Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 304

On Phase Behaviours inLipid/Polymer/Solvent/Water

Systems and their Application forFormation of Lipid/Polymer

Composite Particles

BY

ANNA IMBERG

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2003

Dissertation presented at Uppsala University to be publicly examined in B21, Uppsala Biomedical Centre, Uppsala, Tuesday, December 16, 2003 at 10:15 for the degree of

Doctor of Philosophy (Faculty of Pharmacy).

ABSTRACT

Imberg, A., 2003. On Phase Behaviours in Lipid/Polymer/Solvent/Water Systems and their Application for Formation of Lipid/Polymer Composite Particles. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 304. 58 pp. Uppsala. ISBN 91-554-5816-5.

A new kind of lipid/polymer composite particle, consisting of a biodegradable polymer matrix with well-defined lipid domains, has been created. The lipid used is the water-swelling lipid monoolein (MO),which forms a reversed bicontinuous cubic diamond structure in aqueous solutions. The polymer ispoly(d,l-lactide-co-glycolide) (PLG), which degrades into water-soluble monomers through hydrolysis.This new particle might be interesting for controlled release applications.

To prepare such particles can be difficult. Suitable phase behaviour and a solvent with the rightproperties are needed. For this reason, the phase behaviours of several differentlipid/polymer/solvent/water systems have been explored. From the phase behaviour of a suitable system(i.e. MO/PLG/ethyl acetate/water), a route for formation of lipid/polymer composite particles has beendeduced. Particles have been formed and distinct, water-swelling, lipid domains have been confirmed bycharacterization by means of confocal laser scanning probe microscopy (CLSM).

The sample preparation process has been automated and a method based on using a robotic liquidhandler has been developed. Phase diagrams have been determined by examination of macroscopicbehaviours and the microstructures of the phases have been studied by small- and wide-angle X-rayscattering (L3, V2, L , L), nuclear magnetic resonance self-diffusion (L, L3), viscosimetry (L) andrheology (L). Several different theoretical models have been applied for interpretation of the results. Forexample, the swelling of the reversed bicontinuous cubic phases and the sponge phase have beenmodelled by applying the theory of infinite periodical minimal surfaces, the sponge phase has been shownto be bicontinuous according to the theory of interconnected rods and the phase behaviour of the polymerhas been described by the Flory-Huggins theory. The main focus of this work (4/5) concerns phasestudies in multicomponent systems from a physical-chemical point of view.

Keywords: Biodegradable, CLSM, Composite Particles, Controlled Release, Cubic Phases, Drug Delivery, FT-PGSE-NMR, Liquid phase, Lipid domains, Liquid-handler, Microspheres, Microstructure,Phase behaviour, Polymer matrix, SAXS, Segregation, Sponge Phase, Swelling

Anna Imberg, Department of Pharmacy, Box 580, Uppsala University, SE-751 23 Uppsala, Sweden

© Anna Imberg 2003

ISSN 0282-7484 ISBN 91-554-5816-5 Printed in Sweden by Universitetstryckeriet, Uppsala 2003

To my family

LIST OF PAPERS

This thesis is based on the following papers, which will be referred to by their Roman numerals in thesummary. A. Imberg´s former name was A. K. Johansson.

I. Phase Behaviour of the Quaternary Poly(d,l-lactide-co-glycolide)/Monoolein/Water System: - An Experimental and Theoretical Study Anna K. Johansson, Per Linse, Lennart Piculell, Sven Engström, J. Phys. Chem. B. 2001, 105,12157-12164.

II. On the Self-Assembly of Monoolein in Mixtures of Water and a Polar Aprotic SolventAnna Imberg, Hans Evertsson, Peter Stilbs, Manfred Kriechbaum, Sven Engström, J. Phys. Chem. B. 2003, 107, 2311-2318.

III. An Increased Throughput Method for Determination of Phase Diagrams – Method Development and Validation Anna Imberg and Sven Engström, Colloids and Surfaces A: Physicochem. Eng. Aspects, 2003, 221, 109-117.

IV. Segregation of Lipid and Polymer in Emulsion Droplets Captured by Confocal Laser Scanning Probe Microscopy Anna Imberg and Per Hansson, Submitted 2003.

V. Microstructures of Ethyl Acetate/Monoolein/Water Mixtures and Related SystemsAnna Imberg and Per Hansson, Submitted 2003.

The papers are included in the thesis with permissions from the journals.

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ABBREVIATIONS

AN acetonitrileCLSM confocal laser scanning probe microscopyCMC critical micelle concentrationCR controlled releaseDHPE 1,2-dihexadecanoyl-sn-glycero-3-

phosphoethanolamine (triethyl ammonium salt)DMSO dimethylsulphoxideDNA deoxyribonucleic acidDSC differential scanning calorimetryEtAc ethyl acetateFDA food and drug administrationFT-PGSE NMR fourier transform pulsed field gradient spin

echo nuclear magnetic resonance ICR interconnected rodsMO monooleinNMP 1-methyl-2-pyrrolidinonePEG polyethylene glycolPET polyethylenePG poly(d,l-lactide)PLG poly(d,l-lactide-co-glycolide)PVC polyvinyl chlorideRNA ribonucleic acidSAXS small angle X-ray scattering SEM scanning electron microscopyVBA visual basic for applications WAXS wide angle X-ray scattering

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PREFACE

This thesis is divided into two parts. The first part is an introduction to the papers and the second partconsists of the scientific papers, of which all have been, or is about to be, published in physico-chemicaljournals.

The central issues of this work concern phase behaviours of multi-component lipid/polymer/solvent/watersystems and their applications for formation of lipid/polymer composite particles. In order to formlipid/polymer composite particles, one main question has to be considered: namely, the phase separationprocess between the lipid and the polymer.

In the first part of this summary, I will, for simplicity, present the lipid phase behaviour and the polymerphase behaviour separately. Then, we are ready to proceed and simultaneously study the lipid and thepolymer behaviour. It will be demonstrated that the choice of solvent is crucial for formation oflipid/polymer composite particles. In the second part of the summary the formation and characterizationof such lipid/polymer composite particles are discussed.

This work hopefully brings new insights into phase behaviours of multi-component systems as well as possible composite particle drug delivery systems. As far as I know, this is the first time lipid andpolymers, of the types described here, have been combined in phase studies. Moreover, the approach forformation of composite particles gives new insights into how drug delivery systems can be designed onthe basis of appropriate phase behaviours.

Anna Imberg Uppsala, November 12th, 2003

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CONTENTS

1 INTRODUCTION ............................................................................................................... 91.1 Purpose of the Study................................................................................................... 91.2 Background................................................................................................................. 91.3 Polymer Architecture................................................................................................ 101.4 Lipid Classification................................................................................................... 111.5 Amphiphilic Molecules ............................................................................................ 111.6 Self-Assembly .......................................................................................................... 11

2 PHASE EQUILIBRIA ....................................................................................................... 132.1 Phase Diagrams ........................................................................................................ 132.2 Gibbs Phase Rule...................................................................................................... 132.3 Working Strategy and Sample Preparation............................................................... 14

3 PHASE BEHAVIOURS OF LIPIDS................................................................................. 153.1 The Origin of Lipid Self-assembly ........................................................................... 153.2 Self-assembly into Liquid (Crystalline) Phases........................................................ 153.3 The Lipid/Water Interface ........................................................................................ 173.4 Theoretical Considerations of Phases ....................................................................... 17

3.4.1 Structure and Swelling of Phases......................................................................... 183.4.2 Dynamics ............................................................................................................. 223.4.3 Interplay Between Structure and Dynamics......................................................... 23

3.5 Phase Behaviours of Lipid/Solvent(s)/Water Systems ............................................. 233.6 Protein Encapsulation ............................................................................................... 27

4 PHASE BEHAVIOURS OF POLYMERS........................................................................ 284.1 Polymers in Solution................................................................................................. 28

4.1.1 Concentration Regimes of Polymer Solutions ..................................................... 284.1.2 Good and Bad Solvents........................................................................................ 29

4.2 The Regular Solution Theory ................................................................................... 294.3 The Flory-Huggins Theory ....................................................................................... 29

4.3.1 Mixing a Polymer and a Solvent.......................................................................... 304.3.2 Mixing Two Polymers ......................................................................................... 304.3.3 The Main Equations ............................................................................................. 30

4.4 Polymer Phase Behaviours in Theory and in Practice .............................................. 324.4.1 Polymer 1/Solvent................................................................................................ 334.4.2 Polymer 1/Non-Solvent/Solvent .......................................................................... 334.4.3 Polymer 1/”Polymer 2”/Solvent........................................................................... 334.4.4 “Polymer 2”/Non-Solvent/Solvent....................................................................... 334.4.5 Polymer 1/”Polymer 2”/Non-Solvent/Solvent ..................................................... 33

7

5 LIPID/POLYMER COMPOSITES ................................................................................... 355.1 Phase Behaviours of the Lipid/Polymer/Solvent(s)/Water Systems......................... 355.2 Lipid/Polymer Composite Particles .......................................................................... 37

5.2.1 The Route to Lipid/Polymer Composite Particles................................................ 375.2.2 Interpretation of the Particle Formation Process.................................................. 385.2.3 Characterisation of Lipid/Polymer Composite Particles ...................................... 385.2.4 Interplay Kinetics-Properties ............................................................................... 395.2.5 Phase Separation .................................................................................................. 40

5.3 Simultaneous use of Lipid and Polymer ................................................................... 40

6 CONTROLLED DRUG DELIVERY................................................................................ 426.1 Controlled Release Technology................................................................................ 426.2 Properties of Polymer Matrices ................................................................................ 436.3 Factors that Affect the Release Rate......................................................................... 43

7 HIGHLIGHTS OF THE PAPERS..................................................................................... 45

8 CONCLUDING REMARKS............................................................................................. 46

9 EXPERIMENTAL TECHNIQUES................................................................................... 47

10 SUMMARY IN SWEDISH............................................................................................... 48

11 REFERENCES .................................................................................................................. 50

12 ACKNOWLEDGEMENTS............................................................................................... 57

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1 INTRODUCTION

1.1 Purpose of the Study

The main purpose of this work has been to investigate the phase behaviours of multi-component,pharmaceutically interesting systems containing lipid/polymer/solvent/water and to investigate thepossibilities to form lipid/polymer composite particles for sustained or controlled release applications.

The main focus, i.e. 4/5 of the work, has been on phase studies. In the final year of the project some time has been dedicated to the deduction of an appropriate route for formation of the above-mentionedlipid/polymer composite particles.

1.2 Background

As the human genome now is surveyed, the interest in different protein-based pharmaceuticalpreparations has increased. Therapeutic proteins and peptides are now becoming available in hugequantities. Since proteins are sensitive molecules that easily are inactivated under adverse conditions, theyneed to be packaged in appropriate drug delivery systems. Today there is therefore a great need for newdrug delivery techniques/systems. One kind of such a relatively new way to deliver drugs is the, so-called,controlled or sustained drug delivery technology, which is concerned with the systematic release of pharmaceutical agents at appropriate therapeutic levels for prolonged periods of time (Ottenbrite, 1990).

The molecules of the systems studied here (i.e. polymers, lipids, solvents and water) are found in, forexample, plastic bags, cell membranes and nail-colour-removers. The main components of the study, thelipid and the polymer, deserve a general introduction.

Polymers are present practically everywhere: in nature (e.g. rubber), in vivo (e.g. DNA, RNA, proteins),in plastics (e.g. PVC, PET, etc). They are really fascinating substances.

For the pharmaceutical industry, polymers constitute an important group of substances with attractive properties. Examples of polymer-based pharmaceutically related applications are: gene delivery systems(Huang, 2003), controlled release applications using thermo-responsive polymers (Eckman, 2002) andswelling controlled drug release applications (Gupta, 2002).

Lipids normally perform many essential functions and are common both in plants and in animals. In animals and humans the lipids are for example used as sources of energy. Anyway, for this thesis thatmakes use of polar lipids, maybe the most important property is that they are insoluble in water. Theyform, together with water, different types of structures (which soon will be more thoroughly described)with separate compartments for oily and water-like domains. The cell membranes in the human skin arefor example built of lipids, just to mention a quite easily grasped example. In human skin, and in fact in cell membranes in general, the lipids form structure elements that function as a ”barrier” (Sparr, 2001) that regulates transport between in- and outside. Therefore lipids are essential for proper functioning ofbiological systems.

Lipids can be natural or synthesized and are regularly used by the food and pharmaceutical industries.Examples of lipid-based pharmaceutically related carrier systems are: nanoemulsions, nanosuspensions,

9

mixed micelles (Baskaran, 2003), solid lipid nanoparticles (Jores, 2003), liposomes (Busquets, 2003) andcubosomes, i.e. dispersed cubic phase, (Larsson, 1989). Such colloidal carriers may for example offerbetter bioavailability for poorly water-soluble drugs. In addition, several of these carrier systems displayprolonged release characteristics. The internal structure of the fascinating sub-micron particles of thecubic phase, i.e. cubosomes, was confirmed by cryo-TEM (Gustafsson, 1996, 1997). The cubosomes have been used for drug delivery applications (Turchiello, 2003; Spicer, 2002; Sallam, 2002).

Now, as the two most important groups of substances have been briefly introduced, the next step is topresent the actual subject. Belonging under physical chemistry, surface and colloidal chemistry has beendescribed as the research area, where the length-scale of studied behaviours belongs to the colloidaldomain, which concerns physicians, chemists and biologists (Evans, 1999). Within surface and colloidalchemistry, scientifically and technically interesting systems and their phase behaviours are often studied.If the studied substances have pharmaceutical relevance the subject is normally referred to aspharmaceutical physical chemistry. Generally, components of “pharmaceutical relevance” are also interesting for food applications (Larsson, 1994).

This work is comprised, as the title suggests, of two related parts. The first part, which is covered bypapers I, II, III and V, is on the topic of phase studies in multi-component lipid/polymer/solvent/watersystems with the focus on physical-chemical characterisation and interpretation of ternary phasediagrams. The second part, discussed in papers I, IV, covers the formation and the characterization of lipid/polymer particles.

To, in a good way, deliver active substances to a patient is a challenging task. For the active substance to have the intended effect and to be released in a favourable way, a suitable packing system is essential. Inmany cases it is desirable to release the active substance slowly over a long period of time, which is theobject of controlled/retained/sustained release formulations (LaVan, 2003). In such formulations, polymermatrices (Kumar, 2002; Langer, 1991; Shea, 1999; Edelman, 1996) and lipid-based systems are regularlyused. Lipids that can form ordered liquid crystalline phases have been used in formulations for bothcontrolled and sustained release (Ye, 2000).

1.3 Polymer Architecture

Polymers consist of covalently bound repetitive units, so-called monomers. The polymer chains are either linear or branched. When the chains consist of monomers of one type the polymer is a homopolymerotherwise it is a copolymer. In the latter case the polymers are classified as random, block or graftcopolymers depending on how the monomers are ordered within the chains. If the polymer containscharges the polymer is said to be a polyelectrolyte.

The uncharged, biodegradable polymer used in this thesis is Poly(d,l-lactide-co-glycolide), PLG. Inaqueous solutions, PLG degrades into water-soluble lactide- and glycolide units, which in vivo, by actionof the citric acid cycle, are further degraded into carbon dioxide and water. A polymer consisting ofPoly(d,l-lactide-co-glycolide) with segments of polyethylene glycol, PEG-PLG, has also been used (paperIV). The advantage of using a copolymer instead of pure lactide or glycolide polymers is that the degreeof crystallinity is lower (Reed, 1981). Interestingly, several different types of PLGs are approved by theFDA for use in clinical studies.

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1.4 Lipid Classification

In 1987 Christie proposed the following definition of a lipid (Christie, 1987):

“Lipids are fatty acids and their derivates, and substances related biosynthetically or functionally to thesecompounds.”

Lipids can be further divided into subclasses according to their constituents. However, the most attractivedefinition seems to be according to their functionality and properties. As proposed by Small, lipids can beclassified as polar or non-polar depending on their surface and bulk properties (Small, 1986). Non-polarlipids are of no concern to this work. The polar monoglyceride, monoolein (MO) is, according to Small´sscheme, classified as a polar, water-insoluble, but swelling lipid (Polar Class II). At a gas/liquid interface,it spreads to form a stable monolayer and in aqueous solutions it forms liquid crystals.

At temperatures above the chain-melting temperature a polar lipid is one example of an amphiphilicmolecule.

1.5 Amphiphilic Molecules

By definition, surfactants or amphiphilic molecules contain both a polar hydrophilic, (water-liking) and a non-polar hydrophobic, (water-disliking) part. The former is soluble in water and the latter in organicsolvents. Therefore, amphiphilic molecules are often present at interfaces (e.g. 2-dimensional selfassembly) where they lower the surface energy. For reasons that will be explained, amphiphilic moleculesalso self-assemble when they are completely surrounded by water, (i.e. 3-dimensional self assembly). For amphiphilic molecules to self-assemble, a hydrocarbon chain length of at least 10-12 carbon atoms isnormally required.

If two different types of amphiphilic molecules have hydrocarbon chains of approximately equal length,they can mix, but if the difference in chain length is 4 carbon atoms or more, they tend to segregate, i.e.phase separate, (Engblom, 1996).

1.6 Self-Assembly

Amphiphilic molecules position themselves in such a way that the free energy of the whole system isminimized. Formation of a wide variety of different structures can minimize the free energy depending onthe properties of the amphiphilic molecule and on the volume fractions of polar and non-polar solvent,(normally water and oil).

As mentioned earlier, all amphiphilic molecules contain both hydrophobic and hydrophilic parts. Hence,in contact with a polar or a non-polar solvent, the most favourable way to organize the molecules is toform structures where the hydrophilic- and hydrophobic parts are well separated in different domains.

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Examples of such structures are micellar, hexagonal, lamellar, cubic and sponge phases, which areexamples of lyotropic, i.e. solvent-induced, liquid (crystalline) phases. Most of these phases have beenstudied in papers I-V. The structures of these phases are built of bilayers. The only exception is themicellar phase, which is built of monolayers. Phases can be both normal and reversed. In the former case the interface is curved towards oil and in the latter case the interface is curved towards water. The type ofphase formed depends both on global parameters, such as water to oil ratio of the mixture, and on morespecific properties of the amphiphilic molecule (i.e. spontaneous curvature).

At determination of microstructures of phases there are two concepts that are useful: curvature andpacking. The former will be discussed in section 3 and the latter is discussed here. The critical packingparameter (Israelachvili, 1991) is given by Equation 1:

cla

VCpp

0

(1)

where V denotes the volume of the hydrocarbon chain of the surfactant, a0 is the optimal area of the polarhead group and lc denotes the hydrocarbon chain length. Normal phases (curved towards oil) have Cpp <1. Planar phases have Cpp = 1 and reversed phases have Cpp > 1. The packing parameters of differentphases as well as the morphologies of the phases are presented in Figure 2. During the work with papersII and V the microstructures of the different phases of the phase diagrams were determined and thecritical packing parameters and the curvatures of the interfacial surfaces served as central and usefulconcepts.

For instance, the effect of adding EtAc to binary MO/water is intermediate between two curvature-reducing additives: DO and azone and the curvature-enhancing polar solvents. The additives discussed inpaper V can be arranged in order of increasing contribution to spontaneous curvature of MO monolayers:

DO < azone < EtAc < DMSO < NMP < AN.

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2 PHASE EQUILIBRIA

2.1 Phase Diagrams

Phase diagrams describe the behaviour of systems at thermodynamic equilibrium. At thermodynamicequilibrium a system is at its lowest state of free energy. The free energy (G) depends on energy (H) andentropy (S) according to Equation 2:

STHG (2)

where T denotes the temperature.

The total free energy of the system is defined as the sum of the free energies of all the different components of the system.

To, in practice, decide if thermodynamic equilibrium is reached often requires some common sense. Inthe present work, the phases were assumed to be in thermodynamic equilibrium when the phase behaviours were constant. To distinguish kinetic stability from thermodynamic stability, the glasstransition temperatures, Tg, of the more concentrated polymer solutions were measured. In the PLG/water/solvent system, where PLG is degraded by hydrolysis, the phase behaviour was determined atan early stage. The solvation of the polymer was speeded-up by means of vortexing. In systems withvolatile solvents, the structures and number of phases were determined as soon as possible after samplepreparation.

2.2 Gibbs Phase Rule

Gibbs phase rule (Evans, 1999), given in Equation 3, provides a way to determine the maximum numberof coexisting phases at a given pressure and temperature.

2CPF (3)

F denotes the number of degrees of freedom, P denotes the number of phases and C is the number ofcomponents. The number of degrees of freedom is the number of independent intensive state variablesleft when all possible constraints have been taken into account (Evans, 1999). Extensive variables areproportional to the size of the system and intensive variables are independent of the size of the system.One- and multi-phase regions in phase diagrams are always located in such a way that Gibbs phase rule is fulfilled.

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2.3 Working Strategy and Sample Preparation

To chart complex systems with many different one-phase and multi-phase regions, many samples have tobe prepared and analysed. To prepare samples manually is, however, very time-consuming.

Paper III presents a method for automated preparation of samples; a method that makes use of a Gilsonrobotic liquid handler. Two major advantages obtainable by using a robotic liquid handler are shortpreparation times and small sample volumes. The method has been further used in paper IV and V.

The method of paper III is validated for preparation of samples containing lipid, solvent and water. Sincelipids normally are too viscous to be dispensed at room temperature, a special method had to be developed. The method is based on the idea of dissolving the lipid in a volatile solvent and therebyreducing the viscosity. The method was validated by dispensing an MO/EtAc (40/60 w/w) solution atroom temperature. By using an easily evaporated solvent, such as EtAc, there is no problem to, after thedispensing, reduce the solvent content to below 0.5 wt %. This small residue does not seem to affect the phase behaviour to any significant extent, which is shown by the good agreement with previouslyreported phase behaviours for, for example, the binary MO/water system. To determine if residues ofsolvent affect the lipid crystal structure, pure MO and MO that previously had been dissolved in EtAcwere X-rayed. Data from the wide-angle X-ray scattering analysis, shown in Figure 1 (unpublished data),show that the type of crystal structure is unaffected of the procedure used for sample preparation by therobotic liquid handler.

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Figure 1. SAXS and WAXS spectra of two MO samples. Lipid 1 is MO, which has been dissolved in EtAcand dispensed by the robotic liquid handler and Lipid 2 is pure MO (not dispensed by the robotic liquidhandler).

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3 PHASE BEHAVIOURS OF LIPIDS

Amphiphilic molecules can be in different states. In dilute systems the amphiphilic molecules are eithermolecularly dissolved (i.e. free monomers) or form micelles. In concentrated systems ordered phases areinstead formed. In this section, the theory for lipid self-assembly is presented and examples from thepapers are discussed.

3.1 The Origin of Lipid Self-assembly

To be able to understand why amphiphilic molecules self-assemble in aqueous solutions, the structure of water must be considered. For instance, when apolar molecules are added in water induces local orderingof the water molecules. Thus, water tends, for entropic reason, to induce ordering of the hydrocarbons.This is the reason for amphilic self-assembly in aqueous solution and it is also called the hydrophobiceffect (Tanford, 1973).

It is possible to induce and affect self-assembly in different ways. By increasing the concentration or thetemperature, phase transitions can be induced since the interactions between, for example, singlemonomers or lipid aggregates increase. Let us start by discussing the effect of changing the amphiphileconcentration when considering the transition from free amphiphile molecules to micelles. At lowconcentrations of the amphiphile, it can be molecularly dissolved. In this state, both the hydrophilic andhydrophobic part of the molecule is in contact with water. Hence some unfavourable interactions are present but the entropy of mixing is high. By increasing the concentration, the critical micelleconcentration, CMC, is reached and micelles start to form. The entropy of mixing is lower in the micellarstate compared to in the dissolved state. However, by forming micelles, less hydrophobic domains get incontact with water, i.e. the disturbance of favourable water-to-water interactions is reduced and that is thereason for why micellisation takes place. The aggregation is counteracted by head group repulsion(Lindman, 1980). Micelles can, for instance, be spherical, cylindrical, oblate-shaped or thread-like,depending on the preferred curvature. The latter can form reversible self-intersections and hence a continuous “living” network may form with interesting diffusion and rheological properties (Ambrosone,2001).

3.2 Self-assembly into Liquid (Crystalline) Phases

Repulsive and attractive intra- and inter-micellar interactions determine if and how micellar solutionstransform into ordered phases. The kind of phase formed depends on the relative volumes of amphiphileand water and on the preferred molecular shape of the amphiphilic molecule, i.e. the spontaneouscurvature (defined later).

In Figure 2 the effect of increasing either the amphiphile concentration or the temperature is given. Oneeffect of increased temperature is that the conformation of the hydrophobic tail is altered. For instance,for reversed phases more curved phases are favoured and for normal phases more planar phases arefavoured with increased temperature. This can be observed in, for example, the well-known MO/water

15

system, where L (10% water w/w) transforms to L2 at approximately 40°C and QG (25 % water w/w)transforms to HII at around 70˚C. Another effect is that the head groups change their conformation. Forinstance, the size of common non-ionic head group shrinks with increased temperature (Israelachvili1991). The packing parameters of the phases are given in Figure 2.

Figure 2. Preferred phases and their corresponding packing parameters (Jönsson, 1998). The picture isreproduced with permission from John Wiley & Sons.

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3.3 The Lipid/Water Interface

Common for all the aggregates formed in all phases is that they have well-defined interfacial surfacesbetween polar and apolar domains. Note, for example in Figure 2 above, how the packing parameters vary between different phases. Below some important parameters defining the curvature of the interfacialsurface are presented.

The mean curvature (H) of a semi-flexible elastic film is defined according to Equation 4:

)11

(2

1

21 rrH (4)

where and r are the two radii, characterizing the surface. By convention, is negative when thesurface is curved towards water, i.e. in reversed phases. The free energy per unit area of a film depends onhow much it is curved. In fact, a global minimum in the free energy exists and the correspondingcurvature is called the spontaneous curvature (H

1r 2 ir

0). Equation 5 defines the spontaneous curvature, whichis a specific property of each amphiphilic component:

0

0 2

1

RH (5)

where R0 is the optimal, i.e. preferred, radius of curvature of the monolayer film. The frustration energyof a film is proportional to (H-H0)

2. An amphiphilic film tends to bend as to minimize this energy. TheGaussian curvature, , is defined by Equation 6.

21

1

rr (6)

3.4 Theoretical Considerations of Phases

In this section theoretical models for evaluation of structure and dynamics of phases are presented. Theself-diffusion coefficients of the individual components are of course dependent upon the structure of thephase. Therefore, the relation between structure and dynamics is a useful tool when the microstructures ofphases are investigated.

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3.4.1 Structure and Swelling of Phases

The Lamellar Phase

The lamellar phase consists of planar bilayers separated by water. The ideal swelling of a lamellar phaseis given in Equation 7 (Evans, 1999).

bilayer

bilayerd (7)

where denotes the lattice parameter (repetitive distance), bilayer is the volume fraction bilayer and dbilayerdenotes the bilayer thickness. In papers I, II and V the relation has been applied while determining themicrostructure of the L - and the L-phase.

The Cubic Phase(s)

The first reversed lyotropic bicontinuous phases were reported a long time ago (Luzzati, 1966). Later, twodifferent cubic phases from the MO/water system, were reported (Larsson, 1983). The transformationbetween these has also been described (Hyde, 1984). At least three different kinds of reversedbicontinuous phases with the following space group symmetries: Ia3d, Pn3m and Im3m have beenobserved in the MO/water/third component systems.

The reversed bicontinuous cubic phases consists of a lipid bilayer that separates two separate water channel systems. These phases exhibit a fascinating three-dimensional periodicity and are thereforeattractive for many different applications such as, for instance, membrane protein crystallization(Sennoga, 2003; Caffrey, 2003), drug delivery (Ericsson, 1991; Ganem-Quintanar, 2000; Shah, 2001; Rummel 1998) and for a biosensor application (Razumas, 1994).

The structure of the bicontinuous cubic phases can be described approximately by infinite periodicminimal surfaces (IPMS) which by definition have zero mean curvature (Shriven, 1976) An IPMS for areversed phase approximates the location of the centre of the lipid domain. Similarly, an IPMS for a normal phase approximates the location of the centre of the water domain. Characteristic for IPMS is that every point on the surface is a saddle-point.

Three different examples of IPMS are the Gyroid surface, the Diamond surface and Schwartz´ P-surface.The different surfaces have different surface to volume ratios and that is one reason for why they arelocated in different regions in phase diagrams. The homogeneity index ratios are: H(G)/H(D)=1.02 andH(G)/H(P)=1.07 (Hyde, 1997). The thermodynamic behaviour of the phases has elegantly been describedelsewhere (Templer, 1998).

During swelling the G-surface can transform into the D-surface, which is the case in the well-characterized MO/water system (Hyde, 1984). The D-surface has been reported, to during protein-induced swelling transform into the P-surface (Ericsson, 1983). This finding is in agreement with thesolvent-induced transitions, e.g. G-D-P-L3, induced by NMP, reported in paper II, and further discussedin paper V. The reason for why the P-surface is not present in the binary MO/water system is due to two

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things; the swelling is not large enough. In addition the curvature is not suitable for building the P-surface, i.e. the frustration energy is too large. Addition of water does not change the spontaneouscurvature, but addition of NMP or proteins does, and hence the P surface can be induced. The reasonmight be that the surface energy probably changes when NMP or proteins are added, see paper V.Moreover, addition of NMP also affects the swelling, as demonstrated in paper II.

Figure 3. Schematic illustration of the G-, D- and P unit surfaces. The pictures are reproduced from (Andersson 1999) with permission from Elsevier.

The swelling behaviour of the reversed cubic bicontinuous phases is well described by the theory forinfinite periodic minimal surfaces, (Hyde 1997). The model has for instance been used by (Engblom,1995). However, before applying the IPMS theory on X-ray data, some constants need to be identified:the space groups, the Euler-characteristics and the Homogeneity indices, which all are tabulated elsewhere (Engblom, 1996). The swelling of cubic phases is given by Equation 8:

13/1

333

2CosSin

Hl

(8)

wherebilayer

bilayerArc2)1(tan(

and where is the lattice parameter, l denotes the chain length, denotes the so-called Eulercharacteristic, which is a link between the characteristic repetitive distance and the topology. H is thehomogeneity index, defined below. bilayer denotes the volume fraction bilayer.

In papers II and V the above presented theory for swelling has been used for interpretation of data fromtests performed on two cubic phases. The location of EtAc and NMP in the MO domain and in the waterdomain, respectively, has herby been confirmed.

19

As have been stated by others (Engblom, 1995) the swelling of reversed bicontinuous phases may bedescribed by assuming the existence of a neutral surface. The, inextensible, neutral surface is parallelwith, and displaced a distance, t, from the mid-surface of the bilayer. Equation 9 describes how themolecular cross-section area, , varies with the distance, t, from the mid-surface (IPMS):

UCA

tt

221)0()( (9)

Auc is the IPMS area per unit cell.

Under the assumption that a neutral surface exists, Equation 10 gives the swelling. This equation is,however, not restricted to swelling of cubic phases. In fact, this equation also describes the swellingbehaviour of the bicontinuous, liquid analogue of the cubic phase, i.e. the sponge (L3) phase. In paper IIthe swelling of the L3 phase found in the MO/NMP/water system was modelled in accordance with thisrelation.

3211

ccw (10)

denotes the lattice parameter and w is the volume fraction water. C1 and C2 are both constants, definedin Equations 11-12

)(

)16( 3/12

1 t

VHC s (11)

)(

4 2

2 t

tVC s (12)

where the Homogeneity index, H, is defined in Equation 13 where Vuc denotes the volume of the IPMSunit cell.

20

2/1

3/2

))(2(UC

UC

V

AH (13)

The Sponge Phase

The L3 phase is a bicontinuous liquid which microstructure can be described as a melted cubic phase. TheL3 phase, however, contains more solvent (mostly in its water channels) than the cubic phase. In otherwords, the L3 phase is more swollen compared to the cubic phase and thus has a higher volume to surfaceratio. The long-range periodicity, characteristic for the cubic phase, is lost in L3. There is, however, a smallest repetitive distance in L3, i.e. the centre-to-centre distance between two water channels. Whendetermining the characteristic length in L3, by means of SAXS, a rather wide peak is obtained. An L3phase containing a minimal amount of NMP, has a characteristic length of approximately 110 Å and asample from the middle of the L3 phase region has a characteristic length of around 190 Å. This showsthat the L3 phase swells as solvent is added (paper II).

Characteristic for the L3 phase is that it in many systems occupies only a narrow region. For instance, theL3 phase regions present in the MO/water/water-miscible solvent(s) system (Alfons, 1998; Engström,1998; Ekelund, 2000; Imberg, 2003; Imberg, 2003) and in other systems (Skouri, 1991; Strey, 1992) arenarrow. This has been explained by the spontaneous curvature, (i.e. for the phase to exist there can be nolarge variation in the negative curvature of the interface, (Wennerström, 1997)).

The swelling of the sponge phase has been described theoretically in two different ways (Hyde, 1997) andthese theoretical models have been applied in papers I and II. The first way to describe swelling is toassume that a neutral surface is maintained during the swelling (IPMS). In the second way to describeswelling it is, for dilute systems, possible to interpret the swelling geometrically when assuming that thelaws of swelling apply approximately. The swelling at constant molecular shape is given in Equation 14:

)1()1( 1d

kKinSbilayer (14)

where bilayer denotes the volume fraction bilayer. K and k1 are constants. Sin denotes the inner shapeparameter.

21

Figure 4. Schematic illustration of the microstructure of the sponge phase (Snabre 1990). The picturewas kindly provided by Gregoire Porte with permission to reproduce the picture.

In paper II, under the assumption that all solvent was present in the water domain, the swelling of the L3phase was modelled according to the described theories.

3.4.2 Dynamics

The non-ideal behaviour of the NMP/water system is clearly demonstrated, for example by self-diffusionand viscosity tests, in paper II.

The observed diffusion coefficient of a molecule is an average of the diffusion of free and boundmolecules (Evans, 1999) as described in Equation 15:

TOT

AggAgg

TOT

FreeFreeOBS

C

CD

C

CDD (15)

where DOBS is the observed diffusion coefficient, DFree denotes the diffusion coefficient of monomers and DAgg is the diffusion coefficient of the bound monomers. CTot is the total surfactant concentration. CFree is free monomer concentration and CAgg=CTot-Cfree. Equation 15 can be rewritten as

AggAggFreeFreeTotOBS CDCDCD

)( FreeTotAggFreeFreeTotOBS CCDCDCD

)( AggFreeFreeTotAggTotOBS DDCCDCD

CFree is constant at concentrations higher than CMC. If DOBS×CTot is plotted vs. CTot, DAgg and CFree can bedetermined from the slope and the intercept, respectively.

This analysis was used in paper II to study the dynamics of the L-phase in the MO/ NMP/ water (D2O)system. The results indicated that MO aggregates as the water activity increases. However, it should be

22

noted, that the critical point, of the MO/NMP/water phase diagram is located in the vicinity of the regionin the L phase where micelles probably form.

The self-diffusion of MO, water and NMP was also examined in the L3 phase. By assuming that all NMPwas located in the water domain it was possible to model the L3 phase as bicontinuous according to theapproximate ICR model (Anderson, 1989). The slightly larger than expected, obstruction factor for NMPmight though indicate that maybe not all NMP molecules are located in the water domain. Previously, theL3 phase of the MO/PEG 400/water system (Evertsson, 2002) and cubic phases (Ericsson, 1993) havebeen described as bicontinuous in accordance with the ICR model.

In paper II it was shown that the strong NMP(water)2 complex, that has been suggested in the literature(Assarsson, 1968; Hong, 2000), affects the dynamics. A variation in the diffusion coefficients for NMPand water, in the L3 phase is for example observed. The observed diffusion coefficients are mean values.The distribution between free and complex-bound NMP and water molecules vary by NMP/water ratiosthroughout the entire L3 phase region and therefore affecting the observed diffusion coefficients of waterand NMP, respectively. In addition, the water diffusion in L3 was found to be lower compared to thewater diffusion in the most swollen cubic D phase (Eriksson, 1993), indicating that water is obstructed by,or bound to, NMP. Similar behaviour has earlier been observed in the L3 phase of the MO/water/PEG 400system (Evertsson, 2002).

3.4.3 Interplay Between Structure and Dynamics

The mutual dependence between structure and dynamics has been shown in a number of previous studies.FT-PGSE-NMR, which gives important information of dynamics of a system, is therefore an importanttool to combine with structure-determining techniques like SAXS. In paper II, for example, both thestructures and dynamics of the L and the L3 phase were studied. The L3-phase was found to be possible todescribe as bicontinuous according to the ICR model and its swelling could, from SAXS data, bemodelled in accordance with the theory for IPMS. In addition, analysis according to the two-stage model,see Equation 15, indicated aggregation within the L phase, which was also confirmed by SAXS.

3.5 Phase Behaviours of Lipid/Solvent(s)/Water Systems

Monoolein exhibits a rich behaviour in water. The following phases are present, ordered after increasinghydration: L2, L , V2 (Gyroid), V2 (Diamond).

If completely water-miscible organic solvents are introduced into the binary MO/water system (paper I, IIand III), the phase behaviours presented in Figure 5 are obtained.

23

NMP

L

L3

LV2

Water MO

DMSO

rWater MO

L

L

V2

L3

Figure 5. Phase Behaviours of Lipid/Solvent/Water systems at room temperature. One-phase regionborders (L, L3, L , V2) are drawn with thick solid lines. Three-phase region borders are drawn with thinsolid lines.

24

AN

Water MO

L

L

V2

L3

Figure 5. Phase Behaviours of Lipid/Solvent/Water systems at room temperature. One-phase regionborders (L, L3, L , V2) are drawn with thick solid lines. Three-phase region borders are drawn with thinsolid lines.

Qualitatively, several ternary systems with MO, water and water-soluble organic solvents exhibit thesame kind of phase behaviour (Alfons, 1998; Ekelund, 2000; Engström, 1998; Imberg, 2003; Imberg,2003). In general, the positions of phase boarders and hence the size of one-phase regions differ sincedifferent solvents interact with water and with the MO/water interface to varying extents (log P – specificinteractions). NMP, for example, interacts with water by forming NMP(water)2 complexes (Assarsson,1968; Hong, 2000), which explains the phase behaviour at addition of MO to the binary NMP/watersystem (paper II). Likewise, DMSO and AN have been reported to interact favourably with water (Shin,2002). The microstructures of the binary solvent/water system can therefore be assumed to affect thesolubility of MO.

As discussed in paper V, the swelling behaviour can be explained in the following way. Every solventmolecule added contributes to the swelling of the water domain and has, in addition, an inherent tendencyto change the spontaneous curvature of the MO monolayer. The sponge phases are, as presented in thephase-diagrams of papers II, III, and V, located at fixed water contents. Therefore, the spontaneouscurvature and thus the swelling of the sponge should increase with increasing solvent/water ratio. Thealmost constant water content in L3 can be related to how the solvent affects the spontaneous curvature.There can be several different reasons to why the curvature of the MO monolayer is affected:

(i) The solvent may induce flattening on the curvature in a direct way by acting on theinterface and thus affecting the size of the head-groups.

(ii) Solvent induces swelling of the water domains.

(iii) The water activity is changed when varying the solvent/water ratios within the L3 phase(see paper II), which may affect the effective area of the head group.

25

To sum up, the spontaneous curvature is affected by changes in head group area and by changes inhydrocarbon chain volume. The former is affected by (i, ii, and iii) and the latter by, for instance,temperature.

Notably, as discussed above, different water-miscible organic solvents interact with water and MO indifferent ways. In a recent study, log P was used for prediction of the water content of sponge phases(Ekelund 2000; Ridell 2003).

If a solvent with only limited solubility in water is added to the binary MO/water system, a phasebehaviour like the one presented in Figure 6 is obtained.

EtAc

L

Water MO LD

G

Figure 6. The phase behaviour of the Monoolein/Ethyl acetat/Water system (paper IV and V).

The phase diagram looks different compared to the phase behaviours of systems with completely water-miscible solvents. These differences are of vital importance for being able to form lipid/polymercomposite particles. From the phase diagram it is clear that the L3 phase is not formed. This seemsreasonable since ethyl acetate, does not, to any greater extent, act in the water domain, as furtherdiscussed in paper V. The solubility of ethyl acetate in water is only 8%. Addition of ethyl acetate to binary MO/water mixtures does not affect the spontaneous curvature of MO. This statement is based onthe following observations: (i) the liquid one-phase area is large, (ii) the D-surface transforms into the G-surface as ethyl acetate is added, and (iii) no region with reversed hexagonal structure is present in the system. These facts taken together imply that increasing the EtAc content primarily increases theinterfacial area over which MO spreads. The effect of adding EtAc to binary MO/water is intermediatebetween curvature-reducing additives (such as DO and azone) and curvature-enhancing water-miscible

26

solvents. The additives discussed in paper V, can be arranged in order of increasing contribution topositive spontaneous curvature of MO monolayers. EtAc see

DO < AN < EtAc < DMSO < NMP < AN

3.6 Protein Encapsulation

To determine microstructures has a general scientific value, and well-known microstructures are alsouseful when considering different applications. The bicontinuous cubic phases are attractive forencapsulation of water-soluble proteins (Chang, 1997). In addition, there is today a great interest in usingthe three-dimensional long ranged ordered cubic phase for crystallisation of membrane proteins (Ai,2000; Caffrey, 2003; Cherezov, 2002; Nollert, 2000; Nollert, 2001; Pebay-Peyroula, 2000; Sennoga,2003). Also the bicontinuous L3 phase may be interesting to use in such applications. However, water-soluble proteins can by action of water-soluble organic solvents, like NMP, be induced to crystallize(since the solvent content is higher in L3 compared to the corresponding solvent content of the cubicphase (paper II, III). This mechanism is probably due to that the solvents interact through hydrogenbonding with water, which makes it more difficult for the proteins to interact with water.

Lysozyme has in this study been dissolved in the L3 phase of the MO/NMP/water system and cubosomeshave then been formed by emulsification of L3 in aqueous solution. However, during storage of the L3phase, a precipitation became visible. The precipitation was dissolved in water and the activity ofLysosyme was demonstrated in the solution (Samuelsson, 2001).

Lysozyme is an enzyme of ellipsoidal shape (45x30x30 Å) enzyme fits in the water channels of the L3phase and has even been reported to fit in the water channels of the cubic phase (Razumas, 1996). If, forexample, proteins should be encapsulated into the bilayer of the cubic phase it is important to understandhow the curvature is affected in order to preserve both the phase itself and the activity of the protein. Inpaper V, the effect on the microstructure (i.e. swelling and curvature) by addition of a third component to the MO/water system is discussed.

27

4 PHASE BEHAVIOURS OF POLYMERS

The Flory-Huggins Theory describes the mixing process of systems containing polymer and solvent(Flory, 1953). Since the Flory-Huggins theory, which here has been applied to multi-component systems,can be derived from the regular solution theory (see for example (Evans, 1999), it is a good idea to firstpresent the regular solution theory that describes liquid-liquid phase separation for non-ideal mixing (i.e.when components interact).

4.1 Polymers in Solution

The behaviour of polymers in solutions depends on, for example, the solvent used, the temperature, thepolymer concentration and the polymer architecture. The radius of gyration, which is a measure of thesize of a polymer coil, defined elsewhere (Wennerström, 1997), can be used to predict properties ofpolymer solutions. Below the effects of concentration and solvent properties are further discussed.

4.1.1 Concentration Regimes of Polymer Solutions

A polymer solution is classified as dilute as long as the mean distance between the coils is large comparedto Rg. By increasing the polymer concentration, C, the coils start to overlap. The concentration where thisstarts to occur is denoted C*. By increasing the polymer concentration beyond C*, the semi-dilute regimeis reached. And finally, by further increasing the polymer concentration, the concentrated regime is reached. The different concentration regimes are presented in Figure 7.

Dilute Semi-dilute Concentrated

Figure 7. Schematic illustration of the concentration regimes of polymer solutions The picture is reproduced from (Evans, 1999).

28

4.1.2 Good and Bad Solvents

One amazing property of polymers is their behaviour in solutions. The behaviour is strongly dependent on the properties of the solvent. A polymer occupies a large volume when a good solvent surrounds it. In abad solvent the polymer instead occupies a smaller volume, as illustrated in Figure 8.

Figure 8. Schematic illustration of polymer behaviour in solutions, i.e. in bad and in good solvents. Thesolvent is neither bad nor good at theta conditions, where the polymer solution behaves as an idealsolution with respect to e.g. osmotic pressure.

4.2 The Regular Solution Theory

The regular solution theory provides a description of the non-ideal mixing process of two liquidsubstances. Liquids are, more or less, incompressible and hence H= U and G= A.

Gibbs free energy of mixing for such systems can thus be calculated from Equation 16:

BBAABABAmix XXXXRTwXXG lnln)( (16)

where R and T carry their usual meaning. The number of moles of substance A and B are labelled A

and B , respectively. is the free energy of mixing for the two substances. The effective

interaction parameter is labelled w and XmixG

A and XB are the molar ratios for substances A and B,respectively. The regular solution theory assumes that molecules mix in a random way and that mixingonly affects the positional order at constant density.

4.3 The Flory-Huggins Theory

The Flory-Huggins Theory describes the mixing process between, for example, a polymer and a solvent.Random mixing and linear and flexible polymers are assumed. The theory is valid for the semi-dilute case and only interactions between nearest neighbours are considered.

29

4.3.1 Mixing a Polymer and a Solvent

According to the Flory Huggins Theory, Equation 17 below describes the free energy of mixing for theprocess of mixing one polymer and one solvent.

spppsppssmix nNnnnRTG )()lnln( (17)

R and T carry their usual meaning. The volume fractions and the molar fractions of the solvent and thepolymer are s, p, ns and np, respectively. Np is the degree of polymerisation of the polymer anddenotes the effective interaction parameter.

4.3.2 Mixing Two Polymers

According to the Flory Huggins Theory, Equation 18 gives the free energy of mixing for two polymers,denoted A and B, of equal size.

))(lnln( BABApBBAAmix nnNnnkTG (18)

k and T carry their usual meaning. The numbers of molecules of polymer A and B are nA and nB,respectively. Np is the common degree of polymerisation. The volume fractions of the two polymers are

A and B, respectively and is the interaction parameter.

4.3.3 The Main Equations

From a set of interaction parameters, a set of relative lengths (i.e. the number of segments per molecule)and the relative amounts of each substance, the composition of each existing phase can be calculatedaccording to the Flory-Huggins Theory. In this work, a computer program, developed by Per Linse atphysical chemistry 1 in Lund, has been used for the calculations. The program has been used to find a setof interaction parameters, which describe the experimentally determined phase behaviour. The way toperform the calculations is to assume a set of interaction parameters and then iteratively carry out thecalculations. The calculated result is compared to the experimentally determined phase behaviour, theinteraction parameters are slightly adjusted and the process is repeated until the discrepancy is minimized.The relative lengths have been calculated approximately from the molecular weights of the components.Below, the equations used for multi-component systems are presented.

By minimizing the total free energy per lattice site, ATOT, the equilibrium distribution of the substancesbetween different phases, , can be determined. The total free energy per lattice site is given in Equation19:

30

)( iTOT AA (19)

The set i , which is the set of volume fractions of every substance in phase , is calculated from

Equation 20:

)/()(j

jjiii rnrn (20)

The phase volume fraction is defined according to Equation 21:

jjji

ii rnrn /()( ) (21)

A , which is the free energy of mixing per lattice site in phase , is calculated from Equation 22

i jjiiji

iii rkTA )5.0ln)/( (22)

The final equation of importance is Equation 23, which sets the limit for when a binary mixture will phaseseparate. If ij exceeds the critical value of the interaction parameter, ij

*, phase separation occurs.

2/))()(( 22/12/1* jiij rr (23)

Phase equilibrium is established when the total free energy per lattice site is minimal. This is anoptimisation problem since the total free energy per lattice site is dependent both on the composition andon the relative number of lattice sites of each phase.

To get a better understanding of the calculations, a more detailed description of a ternary system ispresented. For a ternary system the phase index, , is 1,2 and 3. The volume fractions of phase 1,2 and 3 are 1, 2, and 3, respectively. Note that any can be zero, which would indicate a non-existing phase.For example, 1 can be calculated from Equation 21:

jjj

iii rnrn )/()( 11

333223113332222112331221111331221111 /()( rnrnrnrnrnrnrnrnrnrnrnrn )

The free energy per lattice site of phase 1 can be calculated according to Equation 22:

31

i i jjiijiiii rkTA 111111 5.0/ln()(

So, in the ternary case, when i can be either 1,2 or 3, the free energy per lattice site in phase 1 iscalculated as below:

311113211112

2

11113313122121111111 (2

1/ln/ln/ln( rrrkTA

))231332131321131313121232

2122112121

312123311113211112331312212111111 /ln/ln/ln( rrrkT

When it comes to the final minimising problem, ATOT must be minimised. This might look simple, butnote for the ternary case, according to Equation 19, that:

22121111111332211 /ln/ln(( rrkTAAAAATOT +

()/ln 231212331111321111233131 r 2222211212 /ln/ln rr +

33232 /ln r 22323113133322223321213221212 /ln/ln() rr

))//ln 3323233313133131233333 rr

Of course there are a couple of constraints. The sum of the volume fractions can never be anything else than 1. The totalvolume fraction of each substance must be constant throughout the optimisation process; it is only between different phases that volume fractions of the substances are allowed to change.

4.4 Polymer Phase Behaviours in Theory and in Practice

In paper I the calculations were carried out in the same order as the systems are presented here. MO,which aggregates in aqueous environments, is here referred to as “polymer 2”.

32

4.4.1 Polymer 1/Solvent

The interaction parameters of the binary systems must first be estimated since they are used as inputparameters for the calculations. To be able to distinguish equilibrium from non-equilibrium (i.e. theglass transition state) the glass transition temperatures of concentrated polymer/solvent samples must beconsidered when determining the phase behaviour.

4.4.2 Polymer 1/Non-Solvent/Solvent

PLG/water/NMP: PLG is slowly degraded by hydrolysis. NMP prefers water compared to PLG, whichmakes the polymer phase concentrated. There exists a deviation between theoretical calculations andexperimental results.

4.4.3 Polymer 1/”Polymer 2”/Solvent

PLG/MO/NMP: MO and PLG segregate unless the common solvent NMP is present. Note that thepolymer phase can incorporate some lipid, but that the lipid phase cannot incorporate polymer.

4.4.4 “Polymer 2”/Non-Solvent/Solvent

MO/water/NMP: The system contains many ordered phases. Good correlation between theory andexperiments.

4.4.5 Polymer 1/”Polymer 2”/Non-Solvent/Solvent

The experimentally determined phase diagrams, with the results obtained from the modelling according tothe Flory-Huggins Theory (paper I), are shown in Figure 9

33

PLG NMP PLG

Water MO

Figure 9. The phase behaviours of the Polymer 1 (PLG)/Solvent (NMP)/Non-solvent (water), the“Polymer 2” (MO)/Solvent (NMP)/Non-solvent (water), and the Polymer 1 (PLG)/”Polymer 2”(MO)/Solvent (NMP) systems in theory (solid grey lines) and in practice (dashed thick black lines).Underlying samples are marked with dots. Three-phase regions and one-phase regions are presented aswell (thin solid black lines).

Paper I shows that the phase separation between PLG and the aggregating lipid, MO, which in some ways behaves like a short polymer, is strong. This is possible to model with the Flory-Huggins Theory.Furthermore, the phase study shows, by the slopes of the tie-lines of the PLG/NMP/water system thatNMP strongly prefers to mix with water instead of mixing with PLG. Since NMP is a fairly good solventfor PLG, the polymer would have benefited entropically from formation of a more diluted phase, but astrong interaction (Assarsson, 1968; Hong, 2000), studied in paper II, dominates the phase behaviour ofboth the ternary PLG/NMP/water system and the quaternary PLG/MO/NMP/water system.

34

5 LIPID/POLYMER COMPOSITES

This section presents the phase behaviour of lipid/polymer/solvent water systems examined in paper I andIV. Moreover, this section deals with the formation and the characterisation of the lipid/polymercomposite particles. In addition, other lipid/polymer materials, with applications not restricted to drugdelivery but involving simultaneous use of MO and PLG, are also discussed.

5.1 Phase Behaviours of the Lipid/Polymer/Solvent(s)/Water Systems

To explore the possibilities to form and characterize lipid/polymer composite particles, the phasebehaviours of quaternary systems with PLG, MO, solvent (NMP or EtAc) and water were examined in papers I and IV, respectively.

With particle formation in mind, the crucial difference between NMP and EtAc is the complete water-miscibility of NMP in water compared to the limited solubility of EtAc, i.e. 8 % by weight at 20 C. Thephase diagrams of the two quaternary systems are given in Figure 10, where the sides of the phasetetrahedrons are folded out in order to present the phase behaviours in a suitable way. Note how the slopes of the tie-lines, of the ternary polymer/solvent/water systems, differ between the systems. When it comes to particle formation by using emulsion techniques, presented below, this difference is extremelyimportant.

35

PLG NMP PLG

Water MO

PLG

PLG-PEG EtAc PLG-PEG

Water MO

Figure 10. The phase behaviours of the lipid/polymer/solvent/water systems.

Note that the slopes of the tie-lines of the polymer/solvent (EtAc)/water system reveal that the PLG/EtAcmixtures can be in equilibrium with an EtAc/water mixture which is also true for MO/EtAc mixtures.This is necessary for successful formation of lipid/polymer composite particles using an emulsiontechnique.

36

When the completely water-soluble solvent NMP is used, it is not possible to form lipid/polymercomposite particles since the polymer/NMP phase cannot be in equilibrium with water (i.e. at homogenisation, NMP instantly leaves the polymer-rich phase since NMP prefers water). In water, NMPforms strong (NMP)water2 complexes (paper II). In paper IV it is shown that ethyl acetate is a verysuitable solvent for a system intended for creation of lipid/polymer composite particles. There are twomajor reasons for that: (i) EtAc is volatile, which means that the system concentrates as a consequence ofevaporation while the particles are formed and (ii) the polymer-rich phase can coexist with an aqueoussolution under thermodynamic “quasi” equilibrium, which stabilizes the emulsion.

5.2 Lipid/Polymer Composite Particles

The following section is on the topic of the here-developed lipid/polymer composite particles. The focusis on the relation between the properties of the particles (paper IV) and the phase behaviours of thelipid/polymer/solvent/water system that the particles originate from (paper IV).

5.2.1 The Route to Lipid/Polymer Composite Particles

Briefly, particles were, in one work (paper IV) formed by emulsifying a small amount of the L phase intoan outer aqueous phase, consisting of 0%, 4%, 6% or 8% EtAc solved in water. Both phases were room-temperatured when the emulsion was created by means of a Polytron mixer. The particle formationprocess is schematically presented in Figure 11.

37

Lipid/PolymerComposite Particles

Solvent Evaporation

The L-phase

Formation of the O/W emulsion by homogenisation

AqueousSolution

Figure 11. Schematic illustration of lipid/polymer composite particle formation by an oil-in-water (O/W) solvent evaporation technique.

By varying the concentration of EtAc in the outer aqueous phase during the particle formation process,the kinetics of the particle formation process can be controlled (paper IV).

5.2.2 Interpretation of the Particle Formation Process

When a small amount of the L phase is emulsified into an aqueous phase, the solvent, which to someextent is miscible in water, will diffuse out to the aqueous phase. Moreover, the solvent will start toevaporate from the system. As the solvent then leaves the droplets, the polymer concentrates and formsspherical polymer particles. At the same time, the lipid separates from the polymer phase, and forms lipiddomains within the polymer particles. As water enters inside the particles, the lipid domains start to swell(paper IV and V)). First a swollen liquid phase (L phase) is formed and after further swelling (i.e.incorporation of water) the cubic G-surface is formed. As a result of further swelling, the cubic G-surfaceis then expected to be transformed to a cubic D-surface. The latter exists in equilibrium with excess water.The swollen lipid phase finally separates from the polymer as the lipid domains leave the polymerparticles.

5.2.3 Characterisation of Lipid/Polymer Composite Particles

Several different methods for characterisation have been used. Figure 12 shows representative confocalmicroscopy pictures, which clearly show that the lipid segregates, and form distinct lipid domains (paper

38

IV). The strength of phase separation is different in the two cases, as expected from Flory Hugginstheory.

Figure 12. CLSM pictures of composite particles created with 0% EtAc in the aqueous phase. Swollenlipid domains detach from the PEG-PLG matrix left (after approximately 150 minutes) and from the purePLG matrix right (after around 25-45 minutes), see paper V. Scale bars indicate 10 m.

5.2.4 Interplay Kinetics-Properties

The final properties of the particles depend on how they have been formed. As pointed out in paper IV,several different processes take place at the same time:

The solvent first leaves the particle, which results in phase separation between lipid and polymer. The rateat which the solvent leaves the particle can be affected by changing the solvent content of the outer phase.The driving force for phase separation can in analogy with Flory-Huggins theory be adjusted by eitherchanging the molecular weight of the polymer or by modifying it. In paper IV a PEGylated polymer was used to reduce the system’s desire to phase separate. PEGs with low molecular weight act as solvents forMO.

The polymer incorporates water and hydrolysis begins. The degradation is however slow and dependenton several parameters, such as polymer composition (hydrophilicity/hydrophobicity), device morphology,temperature, molecular weight, and particle size (Panyam, 2003). PLG degrades due to autocatalytichydrolysis of the ester-bounds (Anderson, 1997). Components present in the microspheres may affect the water-uptake and the corresponding degradation. For instance, if MO is present within the polymermatrix, there is a driving force to hydrate the lipid; hence water probably enters a lipid/polymer hybridparticle more quickly compared to how quickly it diffuses into a pure polymer particle.

39

The lipid swells according to the phase behaviour presented in papers IV and V. To be able to swell,water and a free volume to occupy are needed. The pressure the polymer matrix exercises on the lipiddomains can restrain the swelling of the lipid but if the driving force for swelling is strong enough, thepolymer matrix can be “broken”. In paper IV the possible formation of a double-emulsion duringhomogenisation is discussed. In that case there will already be water within the polymer matrix and thelipid will thus be able to swell quickly and occupy, more or less, the entire volume of the inner emulsiondroplet.

To sum up, the system (presented in paper IV) offers possibilities to form particles of a certain type. The understanding of the processes that take place is however important for obtaining the desired result.Particle sizes, lipid content, choice of polymer (molecular weight and modification) and EtAc content ofthe outer phase very much determine the properties of the particles.

5.2.5 Phase Separation

There is today a scientific interest for studying phase separation and the underlying mechanisms, i.e.spinodal decomposition or nucleation, (Cahn, 1958; Cahn, 1965; Binder, 1983). The number of suitablesystems is however limited since phase separation has to be slow in order to be possible to examine bydirect imaging. Today, systems with slow kinetics, i.e. colloidal glasses or supercooled liquids, aretherefore used (Weeks, 2000). The system presented in paper IV might be interesting for such an application assuming a time-resolved confocal microscopy is used. A limitation might, however, be theresolution. The CLSM picture, on the cover-page, presents lipid (red)/polymer (green) phase separation.However, from this experiment no conclusions regarding the mechanism behind the phase separationcould be drawn, i.e. the equipment used is a traditional CLSM microscope.

5.3 Simultaneous use of Lipid and Polymer

The following patents exemplifies simultaneous use of MO and PLG:

US Pat. No. 6,277,413 issued in the name of Sankaram et al, describes howthe release rates, of encapsulated substances can be controlled by varying theratio between lipid/polymer in lipid/polymer particles.

US Pat. No. 6,488,952 issued in the name of Kennedy et al, describes how a multiparticulate system, for injection, deposition or implantation, can beformed by incorporation of polymer micro-particles in a hydrogel (i.e. cubicphase).

International Patent Application No. WO02/19988 A2 issued in the name of Bodmeier et al, describes how sustained release particles can be formed, from liquid components.

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International Patent Application No. WO99/47588 issued in the name of Dawson et al, describes a method for preparation of polymer microparticles, over a wide range of preparation conditions, by using an emulsifier, i.e. MO.

These examples show that both MO and the PLG have received some interest.

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6 CONTROLLED DRUG DELIVERY

Active substances can be delivered in several different ways. In many cases, as for example when the active substance is given in form of injections, the whole dose is given in one portion. However, manysubstances should preferably be released slowly and over a long period of time. This thesis does notinclude any experimental release studies. In spite of this the controlled/sustained/retained release, CRtechnology, will be briefly presented here since lipid/polymer composite particles are supposed to release encapsulated substances in a similar way.

The CR technology is a relatively new technology even though the diffusion of small molecules through a silicon wall was reported long time ago (Folkman, 1966). Since then, progresses have been made withinthe field. The following are important events regarding PG and PLG:

In 1970 PG sutures were commercialised under the name Dexon ™.

In 1975 there were sutures made from the co-polymer PLG, commercialised under the name

Vicryl ™.

However, relatively few CR-based products have until today been commercialised. One example thoughis Lupron Depot®, one-month injectable PLG microspheres encapsulating leuprorelin acetate fortreatment of prostate cancer (Langer 2003).

6.1 Controlled Release Technology

Controlled/sustained/retained release implies that the active substance is released slowly over a longperiod of time. By using a controlled release technology the side effects are reduced since lower concentrations of the active substance can be used, see Figure 13.

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Time

Pla

sm

ac

on

ce

ntr

ati

on

of

dru

g Toxic

Therapeutic

Non-effective

Figure 13. Controlled release: A constant concentration of the active substance is obtained over a longperiod of time (solid line). The plasma concentration obtained after repeated intake of a traditionalformulation is presented as well (dashed line). The picture is reproduced from (Edlund 2000).

One advantage of the controlled release technology is the more levelled concentration obtained of theactive substance in the patient’s body. This greatly reduces the risk for side effects. Furthermore, thetreatment gets more effective since the therapeutic window is better utilized over a longer period of timeand from the patient’s point of view the pharmaceutical is more easily handled since it has to beadministrated less often.

There are many different kinds of devices used for CR applications. Examples are: patches fortransdermal delivery, prodrugs, hydrogels, lipid-based systems and biodegradable polymer systems.

6.2 Properties of Polymer Matrices

During degradation of a polymer, the main chain is cut shorter into oligomers or monomers (Göpferich,1996). Degradation can be due to chemical processes (like hydrolysis or oxidation) or it can be due tobiocatalytic processes (induced by bacteria or enzymes). If degradation takes place in vivo, the polymer issaid to be biodegradable.

The polymer must also be biocompatible. Biocompatibility is defined as ”the ability of a material toperform with an appropriate host response in a specific application” (Williams, 1989).

6.3 Factors that Affect the Release Rate

No release experiments have been performed with the lipid/polymer composite particles. A few factorscan, however, be identified that will affect the total release rate from an ordinary PLG particle (a particlewithout MO-domains). They are:

The hydrophilicity/hydrophobicity of the active substance.

The molecular weight off both the polymer and the active substance.

The degree and type of modification of the polymer, i.e. hydrophilicity/hydrophobicity of thepolymer.

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The concentration of EtAc in the outer phase during particle formation.

Temperature and pH.

Particle size distribution

With a lipid/polymer composite particle, the release mechanism may be more complicated since differentmechanisms can be assumed to dominate depending on how the particles were formed.

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7 HIGHLIGHTS OF THE PAPERS

Interesting results from the papers are highlighted here.

Paper I: The phase behaviour of the quaternary NMP/MO/PLG/water system is presented.

Paper II: The swelling of L3 was well described by the IPMS theory. The calculations showed thatthe microstructure of the L3 phase reminds about the P-surface. SAXS tests performed on Q indicate that it is probably the P-surface that is in equilibrium with the L3 phase. Thephase diagram can be understood and explained by considering the formation ofNMP(water)2 complexes. In summary, the complex is so strong that it makes thesolubility of MO vary by NMP/water ratio. Interestingly, the entire phase behaviour canbe explained by regarding all NMP as present in the water domain.

Paper III: The method to automate the preparation of samples containing lipid/solvent/water worksnicely. Ethyl acetate is a suitable solvent for dissolving the lipid since crystals are formedin the binary MO/EtAc system, which probably promotes evaporation of EtAc. Remaining EtAc is not significantly affecting the phase behaviour.

Paper IV: The phase behaviour itself is interesting. There are, to our knowledge, no other systemsreported where a reversed bicontinuous cubic phase is in equilibrium with an L2 phase.The phase behaviour is very suitable from a particle formation point of view (with cubicphase in equilibrium with water even with solvent present to the point where the EtAc concentration of the water and MO domains are identical, which is at about 8% EtAc).

Paper V: The paper extends the understanding of other lipid-based MO/water/third-componentsystems. In the case with NMP being the third component, NMP mainly acts in the waterdomain, where it increases the spontaneous curvature of MO-monolayers, which resultsin formation of an L3 phase as NMP is added to the cubic diamond phase. The water-insoluble substances azone and diolein act in the MO domain, which promotes theformation of reversed phases. Ethyl acetate mainly acts in the MO domain, but is also partly soluble in water. Addition of EtAc therefore promotes formation of planarstructures, i.e. additions of EtAc to MO/water samples do not affect the spontaneouscurvature of MO.

Interestingly, the effect of adding EtAc to binary MO/water is intemediate between two curvature-reducing additives and the curvature enhancing polar solvents. The additivesdiscussed in paper V, can be arranged in order of increasing contribution to positivespontaneous curvature of MO monolayers: DO

8 CONCLUDING REMARKS

The phase studies have resulted in a number of phase diagrams that are interesting from a pure scientificpoint of view. Noteworthy is for example that in lipid/solvent/water systems the self-aggregation of the lipid is strongly dependent on the properties of the solvent. As presented in paper V, the explanation canbe found in the interaction between the lipid and the polar media. If, for example the solvent is NMP, thelipid is increasingly soluble at higher NMP contents, since the water activity then is reduced (paper II),which is due to the formation of specific NMP(water)2 complexes.

The preparation of samples containing lipid/solvent/water has been automated by means of a roboticliquid handler. For this purpose a method has been developed and validated (paper III).

The lipid and the polymer segregate, as expected, but mix in a common solvent (papers I and IV). Ifwater is added to an L phase containing lipid, polymer and solvent, phase separation is induced. In the case with NMP (paper I) the polymer phase gets very concentrated since NMP so strongly prefers thewater phase, as shown in paper II. This is one of the reasons to why NMP is unsuitable as a solvent in a system for preparation of lipid/polymer composite particles (see paper IV).

The microstructures of phases present in systems of monoolein, water and ethyl acetate have beenexamined in paper IV. In contrast to NMP, which mainly acts in the water-domain, ethyl acetate acts in the MO domain. Adding ethyl acetate to V2 thus has the same effect as dehydration and the L3 phase isconsequently not induced. In fact, when ethyl acetate is added to theV2 phase, coexisting with excesswater, i.e. the cubic D phase, the cubic phase transforms into cubic G phase. This is precisely the oppositecompared to when NMP is used. Adding NMP to V2 instead swells the water domain of the cubic D-surface and L3 is induced.

Novel lipid/polymer composite particles have been created from the lipid/polymer/ethyl acetate/watersystem by using an emulsion technique. The route to particles has been deduced from the phase behaviourof the system (paper IV). Distinct lipid domains that swell in water have been demonstrated by means ofCLSM. By considering the phase behaviour, it is possible to understand how the emulsion droplets are transformed into polymer particles as the solvent evaporates (paper IV). In aqueous solutions lipiddomains leave the polymer matrix of the particle. Interestingly, there is a difference if the polymer isPEGylated or not. When PLG is PEGylated there are a thin polymer shells around the lipid domainsleaving the matrix, probably due to favourable interaction between MO and PEG. By introducingPEGylation to PLG, the Flory-Huggins interaction parameter lipid-polymer is lowered. With non-PEGylatedPLG it seems like pure MO domains are leaving the matrix. The structure of the lipid domains has notbeen determined by means of SAXS but according to the phase diagram the lipid phase should swellaccording to: L, L , V2 (G) to V2 (D).

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9 EXPERIMENTAL TECHNIQUES

Scientifically interesting experimental techniques have been utilized. Since standard methods have been used, the techniques have been described elsewhere and there is no need to describe them in detail again.Anyway, the different techniques utilized during the work towards this thesis are listed:

Confocal Laser Scanning Probe Microscopy, presented by (Sandison, 1990), has been used forcharacterisation of lipid/polymer composite particles (paper IV).

Differential Scanning Calorimetry has been used to determine the glass transition temperature ofconcentrated polymer solutions (paper I).

Fourier Transformed Pulsed Gradient Spin Echo Nuclear Magnetic Resonance (Stilbs 1987;Stilbs 1996; Tanner 1970), for instance presented by (Evertsson, 1999), has been used tomeasure self-diffusion coefficients of lipid, water and solvent (paper II).

Microsphere preparation by an O/W technique, deduced from the phase behaviour (paper IV)

Phase diagrams determination and sample preparation (paper I, II, III, IV, V).

Rheology, for instance presented by (Marriott, 1988), has been used to determine the viscosity ofthe L phase of the NMP/MO/water system (paper II).

Small and wide-angle X-ray scattering, for instance explained by (Svensson, 2003), have beenused for determination of structural properties of phases (paper II and V).

Scanning Electron Microscopy, presented by (Forslind, 1981), has been used for characterisationof lipid/polymer composite particles (unpublished data).

Fluorescence Microscopy has been used for 2D-characterisation of lipid/polymer compositeparticles (unpublished data).

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10 SUMMARY IN SWEDISH

En populärvetenskaplig sammanfattning av arbetet följer nedan. För att underlätta läsandet läggstonvikten i det här avsnittet på lipid/polymer hybrid partiklar, som i sig kanske är lite konkretare än självafasstudierna. Noterbart är ändå, att den största biten av arbetet omfattar fasstudier i fler komponentsystem-ur ett grundläggande fysikal-kemiskt perspektiv. Studien baseras på 5 vetenskapliga arbeten somalla, på ett eller annat sätt, omfattar fasdiagram.

Lipider och polymerer, som använts flitigt i den här studien, är två vanliga molekylslag som vi stöter pådagligen. Lipider är medelstora molekyler som t.ex. finns i huden och i cellmembran. Polymerer består avmindre byggstenar, s.k. monomerer, som är sammanbundna till långa kedjor. Vårt DNA är ett intressantexempel på en polymer.

Den mänskliga DNA-sekvensen har nyligen kartlagts och därmed är det möjligt att tillverka nya effektivare proteinbaserade läkemedel. När man tillverkar ett läkemedel med ett visst protein, behöverofta andra substanser, s.k. hjälpämnen, tillsättas för att läkemedlet ska ha önskad effekt. Med önskadeffekt menas att den verksamma substansen har bibehållen aktivitet (är effektiv), har möjlighet att verkapå önskvärd plats i kroppen samt att den utsöndras i lagom takt.

I många fall är det önskvärt att uppnå en jämn koncentration av den verksamma substansen i kroppen,vilket kan tillgodoses genom att läkemedlet släpper ifrån sig den verksamma substansen sakta, saktaunder en längre tid (s.k. kontrollerad eller fördröjd frisättning). Det ställs stora krav på en sådan formulering, t.ex.: (i) inget av de ingående ämnena får vara giftigt eller får brytas ned till giftigaprodukter, (ii) formuleringen måste vara stabil (den får ej brytas ned under t.ex. lagring) och (iii)formuleringen måste frisläppa den verksamma substansen under önskad tidsperiod och på rätt plats i kroppen.

I avhandlingsarbetet har en ny typ av lipid/polymer kompositpartikel tagits fram. Partikeln består av enbiologiskt nedbrytbar polymermatris med lipiddomäner jämnt fördelade inuti. Vatten löser inte polymerenutan bryter istället efter ett tag ner den till små vattenlösliga enheter som i kroppen sedan omvandlas tillkoldioxid och vatten. Lipiden, som finns naturligt i vår kropp, är ett ytaktivt ämne som består av envatten-gillande del och en vatten-skyende del. Dessa gör att lipiden, i närvaro av vatten, bildar olika typerav strukturer (faser). Anledningen till att faserna bildas är att lipiden organiserar sig så att separata vatten-och olje-domän