2nd ed.).

[2℄ M. J. Rosen.

John Wiley

& Sons, 1989.

[3℄ D. F. Evans and H. Wennerström.

The Colloidal Domain.

[4℄ B. Lindman and H. Wennerström.

Wiley-VCH, 1999.

Topi s in Current Chemistry, Vol. 87.

Springer-Verlag, Germany, 1980.

[5℄ K. Meguro, M. Ueno, and K. Esumi.

istry.

Nonioni Surfa tants, Physi al Chem-

Mar el Dekker, New York, 1987.

[6℄ K. Shinoda, T. Nakagawa, B. I. Tamamush, and T. Isemura.

tants, Some physi o- hemi al properties.

Colloidal surfa -

A ademi Press, London, 1963.

[7℄ G. Gunnarsson and H. Jönsson, B.and Wennerström.

J. Phys. Chem., 84:3114,

1980.

Colloids and Interfa es with Surfa tants and Polymers.

[8℄ J. Goodwin.

Wiley & Sons, 2004.

[9℄ J. H. Fendler.

Membrane Mimeti Chemistry.

Wiley, New York, 1982.

[10℄ W. Helfri h.

Z. Naturfors h., 28:693, 1973.

[11℄ M. Kléman.

Pro . R. So . London, Ser. A, 347:387, 1976.

[12℄ W. Helfri h.

J. Phys., 47:321, 1986. 137

John

[13℄ S. A. Safran, P. Pin us, and D. Andelman.

S ien e, 248:354, 1990.

[14℄ J. N. Israela hvili, D. J. Mit hell, and B. W. Ninham.

Bio hem. Biophys.

A ta, 470:185, 1977. J. Chem. So ., Faraday

[15℄ J. N. Israela hvili, D. J. Mit hell, and B. W. Ninham.

Trans. 2, 72:1525, 1976. [16℄ D. J. Mit hell and B. W. Ninham.

J. Chem. So ., Faraday Trans. 2, 77:601,

1981.

[17℄ M. Dubois and T. Zemb.

Langmuir, 7:1352, 1991.

[18℄ E. F. Marques, O. Regev, A. Khan, and B. Lindman.

Adv. Coll. Interf. S i.,

100:83, and refs. therein, 2003.

[19℄ D.D. Miller, J.R. Bellare, T. Kaniko, and D.F. Evans.

[20℄ B.W. Ninham, D.F. Evans, and G.J. Wei.

Langmuir, 4:1363, 1988.

J. Phys. Chem., 87:5020, 1983.

[21℄ Y. Talmon, D. F. Evans, and B. W. Ninham.

S ien e, 221:1047, 1983.

[22℄ M.I. Viseu, M.M. Velazquez, C.S. Campos, I. Gar ia-Mateos, and S.M.B. Costa.

Langmuir, 16:4882, 2000.

[23℄ B. A. Coldren, H. Warriner, R. van Zanten, and J. A. Zasadzinski.

Langmuir,

22:2465, 2006.

[24℄ E. W. Kaler, A. K. Murthy, B. E. Rodriguez, and J. A. Zasadzinski.

S ien e,

245:1371, 1989.

[25℄ A. Kihisa and T. A. Hatton.

Langmuir, 18:7341, 2002.

[26℄ M. T. Yat illa, K. L. Herrington, L. L. Brasher, E. W. Kaler, S. Chiruvolu, and J. A. Zasadzinski.

J. Phys. Chem., 100:5874, 1996.

[27℄ M. Bergmeier, H. Homann, and C. Thunig. 1997. 138

J. Phys. Chem. B,

101:5767,

[28℄ M. Bergmeier, H. Homann, F. Witte, and S. Zouraub.

J. Colloid Interfa e

S i., 203:1, 1998. [29℄ M. Gradzielski, M. Bergmeier, M. Mueller, and H. Homann.

J. Phys. Chem.

B, 101:1719, 1997. [30℄ M. Gradzielski, M. Mueller, M. Bergmeier, H. Homann, and E. Hoinkis.

J.

Phys. Chem. B, 103:1416, 1999. [31℄ J. Hao, H. Homann, and K. Horbas hek.

Langmuir, 17:290, 2001.

[32℄ J. Huang, Y. Zhu, B. Zhu, R. Li, and H. Fu.

J. Colloid Interfa e S i., 236:201,

2001.

[33℄ R. Oda and L. Bourdieu.

J. Phys. Chem. B, 101:5913, 1997.

[34℄ M. Andersson, L. Hammarström, and K. Edwards.

J. Phys. Chem., 99:14531,

1995.

[35℄ A. M. Carmona-Ribeiro.

Chem. So . Rev., 21:207, 1992.

[36℄ J.B.F.N. Engberts and D. Hoeskstra.

Bio him. Biopphys. A ta,

1241:323,

1995.

[37℄ E. Feitosa and G. Barreleiro, P.C.A and; Olofsson.

Chem. Phys. Lipids,

105:201, 2000.

[38℄ E. Feitosa and W. Brown.

Langmuir, 13:4810, 1997.

[39℄ P. Saveyn, J. Co quyt, P. Bomans, P. Frederik, M. De Cuyper, and P. Van der Meeren.

Langmuir, 23:4775, 2007.

[40℄ H. Hauser.

Chem. Phys. Lipid, 43:283, 1987.

[41℄ N. E. Gabriel and M. F. Roberts.

Bio hemistry, 23:4011, 1984.

[42℄ A. K. Murthy, E. W. Kaler, and J. A. Zasadzinski. 145:598, 1991. 139

J. Colloid Interf. S i.,

[43℄ J. E. Brady, D. F. Evans, B. Ka har, and B. W. Ninham.

J. Am. Chem. So .,

106:4279, 1984.

[44℄ S. Hashimoto, J. K. Thomas, D. F. Evans, S. Mukherjee, and B. W. Ninham.

J. Colloid Interfa e S i., 95:594, 1983. [45℄ B. W. Ninham and D. F. Evans.

Faraday Dis uss. Chem. So ., 81:1, 1986.

[46℄ Z. Lin, M. He, L. E. S riven, and H. T. Davis.

J. Phys. Chem., 97:3571, 1993.

[47℄ L. L. Brasher, K. L. Herrington, and E. Kaler.

Langmuir, 11:4267, 1995.

[48℄ K. L. Herrington, E. W. Kaler, D. D. Miller, J. A. Zasadzinski, and S. Chiruvolu.

J. Phys. Chem., 97:13792, 1993.

[49℄ E. W. Kaler, K. L. Herrington, A. K. Murthy, and J. A. N. Zasadzinski.

J.

Phys. Chem., 96:6698, 1992. [50℄ Y. Kondo, H. U hiyama, N. Yoshino, K. Nishiyama, and M. Abe.

Langmuir,

11:2380, 1995.

[51℄ G. Gregoriadis.

Liposome Te hnology. Volume II: Entrapment of Drugs and

Other Materials, 2nd ed. [52℄ D. D. Lasi .

CRC Press: Bo a Raton, FL, 1988.

Liposomes: From Physi s to Appli ations.

Elsevier Publishing

Co., Amsterdam, 1993.

[53℄ M. Dubois and T. Zemb.

Curr. Opin. Colloid Interfa e S i., 5:27, 2000.

[54℄ R.D. Koehler, S.R. Raghavan, and E.W. Kaler.

J. Phys. Chem. B, 104:11035,

2000.

[55℄ X. Li and H. Kunieda.

Curr. Opin. Colloid Interfa e S i., 8:327, 2003.

[56℄ E. Marques, A. Khan, M. Miguel, and B. Lindman.

J. Phys. Chem., 97:4729,

1993.

[57℄ S.R. Raghavan, G. Fritz, and E.W. Kaler. 140

Langmuir, 18:3797, 2002.

[58℄ A. Khan.

Curr. Opin. Colloid Interfa e S i., 1:614, 1996.

[59℄ P. K. Yuet and D. Blanks htein.

[60℄ C. Tanford.

Langmuir, 12:3802, 1996.

The Hydrophobi Ee t: Formation of Mi elles and Biologi al

Membranes (2nd ed.).

John Wiley & Sons, New York, 1980.

[61℄ W. R. Hargreaves and D. W. Deamer.

[62℄ C. Tondre and C. Caillet.

Bio hemistry, 17:3759, 1978.

Adv. Colloid Interfa e S i., 93:115, 2001.

[63℄ P. M. Gullino, F. H. F. H. Grantham, S. H. Smith, and M. Haggerty.

J. Natl.

Can er Inst., 34:857, 1965. [64℄ H. Kahler and W. V. B. Robertson.

J. Natl. Can er Inst., 3:495, 1943.

[65℄ K. A. Meyer, E. M. Kummerling, L. Altman, M. Koller, and S. J. Homan.

Can er Res., 8:513, 1948. [66℄ J. Naeslund and K. E. Swenson.

A ta Obstet. Gyne ol. S and., 32:359, 1953.

[67℄ M. Stubbs, P. M. J. M Sheehy, J. R. Griths, and C. L. Bashford.

Mol. Med.

Today, 6:15, 2000. [68℄ M. C. De Oliveira, E. Fattal, C. Ropert, C. Malvy, and P. Couvreur.

Journal

of Controlled Release, 48:179, 1997. [69℄ I. M. Hafez, S. Absell, and P. R. Cullis.

[70℄ L. Huang and C. Y. Wang.

[71℄ X. Li and M. S hi k.

Biophys. J., 79:1438, 2000.

Bio hemistry, 28:9508, 1989.

Biophys. J., 80:1703, 2001.

[72℄ C. Ropert, M. Lavignon, C. Dubernet, P. Couvreur, and C. Malvy.

Bio hem.

Biophys. Res. Commun., 183:879, 1992. [73℄ S. Simões, J. Nuno Moreira, C.; Fonse a, N. Duzgunes, and M. C. Pedroso de Lima.

Adv. Drug Deliv. Rev., 56:947, 2004. 141

[74℄ S. Simões, V. Slepushkin, N. Duzgunes, and M. C. Pedroso de Lima.

Bio him.

Biophys. A ta, 1515:23, 2004. [75℄ J.J. Sudima k, W. Guo, W. Tjarks, and R.J. Lee.

Bio him. Biophys. A ta,

1564:31, 2002.

[76℄ V.P. Tor hilin, F. Zhou, and L. Huang.

J. Liposome Res., 3:201, 1993.

[77℄ P. Venugopalan, S. Jain, S. Sankar, P. Singh, A. Rawat, and S. P. Vyas.

Pharmazie, 57:659, 2002. [78℄ M. B. Yatvin, W. Kreutz, B. A. Horwitz, and M. Shinitzky.

S ien e, 210:1253,

1980.

Pro . Natl. A ad. S i., 101:7965, 2004.

[79℄ I. A. Chen and J. W. Szostak.

[80℄ I. Chen and J. W. Szostak.

Biophys. J., 82:988, 2004.

[81℄ K. Morigaki, S. Dallavalle, P. Walde, S. Colonna, and P. L. Luisi.

J. Am.

Chem. So ., 119:292, 1997. [82℄ C.G. Goltner and M. Antonietti.

[83℄ R. Hoss and F. Voegtle.

Adv. Mat., 9:431, 1997.

Angew. Chem., 106:389, 1994.

[84℄ S. Mann, S.L. Burkett, S. A. Davis, C.E. Fowler, N.H. Mendelson, S.D. Sims, D. Walsh, and N.T. Whilton.

Chemistry of Materials, 9:2300, 1997.

[85℄ C.A. M Kelvey, E.W. Kaler, J.A. Zasadzinski, B. Coldren, and H.-T. Jung.

Langmuir, 16:8285, 2000. [86℄ Z. Yuan, Z. Yin, S. Sun, and J. Hao.

J. Phys. Chem. B, 112:1414  1419, 2008.

[87℄ A. Bonin ontro, M. Falivene, C. La Mesa, G. Risuleo, and M. R. Pena.

Lang-

muir, 24:1973, 2008. [88℄ J. D. Bernal and R. H. Fowler.

[89℄ R. W. Gurney.

J. Chem. Phys., 1:515, 1933.

Ioni Pro esses in Solution. 142

M Graw-Hill Book Co., 1953.

Thermodynami s of Solvation.

[90℄ G. A. Krestov.

Ellis Horwood:

New York,

1990.

[91℄ R. A. Robinson and R. H. Stokes.

Ele trolyte Solutions. Butterworth S ienti

Publi ations: London, 1959.

[92℄ O. Y. Samoilov.

Water and Aqueous Solution: Stru ture, Thermodynami s,

and Transport Pro esses, pages 597612. Wiley-Inters ien e: [93℄ O. Y. Samoilov.

New York, 1972.

Dis uss. Faraday So , 24:141, 1957.

[94℄ G. Galli and M. Parrinello.

Computer Simulations in Materials S ien e.

Kluwer: Dordre ht, 1991.

[95℄ K. Heinzinger and P. C. Vogel.

Z. Naturfors h., 29:1164, 1974.

[96℄ G. Hummer, L. R. Pratt, and A. E. Gar ia.

J. Phys. Chem. A, 102:7885, 1998.

[97℄ D. Marx, M. Sprik, M. Sprik, and M. Parinello.

Chem. Phys. Lett.,

273:360

and refs therein, 1997.

[98℄ M. C. Payne, M. P. Teter, D. C. Allan, T. A. Arias, and J. D. Joannopoulos.

Rev. Mod. Phys., 64:1045, 1992. [99℄ W. Kauzmann.

Adv. Protein Chem., 14:1, 1959.

[100℄ M. Kaminsky.

Dis uss. Faraday So ., 24:171, 1957.

[101℄ K. D. Collins.

Methods, 34:300, 2004.

[102℄ N. T. Skipper and G. W. Neilson.

[103℄ K. D. Collins.

Biophys. J., 72:65, 1997.

[104℄ P. Debye and B. Hü kel.

[105℄ K.D. Collins.

J. Phys.: Condens. Matter, 1:4141, 1989.

Phys. Z., 24:185, 1923.

Pro .Natl.A ad. S i., 92:5553, 1995.

[106℄ K. D. Collins and M. W. Washabaugh.

J. Biol. Chem., 261:12477, 1986.

[107℄ K. D. Collins and M. W. Washabaugh.

Q. Rev. Biophys., 18:323, 1985.

143

[108℄ S. Gopalakrishnan, D. Liu, H.C. Allen, M. Kuo, and M. J. Shultz.

Chem.

Rev., 106:1155, 2006. [109℄ P.B. Petersen and R.J. Saykally.

Annu. Rev. Phys. Chem., 57:333, 2006.

[110℄ J. Cheng, C.D. Ve itis, M.R. Homann, and A. J. Colussi.

J. Phys. Chem. B,

110:25598, 2006.

[111℄ P. Jungwirth and D. J. Tobias.

Chem. Rev., 106:1259, 2006.

[112℄ L. Vrbka, M. Mu ha, B. Minofar, P. Jungwirth, E. C. Brown, and D. J. Tobias.

Curr. Opin. Colloid Interfa e S i., 9:67, 2004. [113℄ M. Bostrom, F.W. Tavares, B.W. Ninham, and J.M. Prausnitz.

J. Phys.

Chem. B, 110:24757, 2006. [114℄ F. Hofmeister.

Ar h. Exp. Pathol. Pharmakol., 24:247, 1888.

[115℄ W. Kunz, P. Lo Nostro, and B. W. Ninham.

Curr. Opin. Colloid Interfa e

S i., 9:1, 2004. [116℄ Y. Mar us.

Ion Solvation.

[117℄ Y. Mar us and G. Hefter.

Wiley: Chi hester, U.K., 1985.

Chem. Rev., 106:4585, 2006.

[118℄ Y. V. Kalyuzhnyi, V. Vla hy, and K. Dill.

A ta Chim. Slov., 48:309, 2001.

[119℄ T. V. Chalikian, J. Volker, E. Plum, and K. Breslauer.

Pro . Natl. A ad. S i.,

96:7853, 1999.

[120℄ J. Darnell, H. Lodish, and D. Baltimore.

Mole ular Cell Biology.

S ienti

Ameri an Books, New York, 1990.

[121℄ K. A. Dill.

Bio hemistry, 29:7133, 1990.

[122℄ N. Korolev, A. P. Lyubartsev, A. Ruppre ht, and L. Nordenskiold.

J., 77:837, 1999. [123℄ J. Rupley and G. Careri.

Adv. Protein Chem., 41:37, 1991. 144

Biophys.

[124℄ T. P. Lybrand, A. M Cammon, and G. Wi.

Pro . Natl. A ad. S i.,

83:833,

1986.

[125℄ F. Sussman and H. Weinstein.

[126℄ P. C. Jordan.

[127℄ B. Katz.

Pro . Natl. A ad. S i., 86:7880, 1989.

Biophys. J., 58:1133, 1990.

Nerve, Mus le, and Synapse.

M Graw-Hill: London, 1966.

[128℄ M. G. Ca a e, E. M. Landau, and J. J. Ramsden.

Q. ReV. Biophys., 30:241,

1997.

[129℄ M. F. Kropman and H. J. Bakker.

S ien e, 291:2118, 2001.

[130℄ M. Maron elli, J. Ma Innins, and G. R. Fleming.

[131℄ V. L. Larwood, B. J. Howlin, and G. A. Webb.

[132℄ S. Habu hi, H. B. Kim, and N. Kitamura.

S ien e, 243:1674, 1989.

J. Mol. Model., 2:175, 1996.

Anal. Chem., 73:366, 2001.

[133℄ M. Chavez-Paez, K. Van Workum, L. de Pablo, and L. L. de Pablo.

J. Chem.

Phys., 114:1405, 2001. [134℄ K. L. Stellner, J. C. Amante, J. F. S amehorn, and J. H. Harwell.

J. Colloid

Interfa e S i., 123:186, 1988. [135℄ L. S. Romsted.

Langmuir, 23:414, 2007.

[136℄ H. Fukuda, K. Kawata, and H. Okuda.

J. Am. Chem. So ., 112:1635, 1990.

[137℄ Th. Zemb, M. Dubois, B. Demé, and T. Gulik-Krzywi ki.

S ien e,

283:816,

1999.

[138℄ M. Dubois, B. Demé, T. Gulik-Krzywi ki, J.-C. Dedieu, C. Vautrin, S. Désert, E. Perez, and Th. Zemb.

Nature, 411:672, 2001.

[139℄ A. Song, S. Dong, X. Jia, J. Hao, W Liu, and T. Liu. 44:4018, 2005. 145

Angew. Chem. Int. Ed.,

[140℄ P. André, A. Filankembo, I. Lisie ki, C. Petit, T. Gulik-Krzywi ki, B.W. Ninham, and M. P. Pileni.

Adv. Mat., 12:119, 2000.

[141℄ S. T. Hyde, S. Andersson, K. Larsson, Z. Blum, T. Landh, S. Lidin, and B. W. Ninham.

The Language of Shape.

Elsevier, Amsterdam, 1997.

[142℄ P. André, B. W. Ninham, and M. P. Pileni.

New J. Chem., 25:563, 2001.

[143℄ P. André, B. W. Ninham, and M. P. Pileni.

Adv. Coll. Interf. S i.,

89:155,

2001.

[144℄ D. Miller, J. Bellare, D. F. Evans, Y. Talmon, and B. W. Ninham.

J. Phys.

Chem., 91:674, 1987. [145℄ B. W. Ninham, S. Hashimoto, and J. K. Thomas.

J. Colloid Interfa e S i.,

95:594, 1983.

[146℄ B. W. Ninham, Y. Talmon, and D.F. Evans.

S ien e, 221:1047, 1983.

[147℄ J. L. Parker, H. K. Christenson, and B. W. Ninham.

J. Phys. Chem., 92:4155,

1988.

[148℄ E. Z. Radlinska, B. W. Ninham, J. P. Dalbiez, and T. N. Zemb.

J. Coll. Surf.,

46:213, 1990.

[149℄ E. Z. Radlinska, T. N. Zemb, J. P. Dalbiez, and B. W. Ninham.

Lamgmuir,

9:2844, 1993.

[150℄ T. Bramer, M. Paulsson, K. Edwards, and K. Edsman.

Pharm. Res., 20:1661,

2003.

[151℄ H. P. Hentze, S. R. Raghavan, C. A. M Kelvey, and E. W. Kaler.

Langmuir,

19:1069, 2003.

[152℄ M. Kep zynski, F. Gana haud, and P. Hemery.

Adv. Mat., 16:1861, 2004.

[153℄ J. C. Amante, J. F. S amehorn, and J. H. Harwell. 144:243, 1991. 146

J. Colloid Interfa e S i.,

[154℄ N. Kamenka, M. El Amrani, J. Appell, and M. Lindheimer.

J. Colloid Interfa e

S i., 143:463, 1991. [155℄ A. Renon ourt, P. Bauduin, D. Touraud, W. Kunz, N. Azemar, and C. Solans.

Comuni a iones presentadas a las Jornadas del Comite Espanol de la Detergen ia, 34:273, 2004. [156℄ N. Filipovi -Vin ekovi , M. Bujan, I. Smit, Lj. Tusek-Bozi , and I. Stefani .

J. Colloid Interfa e S i., 201:59, 1998. [157℄ A. J. O'Connor, T. A. Hatton, and A. Bose.

Langmuir, 13:6931, 1997.

[158℄ O. Soederman, K. L. Herrington, E. W. Kaler, and D. D. Miller.

Langmuir,

13:5531, 1997.

[159℄ H. Yin, Z. Zhou, J. Huang, R. Zheng, and Y. Zhang.

Angew. Chem., 42:2188,

2003.

[160℄ H. Yin, S. Lei, S. Zhu, J. Huang, and J. Ye.

[161℄ L. Hao, Y. Nan, H. Liu, and Y. Hu.

Chem. Eur. J., 12:2825, 2006.

J. Dispersion S i. and Te hnology, 27:271,

2006.

[162℄ L. Zhai, G. Li, and Z. Sun.

Colloids Surf., A, 190:275, 2001.

[163℄ L. Zhai, M. Zhao, D. Sun, J. Hao, and L. Zhang.

J. Phys. Chem. B, 109:5627,

2005.

[164℄ A. Renon ourt. PhD thesis, University of Regensburg, 2005.

[165℄ M. Bergström and J. S. Pedersen.

Langmuir, 15:2250, 1999.

[166℄ E. F. Marques, A. Khan, and B. Lindman.

Thermo himi a A ta, 394:31, 2002.

[167℄ K. Tsu hiya, H. Nakanishi, H. Sakai, and M. Abe.

Langmuir, 20:2117, 2004.

[168℄ C. Vautrin, M Dubois, Th. Zemb, St. S hmölzer, H. Homan, M. Colloids Gradzielski, and 165. Surfa es A: Physi o hem. Eng. Aspe ts 2003, 217.

loids Surf., A, 217:165, 2003. 147

Col-

[169℄ A. Renon ourt, N. Vla hy, P. Bauduin, M. Dre hsler, D. Touraud, J.-M. Verbavatz, M. Dubois, W. Kunz, and B. W. Ninham Langmuir 23 (2007) 2376.

Langmuir, 23:2376, 2007. [170℄ K. Edwards, J. Gustafsson, M. Almgren, and G. Karlsson.

J. Colloid Interfa e

S i., 161:161, 1993. [171℄ J. Gustafsson, G. Orädd, G. Lindblom, U. Olsson, and M. Almgren.

Langmuir,

13:852, 1997.

[172℄ J. Gustafsson, G. Orädd, M. Nydén, P. Hansson, and M. Almgren.

Langmuir,

14:4987, 1998.

[173℄ H. Homann, C. H. Thunig, U. Munkert, H. W. Meyer, and W. Ri hter.

Langmuir, 8:2629, 1992. [174℄ M. Silvander, G. Karlsson, and K. Edwards.

J. Colloid Interfa e S i., 179:104,

1996.

[175℄ A.-L. Bernard, M.-A. Guedeau-Boudeville, L. Jullien, and J.-M. di Meglio.

Bio him. Biophys. A ta, 1567:1, 2002. [176℄ C. Ménager and V. Cabuil.

J. Phys. Chem. B, 106:7913, 2002.

[177℄ J. B. F. N. Sein, A.and Engberts.

Langmuir, 11:455, 1995.

[178℄ S. Chiruvolu, S. A. Walker, J. Israela hvili, F. J. S hmitt, D. Le kband, and

S ien e, 264:1753, 1994.

J. A. Zasadzinski.

[179℄ S. A. Walker, M. T. Kennedy, and J. A. Zasadzinski.

[180℄ S. A. Walker and J. A. Zasadzinski.

Nature, 387:61, 1997.

Langmuir, 13:5076, 1997.

[181℄ H. Aranda-Espinoza, Y. Chem, N. Dan, T. C. Lubensky, P. Nelson, L. Ramos, and D. A. Weitz.

S ien e, 1999:285, 394.

[182℄ A. A. Gurtovenko.

J. Chem. Phys., 122:244902, 2005.

[183℄ B. Hribar and V. Vla hy.

J. Phys. Chem. B, 101:3457, 1997. 148

[184℄ A. Walter, P. K. Vinson, A. Kaplun, and Y. Talmon.

Biophys. J.,

60:1315,

1991.

[185℄ B. Demé, M. Dubois, T. Gulik-Krzywi ki, and Th. Zemb.

Langmuir, 18:997,

2002.

[186℄ M. Dubois, V. Lizunov, T A. Meister, Gulik-Krzywi ki, J.-M. Verbavatz, E. Perez, J. Zimmerberg, and Th. Zemb.

Pro . Natl. A ad. S i.,

101:15082,

2004.

[187℄ H. W. Meyer, W. Ri hter, and J. Gumpert.

Bio him. Biophys. A ta, 1026:171,

1990.

[188℄ B. Sternberg, J. Gumpert, G. Reinhardt, and K. Gawris h.

Bio him. Biophys.

A ta, 898:223, 1987. [189℄ H. W. Meyer, H. Bunjes, and A. S. Ulri h.

[190℄ E.; Sa kmann and T. Feder.

Chem. Phys. Lipids, 99:111, 1999.

Mol. Membr. Biol., 12:21, 1995.

[191℄ V. S. Kulkarni, W. H. Anderson, and R. E. Brown.

[192℄ M. Antonietti, A. Wenzel, and A. Thünemann.

Biophys. J., 69:1976, 1995.

Langmuir, 12:2111, 1996.

[193℄ H. W. Meyer, K. Semmler, W. Rettig, W. Pohle, A. S. Ulri h, S. Grage, C. Selle, and P. Quinn.

J. Chem. Phys. Lipids, 105:149, 2000.

[194℄ C. Gebhardt, H. Gruler, and E. Sa kmann.

Zeits hrift für Naturfors hung,

32:581, 1977.

[195℄ W. Helfri h.

Liquid Crystals, 5:1647, 1989.

[196℄ P. G. Dommersnes and J. B. Fournier.

[197℄ J. B. Fournier.

Phys. Rev. Lett., 76:4436, 1996.

[198℄ R. Goetz and W. Helfri h.

[199℄ K. D. Collins.

Biophys. J., 83:2898, 2002.

J. Phys. II, 6:215, 1996.

Biophysi al Chemistry, 119:271, 2006. 149

[200℄ K. D. Collins, G. W. Neilson, and J. E. Enderby.

Biophysi al Chemistry,

128:95, 2007.

J. Phys.

[201℄ B. Jagoda-Cwiklik, R. Vá ha, M. Lund, M. Srebro, and P. Jungwirth.

Chem. B (2007), doi: 10.1021/jp709634t, doi: [202℄ I. Boime, E. E. Smith, and F. E. Hunter.

10.1021/jp709634t, 2007.

Ar h. Bio hem. Biophys., 139:425,

1970.

[203℄ G. J. van de Vusse, R. S. Reneman, and M. van Bilsen.

Prostaglandins,

Leukotrienes Essent. Fatty A ids, 57:85, 1997. [204℄ A. Jakubowska.

ChemPhysChem, 6:1600, 2005.

[205℄ M. Yamashita and J. B. Fenn.

J. Phys. Chem., 88:4671, 1984.

[206℄ C. Gamboa and L. Sepulveda.

J. Colloid Interfa e S i., 113:566, 1986.

[207℄ S. Ikeda, S. Hayashi, and T. Imae.

J. Phys. Chem., 85:106, 1981.

J. Am. Chem. So ., 69:683, 1947.

[208℄ M. L. Corrin and W. D. Harkins.

[209℄ N. A. Mazer, G. B. Benedek, and M. C. Carey.

J. Phys. Chem., 80:107, 1976.

[210℄ L. Vrbka, P. Jungwirth, P. Bauduin, D. Touraud, and W. Kunz.

J. Phys.

Chem. B, 110:7036, 2006. [211℄ E. Leontidis.

Curr. Opin. Colloid Interfa e S i., 7:81 and refs. therein, 2002.

[212℄ H. Gustavsson and B. Lindman.

J. Am. Chem. So ., 100:4647, 1978.

[213℄ N. Hedin, I. Furo, and P. O. Eriksson.

[214℄ V. E. Haverd and G. G. Warr.

Langmuir, 16:157 and refs. therein, 2000.

[215℄ H. P. Gregor and M. J. Frederi k.

[216℄ K. Huts hneker and H. Deuel.

[217℄ U. P. Strauss.

J. Phys. Chem. B, 140:8544, 2000.

Polymer S i., 23:451, 1957.

Helv. Chim. A ta, 39:1038, 1956.

Polyele trolytes,

page 79.

Dordre ht-Holland, 1974. 150

D. Reidel Publishing Company,

J. Am. Chem. So ., 87:1476, 1965.

[218℄ U. P. Strauss and Y. P. Leung.

[219℄ H. P. Gregor, M. J. Hamilton, R. J. Oza, and F. Bernstein.

J. Phys. Chem.,

60:263, 1956.

Ion Ex hange.

[220℄ F. Heleri h.

M Graw-Hill Book Co., In ., New York, N.Y.,

1962.

[221℄ H. G. Bungenberg de Jong.

Colloid S ien e, Vol. II,

hapter 9.

Elsevier

Publishing Co., In ., New York, N. Y., 1949.

[222℄ G. Eisenman.

Symposium on Membrane Transport and Metabolism, page 163.

A ademi Press, New York, N. Y., 1961.

[223℄ M. E. Feinstein and H. L. Rosano.

[224℄ I. Weil.

J. Colloid Interfa e S i., 24:73, 1967.

J. Phys. Chem., 70:133, 1966.

[225℄ H. L. Rosano, A. P. Christodoulou, and M. E. Feinstein.

J. Colloid Interfa e

S i., 29:335, 1969. [226℄ R. M. Fuoss, A. Kat halsky, and S. Lifson.

Pro . Natl. A ad. S i. U. S., 37:579,

1951.

[227℄ G. S. Manning.

J. Chem. Phys., 51:924, 1969.

[228℄ H. Daoust and M.-A. Chabot.

[229℄ D. Dolar.

Polyele trolytes,

Ma romole ules, 13:616, 1980.

page 97. Reidel Publishing Company, Dordre ht-

Holland, 1974.

[230℄ I. Lipar, P. Zalar, C. Pohar, and V. Vla hy.

J. Phys. Chem. B,

111:10130,

2007.

[231℄ G. E. Boyd and B. A. Soldano.

Z. Elektro hem., 57:162, 1953.

[232℄ L. Xu, X. Li, M. Zhai, L. Huang, J. Peng, J. Li, and G. Wei.

B, 111:3391, 2007. 151

J. Phys. Chem.

[233℄ H. P. Gregor, L. B. Luttinger, and E. M. Loebl.

J. Am. Chem. So ., 76:5879,

1954.

[234℄ H. Gustavsson and B. Lindman.

J. Am. Chem. So ., 97:3923, 1975.

[235℄ J. M. Chialvo, A. A.; Simonson.

J. Phys. Chem. B, 109:23031, 2005.

[236℄ N. Vla hy, B. Jagoda-Cwiklik, R. Vá ha, D. Tourauda, P. Jungwirth, and W. Kunz. 2008, submitted.

[237℄ M. M. A. E. Claessens.

Do toral dissertation. [238℄ H. Ti Tien.

Size regulation and stability of harged lipid vesi les,

PhD thesis, 2003.

J. Phys. Chem., 68:1021, 1964.

[239℄ M. M. A. E. Claessens, B. F. van Oort, F. A. M. Leermakers, F. A. Hoekstra, and M. A. Cohen Stuart.

Biophys. J., 87:3882, 2004.

[240℄ J. A. W. M. Beena kers, B. F. M. Kuster, and H. S. . van der Baan.

Carbohydr.

Res., 140:169, 1985. [241℄ W. Kunz, P. Calmettes, T. Cartailler, and P. Turq.

J. Chem. Phys., 99:2074,

1993.

[242℄ P. Bauduin, A. Renon ourt, A. Kopf, D. Touraud, and W. Kunz.

Langmuir,

21:6769, 2005.

[243℄ J Narayanan, C. Manohar, D. Langevin, and W. Urba h.

Langmuir,

13:398,

1997.

[244℄ S. R. Raghavan, H. Edlund, and E. W. Kaler.

Langmuir, 18:1056, 2002.

[245℄ J. F. A. Soltero, J. E. Puig, O. Manero, and P. C. S hulz.

Langmuir, 11:3337,

1995.

[246℄ H. Wu, S. Kawagu hi, and I. Koi hi.

[247℄ S. Friberg and I. Blute.

gents 2nd ed.

Colloid Polym. S i., 283:636, 2005.

Hydrotropy, Surfa tant S ien e Series, Liquid Deter-

Dekker, New York, 2006. 152

[248℄ S. Berr, R. R. M. Jones, and J. S. Johnson Jr.

J. Phys. Chem., 96:5611, 1992.

[249℄ G. Biresaw, D. C. M Kenzie, C. A. Bunton, and D. F. Ni oli.

J. Phys. Chem.,

89:5144, 1985.

[250℄ M. S. Vethamuthu, M. Almgren, E. Mukhtar, and P. Bahadur.

Langmuir,

8:2396, 1992.

Biologi al Ma romole ules, Vol. 3.

[251℄ P. H. von Hippel and T. S hlei h.

Mar el

Dekker, New York, N. Y., 1969.

[252℄ S. Gravsholt.

J. Colloid Interfa e S i., 57:575, 1976.

[253℄ T. Imae, R. Kamiya, and S. Ikeda.

J. Colloid Interfa e S i., 108:215, 1985.

[254℄ C. Gamboa, H. Ríos, and S. J. Sepúlveda.

[255℄ A. J. Hyde and D. W. M. Johnstone.

[256℄ J. W. Larsen and L. J. Magid.

[257℄ I. Cohen and T. Vassiliades.

J. Chem. Phys., 93:5540, 1989.

J. Colloid Interfa e S i., 53:349, 1975.

J. Am. Chem. So ., 96:5774, 1974.

J. Phys. Chem., 65:1774, 1961.

[258℄ H. Fabre, N. Kamenka, A. Khan, G. Lindblom, B. Lindman, and G. J. T. Tiddy.

J.Phys.Chem., 84:3428, 1980.

[259℄ N. Jiang, P. Li, Y. Wang, J. Wang, H. Yan, R. K. Thomas, I. Chakraborty, and S. P. Moulik.

J. Phys. Chem. B, 111:3658, 2007.

[260℄ G. Para, E. Jarek, and P. Warszynski.

Adv. Colloid Interfa e S i.,

122:39,

2006.

[261℄ G. Para, E. Jarek, and P. Warszynski.

Colloids Surf., A, 261:65, 2005.

[262℄ U. Henriksson, L. Ödberg, J. C. Eriksson, and L. Westman.

J. Phys. Chem.,

81:76, 1977.

[263℄ G. Lindblom, B. Lindman, and L. Mandell. 1973. 153

J. Colloid Interfa e S i., 42:400,

[264℄ F. Reiss-Husson and V. Luzzati.

J. Phys. Chem., 68:3504, 1964.

[265℄ J. Ulmius, B. Lindman, G. Lindblom, and T. Drakenberg.

J. Colloid Interfa e

S i., 65:88, 1978. [266℄ J. Ulmius and H. Wennerström.

[267℄ A. Abe, T. Imae, and S. Ikeda.

J. Magn. Reson., 28:309, 1977. Colloid Polym. S i., 265:637, 1987.

[268℄ J. K. Ba kus and H. A. S heraga.

J. Colloid Interfa e S i., 6:508, 1951.

[269℄ S. J. Candau, E. Hirs h, and R. Zana.

J. Colloid Interf. S i., 105:521, 1985.

[270℄ S. J. Candau, E. Hirs h, and R. Zana.

J. Physique, 45:1263, 1984.

[271℄ P. Debye and E. W. Ana ker.

J. Phys. Colloid Chem., 55:644, 1951.

[272℄ R. Dorshow, J. Briggs, C. A. Bunton, and D. F. Ni oli.

J. Phys. Chem.,

86:2388, 1982.

[273℄ R. B. Dorshow, C. A. Bunton, and D. F. Ni oli.

J. Phys. Chem.,

87:1409,

1983.

[274℄ S. Ikeda.

Surfa tants in Solution, Vol. 2, page 825.

[275℄ T. Imae, A. Abe, Y. Tagu hi, and S. Ikeda.

Plenum, New York, 1984.

J. Colloid Interfa e S i., 109:567,

1986.

[276℄ T. Imae and S. Ikeda.

J. Phys. Chem., 90:5216, 1986.

[277℄ S. Ozeki and S. Ikeda.

J. Colloid Interfa e S i., 87:424, 1982.

[278℄ H. A. S heraga and J. K. Ba kus.

J. Am. Chem. So ., 73:5108, 1951.

[279℄ N. A. Riegelman, M. K. Allawa, M. K. Hreno, and L. A. Strait.

S i., 12:208, 1958. [280℄ L. Sepulveda.

J. Colloid Interfa e S i., 46:372, 1974.

[281℄ B. C. Smith, L. C. Chou, and J. L. Zakin. 154

J. Rheol., 38:73, 1994.

J. Colloid

[282℄ M. B. Sierra, P. V. Messina, M. A. Morini, J. M. Russo, G. Prieto, P. C. S hulz, and F. Sarmiento.

Colloids Surf., A, 277:75, 2006.

[283℄ V. K. Aswal, P. S. Goyal, and P. Thiyagarajan.

J. Phys. Chem.,

109:2469,

1998.

[284℄ R. Bu hner, C. Baar, P. Fernandez, S. S hrödle, and W. Kunz.

J. Mol. Liq.,

118:179, 2005.

[285℄ M. C. Brum and J. F. Oliveira.

Minerals Engineering, 20:945, 2007.

[286℄ M. Nyman, J. M. Bieker, S. G. Thoma, and D. E. Trudell.

J. Colloid Interfa e

S i., 316:968, 2007. [287℄ B. Wu, S. D. Christian, and J. F. S amehorn.

Prog. Colloid Polym. S i.,

109:60, 1998.

[288℄ A. Patist, S. G. Oh, R. Leung, and D. O. Shah.

Colloids Surf., A, 176:3, 2000.

Mi elles, Mi roemulsions and Monolayers.

Dekker, New York,

[290℄ A. Patist, J. R. Kani ky, P. K. Shukla, and D. O. Shah.

J. Colloid Interfa e

[289℄ D. O. Shah. 1998.

S i., 245:1 and refs. therein, 2002. [291℄ P. C. Hiemenz and R. Rajagopalan.

istry, 3rd ed. [292℄ R. J. Hunter.

Prin iples of Colloid and Surfa e Chem-

Dekker, New York, 1997.

Foundations of Colloid S ien e.

Oxford Univ. Press, New York,

1987.

[293℄ H. Chakraborty and M. Sakar.

[294℄ E. F. Marques.

Langmuir, 20:3551, 2004.

Langmuir, 16:4798, 2000.

[295℄ J. M. Pea o k and J. E. Matijevi .

[296℄ R. G. Laughlin.

J. Colloid Interfa e S i., 77:548, 1980.

Colloids Surf., A, 128:27, 1997. 155

[297℄ D. Chu and J. K. Thomas.

[298℄ K. Kogej and J. Skerjan .

J. Am. Chem. So ., 108:6270, 1986.

Langmuir, 15:4251, 1999.

[299℄ C. Vautier-Giongo and B. L. Bales.

J. Phys. Chem. B, 107 (2003) 5398:5398,

2003.

[300℄ A. R. Tehrani-Bagha, H. Bahrami, B. Movassagh, M. Arami, S. H. Amirshahi, and F. M. Menger.

Colloids Surf., A, 307:121, 2007.

[301℄ M.-P. Nieh, T. A. Harroun, V. A. Raghunathan, C. J. Glinka, and J. Katsaras.

Biophys. J., 86:2615, 2004. [302℄ B. Sohrabi, H. Gharibi, S. Javadian, and M. Hashemianzadeh.

J. Phys. Chem.

B, 111:10069, 2007. [303℄ M. Bergström, J. S. Pedersen, P. S hurtenberger, and S. U. Egelhaaf.

J. Phys.

Chem. B, 103:9888, 1999. [304℄ I. Yaa ob and A. Bose.

[305℄ B. Lindman.

J. Colloid Interfa e S i., 178:638, 1996.

Handbook of Applied Surfa e and Colloid Chemistry. John Wiley

& Sons Ltd, England, 2002.

[306℄ N. Vla hy, K. Kogej, D. Touraud, and W. Kunz.

J. Colloid Interfa e S i.,

315:445, 2007.

[307℄ E. B. Abuin and J. C. S aiano.

J. Am. Chem. So ., 106:6274, 1984.

[308℄ D. S. Karpovi h and G. J. Blan hard.

J. Phys. Chem., 99:3951, 1995.

[309℄ D. H. Kim, S. G. Oh, and C. G. Cho.

Colloid Polym. S i., 279:39, 2001.

[310℄ R. J. Anderson, C. Delgado, D. Fisher, J. M. Cunningham, and G. E. Fran is.

Analyti al Bio hemistry, 193:101, 1991. [311℄ S. Askolin, T. Nakari-Setälä, and M. Tenkanen. 57:124, 2001. 156

Appl. Mi robiol. Biote hnol.,

[312℄ J. B. S. Bonilha, R. M. Z. Georgetto, E. Abuin, E. Lissi, and F. Quina.

J.

Colloid Interfa e S i., 135:238, 1990. [313℄ P. Mukerjee, K. Mysels, and P. Kapauan.

J. Phys. Chem., 71:4166, 1967.

[314℄ W. D. Harkins, R. W. Mattoon, and R. Mittelman.

J. Chem. Phys.,

15:763,

1947.

[315℄ A. E. Alexander.

Trans. Faraday So ., 38:54, 1942.

[316℄ P. F. Grieger and C. A. Krauss.

[317℄ K. Shinoda.

J. Am. Chem. So ., 70:3803, 1948.

J. Phys. Chem., 58:1136, 1954.

[318℄ H. Nakayama, K. Shinoda, and E. Hut hinson.

[319℄ S. Kaneshina, H. Kamaya, and I. Ueda.

J. Phys. Chem., 70:3502, 1966.

J. Colloid Interfa e S i., 83:589, 1981.

[320℄ T. Okubo, K. Hiraiwa, S. Kinoshita, and M. Watanabe.

Alternatives to Animal

Testing and Experimentation, 1:2, 1990. [321℄ M. C. S aife.

International Journal of Cosmeti S ien e, 4:179, 1982.

[322℄ J. Selling and B. Ekwall.

Xenobioti a, 15:713, 1985.

[323℄ A. Trivedi, N. Kitabake, and E. Doi.

Agri ultural and Biologi al Chemistry,

54:2961, 1990.

[324℄ K. Chiba, I. Makino, J. Ohu hi, Y. Kasai, H. Kakishima, K. Tsukumo, T. U hiyama, E. Miyai, J. Akiyama, Y. Okamoto, H. Kojima, H. Okumura, Y. Tsurumi, M. Usami, K. Katoh, S. Sugiura, A. Kurishita, M. Sunou hi, A. Miyajima, M. Hayashi, and Y. Ohno.

[325℄ W. Yang and D. A osta.

[326℄ O. P. Flint.

Toxi ology in Vitro, 13:189, 1999.

Toxi ol. Lett., 70:309, 1994.

Altern. Test. Lab. Anim., 18:11, 1990.

[327℄ M. Cornelis, Ch. Dupont, and J. Wepierre.

Toxi . in Vitro, 6:119, 1992.

[328℄ R. L. Grant, C. Yao, D. Gabaldon, and D. A osta. 157

Toxi ology, 76:153, 1992.

[329℄ H. Kojima, J. Ohu hi, Y. Kasai, J. Okada, K. Tsukumo, H. Kakishima, E. Miyai,

J. Akiyama,

Y. Okamoto,

M. Kotani, K. Inoue,

M. Hibata,

H. Okumura, M. Arashima, T. Atsumi, I. Makino, K. Chiba, Y. Ohno, and A. Takanaka.

Alternatives to Animal Testing and Experimentation,

3:191,

1995.

[330℄ T. Ohno, Y.and Kaneko, T. Kobayashi, T. Inoue, Y. Kuroiwa, T. Yoshida, J. Momma, M. Hayashi, J. Akiyama, T. Atsumi, K. Chiba, T. Endo, A. Fujii, H. Kakishima, H. Kojima, Y. Masamoto, M. Masuda, K. Matsukawa, K. Ohkoshi, J. Okada, K. Sakamoto, K. Takano, T. Suzuki, and A. Takanaka.

Alternatives to Animal Testing and Experimentation, 3:123, 1995. [331℄ E. Owen, J. Cliord, and A. Marson.

J. Cell S i., 32:363, 1978.

[332℄ J. F. Sina, G. J. Ward, M. A. Laszek, and P. D. Gautheron.

Fundam. Appl.

Toxi ol., 18:515, 1992. [333℄ J.-H. S. Kuo, M.-S. Jan, C.-H. Chang, H.-W. Chiu, and C.-T. Li.

Colloids

Surf., B, 41:189, 2005. [334℄ S. Mahiuddin, A. Renon ourt, P. Bauduin, D. Touraud, and W. Kunz.

Lang-

muir, 21:5259, 2005. [335℄ N. Vla hy, M. Dre hsler, J.-M. Verbavatz, D. Touraud, and W. Kunz.

J.

Colloid Interfa e S i., 319:542, 2008. [336℄ D. P. Cistola, J. A. Hamilton, D. Ja kson, and D. M. Small.

Bio hemistry,

27:1881, 1988.

[337℄ K. Fontell and L. Mandell.

Colloid Polym. S i., 271:974, 1993.

[338℄ M. Gebi ki, J. M.and Hi ks.

Nature, 243:232, 1973.

[339℄ C. L. Apel, D. W. Deamer, and M. N. Mautner.

Bio himi a et Biophysi a

A ta, 1559:1, 2002. [340℄ K. Morigaki and P. Walde.

Curr. Opin. Colloid Interfa e S i., 12:75, 2007. 158

[341℄ M. Bergsma, M. L. Fielden, and J. B. F. N. Engberts.

J. Colloid Interfa e

S i., 243:491, 2001. [342℄ M. Johnsson, A. Wagenaar, M. C. A. Stuart, and J. B. F. N. Engberts.

Lang-

muir, 19:4609, 2003. [343℄ S. Fran es hi, A. Moisand.

V. Andreu,

N. de Viguerie,

M. Riviere,

A. Lattes,

and

New J. Chem., 22:225, 1998.

[344℄ Z. Yuan, J. Hao, and H. Homann.

J. Colloid Interfa e S i., 302:673, 2006.

[345℄ A. de Groot, R. W. and; Wagenaar, A. Sein, and J. B. F. N. Engberts.

Re l.

Trav. Chim. Pays-Bas, 114:371, 1995. [346℄ A.

Renon ourt,

P.

Bauduin,

E.

Ni holl,

M. Dubois, M. Dre hsler, and W. Kunz.

[347℄ T. Mosmann.

[348℄ Y. Talmon.

D.

Touraud,

J.M.

Verbavatz,

ChemPhysChem, 7:1892, 2006.

J. Immunol. Methods, 65:55, 1983.

Modern Chara terization Methods of Surfa tant Systems,

page

147. Mar el Dekker: New York, 1999.

[349℄ R. Smith and C. Tanford.

[350℄ T. H. Haines.

Pro . Natl. A ad. S i., 70:289, 1973.

Pro . Natl. A ad. S i., 80:160, 1983.

[351℄ H.-T. Jung, S. Y. Lee, E. W. Kaler, and J. A. Zasadzinski.

Pro . Natl. A ad.

S i., 99:15318., 2002. [352℄ T. M. Weiss, T. Narayanan, C. Wolf, M. Gradzielski, P. Panine, S. Finet, and W. I. Helsby.

Phys. Rev. Lett., 94:38303, 2005.

[353℄ H. Hoekes, K. Nelles, and B. Bergmann, 2000. DE 19835327 A1 200002210.

[354℄ H. Hoekes, M. Seiler, W. Gross, G. Knuebel, D. Oberkobus h, S. Kainz, and A. Rei hert, 2007. DE 102005055340 A1 20070524.

[355℄ S. Kainz, H. Hoekes, M. Krippahl, and C. Kolonko, 2006. WO 2006097167 A1 20060921. 159

[356℄ A. Kleen, M. Akram, S. Hoepfner, and H. Manne k, 2005. WO 2005058260 A1 20050630.

[357℄ A. Kleen and B. Frauendorf, 2007. EP 1762220 A2 20070314.

[358℄ Y. Barenholz.

Curr. Opin. Colloid Interfa e S i., 6:66, 2001.

[359℄ J. Dong, D. R. Whit omb, A. V. M Cormi k, and H. T. Davis. 23:7963, 2007.

160

Langmuir,

A knowledgements From experien e I an tell you that these last pages of a PhD thesis are the most widely read pages of the entire publi ation. It is here where you supposedly nd out to what extent you have inuen ed the life and work of the author. While this may be true to some extent, you have to weigh my verdi t with the disturbingly low level of sanity left in this PhD andidate after several years of studying toxi mole ules, redening the olor blue and sear hing for `berries' in aqueous surfa tant solutions. The work des ribed in this thesis has been arried out under the guidan e of Prof. Dr. Werner Kunz at the Institute for Physi al and Theoreti al Chemistry, University of Regensburg.

I would like to thank him for the trust he showed by letting me

work independently and the opportunity to ollaborate with a number of other groups around the world.

Furthermore, thanks to all of his various personalities

(the arabi one I am espe ially fond of ) for keeping us amused during lun hes and long onferen e rides. Part of the experimental work was performed at the Department of Pharma euti al Biology, University of Regensburg. Thank you to Prof. Dr. Jörg Heilmann, Dr. Birgit Kraus, and Gabi Brunner for their patien e and trust they showed by letting this near-theoreti ian work in their sterile environment. Spe ial thanks to Dr. Didier Touraud for de orating our lab in pink olor and making ammonia the ommonly used air freshener. Furthermore, Didier, thank you for your en ouragement through the years; for knowing when to push me further and when to ba k o and, espe ially, for always taking the time to dis uss new (mostly

razy) ideas. The good basi s were developed already during my undergraduate studies. For that, I am thankful to Prof. Dr. Ksenija Kogej, for tea hing me to pay attention 161

to detail and work in an organized fashion. An indispensable skill that was proven ne essary to survive in the sometimes he ti half-Fren h institute. No s ienti work is done in solitude. Three mi ros opists are responsible for my newly found appre iation for (abstra t) art: Dr. Jean-Mar Verbavatz (CEA, Sa lay), Dr. Mar us Dre hsler (University of Bayreuth), and Prof. Dr. Ishi Talmon (Te hnion-Israel Institute of Te hnology). Thank you for your wonderful pi tures and informative dis ussions. I thank my oworkers that transformed the `supposed-to-be' serious work environment into a fun pla e (although it sometimes resembled more a loony-bin): Alina for showing me the ropes from the rst day on, Chloé for her ability to turn ba k time and make me feel like a kid whenever I'm in her ompany, Geli for the smile I was greeted with every morning, Jeremy for his onversations on and o the OrbiTrek. To my traveling ompanions:

Moj a & Neza.

After the adventures we had,

oming ba k to work seemed like va ation. I am looking forward to all the ities that still await us. Thank you to Jorge Cham, the reator of PhD omi s, for helping me ght insanity with irony. To Daniel: Thank you for reading my thesis, listening to my endless omplaining, helping me pi k out my interview suit, wiping the tears away when the reviewers (wrongfully, of ourse!) reje ted my manus ripts and nursing me after my eye operation. You are my endless sour e of motivation and I am lu ky to have you to open my eyes (despite my newly-found 150% vision) when needed. To my father: thank you for en ouraging me on all my paths. I am still waiting for that Vla hy & Vla hy arti le however

...

162

List of Publi ations Published: 1-N. Vla hy, J. Dolen , B. Jerman, K. Kogej, Inuen e of Stereoregularity of the Polymer Chain on Intera tions with Surfa tants: Binding of Cetylpyridinium Chloride by Isota ti and Ata ti Poly(metha ryli a id).

J. Phys. Chem. B 2006 110,

9061-9071. 2-A. Renon ourt, N. Vla hy, P. Bauduin, M. Dre hsler, D. Touraud, J.-M. Verbavatz, M. Dubois, W. Kunz, B. W. Ninham, Spe i alkali ation ee ts in the transition from mi elles to vesi les through salt addition.

Langmuir 2007 23, 2376-

2381. 3-N. Vla hy, D. Touraud, K. Kogej, W. Kunz, Solubilization of metha ryli a id based polymers by surfa tants in a idi solutions.

J. Colloid Interf. S i. 2007 315,

445-455. 4-N. Vla hy, M. Dre hsler, J.-M. Verbavatz, D. Touraud, W. Kunz, Role of the surfa tant headgroup on the ounterion spe i ity in the mi elle-to-vesi le transition through salt addition.

J. Colloid Interf. S i. 2008 319, 542-548.

5-N. Vla hy, A. Renon ourt, M. Dre hsler, J.-M. Verbavatz, D. Touraud, W. Kunz, Blastulae aggregates: New intermediate stru tures in the mi elle-to-vesi le transition of atanioni systems.

J. Colloid Interf. S i. 2008 320, 360-363.

A

epted: 6-N. Vla hy, A. F. Arteaga, A. Klaus, D. Touraud, M. Dre hsler, W. Kunz, Inuen e of additives and ation hain length on the kineti stability of supersaturated

atanioni systems.

Colloids Surf., A 2008.

7-N. Vla hy, C. Merle, D. Touraud, J. S hmidt, Y. Talmon, J. Heilmann, W. Kunz, 163

Determining the delayed ytotoxi ity of atanioni surfa tant mixtures on HeLa

ells.

Langmuir 2008.

Submitted: 8-N. Vla hy, M. Dre hsler, D. Touraud, W. Kunz, Anion spe i ity inuen ing morphology in atanioni surfa tant mixtures with an ex ess of ationi surfa tant.

Comptes rendus Chimie A adémie des s ien es 2008. 9-N. Vla hy, B. Jagoda-Cwiklik, R. Vá ha, D. Touraud, P. Jungwirth, and W. Kunz, Hofmeister series of headgroups and spe i intera tion of harged headgroups with ions.

J. Phys. Chem. B 2008.

10-N. Vla hy, D. Touraud, J. Heilmann, W. Kunz, Determining the delayed ytotoxi ity of atanioni surfa tant mixtures on HeLa ells.

164

Colloids Surf., B 2008.

List of Oral and Poster Presentations Oral Presentations: th Conferen e of the European Colloid and Interfa e So iety, Genève, 09/2007 21 Switzerland.

Blastula vesi les:

Formation of regular patterns through se ondary

self-assembly of atanioni vesi les.

Poster Presentations: 08/2005 29. International Conferen e of Solution Chemistry, Portoroz, Slovenia. Mixed Solutions of Isota ti Poly(metha ryli a id) and Cetylpyridinium Chloride in water.

08/2006

1st European Chemistry Congress, Budapest, Hungary.

Solubilization of Hydrophobi Polymers with Surfa tants.

09/2007

21th Conferen e of the European Colloid and Interfa e So iety, Genève,

Switzerland. Ee t of hydrophobi  ations on vesi ular self-assembly.

11/2007

Formula V, Potsdam, Germany.

Inuen e of additives on the stability of (eute ti ) supersaturated atanioni systems.

165

Herewith I de lare that I have made this existing work single-handed. I have only used the stated utilities.

Regensburg, June 2008

Nina Vla hy

166