Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 298

Characterisation of AqueousSolutions, Liquid Crystals and

Solid State of Non-ionic Polymersin Association with Amphiphiles

and Drugs

BY

ANNIKA RIDELL

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2003

Printed in Sweden by Universitetstryckeriet, Uppsala 2003

Contents

1 Introduction .................................................................................................71.1 Polymers...............................................................................................81.2 Amphiphiles .......................................................................................10

2 In aqueous systems ....................................................................................122.1 Polymers in aqueous solution.............................................................122.2 Amphiphiles in aqueous solution .......................................................15

Molecular shape...................................................................................15Aggregated phases in water.................................................................16

Micelles ......................................................................................17Lyotropic liquid crystalline phases.............................................19

2.3 Interaction between polymers and surfactants in aqueous solution ...22Effect of polymer.................................................................................23Effect of amphiphile ............................................................................25Effect of surfactant counterion ............................................................26Structure of the aggregates ..................................................................26

2.4 Interaction of polymers and lipids in aqueous environment ..............28The ternary PEG 400/MO/water-system .............................................29The cubic phases..................................................................................29

Polymer molecular weight .........................................................30The sponge phase ................................................................................33

Polymer molecular weight .........................................................33Amphiphilic additives ................................................................34

3 In solid state...............................................................................................373.1 Polymers in solid state .......................................................................373.2 Lipids in solid state ............................................................................393.3 Mixtures of polymers and lipids ........................................................40

4 Conclusions ...............................................................................................454.1 Prospects ............................................................................................45

5 Acknowledgements....................................................................................47

Appendix – Physico-chemical characterisation methods..............................49Visual observations ..................................................................................49Viscometry ...............................................................................................49

Dye solubilisation.....................................................................................50Equilibrium dialysis .................................................................................51Thermal methods......................................................................................51

Titration microcalorimetry ..................................................................51Differential Scanning Calorimetry, DSC.............................................52

X-ray diffraction.......................................................................................52Small angle x-ray diffraction, SAXS...................................................53

Fluorescence spectroscopy.......................................................................55Micropolarity .......................................................................................56Microviscosity .....................................................................................56Aggregation number determination.....................................................57

Nuclear Magnetic Resonance, NMR........................................................59Self-diffusion NMR.............................................................................59

References.....................................................................................................61

Papers discussed

This thesis is based on the following papers, which will be referred to in the text by their roman numerals:

I Ridell, A., Evertsson, H., Nilsson, S. (2002) Influence of counterion on the interaction of dodecyl sulfates and cellulose ethers. Journal of Colloid and Interface Science, 247 (2) 381-388. Reprint is made with permission.

II Ridell, A., Evertsson, H., Nilsson, S., Sundelöf, L.-O. (1999) Amphiphilic Association of Ibuprofen and two Nonionic Cellulose Derivatives in Aqueous Solution. Journal of Pharmaceutical Sciences, 88 (11) 1175-1181.

III Ridell, A., Evertsson, H., Nydén, M., Engström, S. Shrinking and swelling the cubic phase of the polyethylene glycol/monoolein/water-system, manuscript.

IV Ridell, A., Ekelund, K., Evertsson, H., Engström, S. (2003) On the water content of the solvent/monoolein/water sponge (L3) phase. Colloids and Surfaces A: Physicochemical and Engineering Aspects, in press. Reprint is made with permission.

V Mahlin, D., Ridell, A., Frenning, G., Engström, S. Solid-state characterisation of binary PEG 4000/monoolein mixtures, submitted.

Reprint is made with permission.

Abbreviations

[ ] Intrinsic viscosity Brij, CmEn polyoxyethylene alkyl ethers ChloHCl Hydrochloride salt of Chlorpromazine cmc Critical micelle concentration cpp Critical paccing parameter CTAB Cetyl trimethyl ammonium bromide D Diffusion coefficient DSC Differential scanning calorimetry EHEC Ethyl hydroxyethyl cellulose HPMC Hydroxypropyl methylcellulose I1/I3 Micropolarity index Ia3d Space group of a cubic phase IbuNa Sodium salt of ibuprofen IM/IE Microviscosity index Im3m Space group of a cubic phase KDS Potassium dodecyl sulfate L3 Sponge phase LCST Lower critical solution temperature LiDS Lithium dodecyl sulfate L , Lam Lamellar phase Mn Number average molecular weight MO Monoolein, glycerol monooleate Mw Weight average molecular weight Nav Average aggregation number NMR Nuclear magnetic resonance PC Phosphatidylcholine PEG Polyethylene glycol PEO Polyethylene oxide Pn3m Space group of a cubic phase Q Cubic phase SAXS Small angle x-ray scattering SDS, NaDS Sodium dodecyl sulfate WAXS Wide angle x-ray scattering

Viscosity

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1 Introduction

The overall theme of this thesis is physico-chemical characterisation of pharmaceutically relevant excipient systems. The studies concern different types of drug delivery systems, from the undissolved solid state, for example important in tablet formulations, to aqueous solutions of the excipients. Beside the active drug substance most drug formulations contain mixtures of excipients of different types and functions. The physico-chemical behaviour of these mixtures is important to understand both in the formulation, e.g. for stability reasons, and as the formulation enters and dissolves in the body fluids.

Polymers in different forms are used in pharmaceuticals in various ways, such as gelling and viscosity-increasing agents, filling materials, suspending agents, tablet disintegrants, tablet binders, film-formers and extended release matrices (Wade and Weller 1994). Grades with lower molecular weights can also be used as ointment and suppository bases, tablet lubricants and as matrix in solid dispersions. Polymers are thus used in as diverse administation routes as for example oral, transdermal and parenteral delivery.

Surfactants and lipids are also abundant as excipients in pharmaceutical delivery systems, usually as emulsifiers, lubricants, wetting agents, suppository bases and solubilisers (Wade and Weller 1994). So-called water-swelling lipids can form liquid crystalline phases such as cubic or hexagonal phases that also might be used as specified drug delivery systems. Liposomes, consisting of curved lipid bilayers enclosing parts of the solvent, have also successfully been used as drug delivery vehicles.

The behaviour of many excipients and active drug substances in water are known but much is still unknown about the mixing of excipients of different types or excipient/drug mixtures. Interactions of polymers and surfactants in dilute aqueous solutions have thus been studied quite intensely over the last three decades and also in this thesis. Two aims of this thesis were to describe the specifics of the interaction between non-ionic cellulose ethers and an anionic surfactant with different counterions (paper I) as well as the interaction with an amphiphilic drug molecule (paper II).

Polar lipids can be used to solubilise or protect low-solubility drugs and can form a variety of swollen systems, liquid crystals, in aqueous

8

environment. Such liquid crystals have been proposed as drug delivery systems (Engström 1990; Ganem-Quintanar et al. 2000; Shah et al. 2001). It is thus important to study these systems and how they are regulated by different additives such as polymers of varying molecular weight(paper III) and amphiphilic components of differing molecular shape (paper IV), which also was within the scope of the present thesis.

Solid lipids are usually sticky at room temperature due to their low melting temperatures. Due to this stickiness, a formulation can be hard to deliver and manufacture as an oral dosage form. One way of overcoming these inconveniences and at the same time disperse the lipid with the drug content in the intestinal fluid is to deliver the lipid in a solid dispersion with a water-soluble matrix. The dissolution rate of the drug would thus be increased and the transportation of the drug over the intestinal membranes can follow. In this thesis one aim was to characterise the solid state of one such system (paper V).

This thesis can be viewed as composed of three main parts. The first and also the largest part, chapter 2, deals with polymers and amphiphilic substances in aqueous surroundings. Both polymer-amphiphilic interactions in dilute aqueous solutions and interactions in liquid crystalline environment are discussed. In the second part, chapter 3, polymer-polar lipid interactions in solid state are examined. The last part of the thesis contains the five original papers, referred to by their roman numbers, on which the summary is based. A short introduction to polymers and surfactants will be given as a background to the following summary.

1.1 PolymersPolymers are large molecules consisting of many subunits, monomers, covalently linked to each other. The monomers can be of the same type all over the polymer chain, thereby forming a homopolymer, or the monomers can change randomly, resulting in a random copolymer, or in blocks, giving a blockcopolymer, over the chain. The

OHO n

Fig 1.1. The molecular structure of PEG.

9

polymer chain can be linear or grafted. The polymer can also contain charges and is then called polyelectrolyte.

The polymers in this thesis are all uncharged. Polyethylene glycol, PEG, is a homopolymer with ethylene oxide groups as monomers, fig 1.1. PEG´s are often used as solubility enhancers in solid dispersions for low-solubility drugs and as “protectors” for liposomes and proteins forming so called “stealth liposomes” with the PEG chain covalently linked to lipids in the liposome (Lasic and Needham 1995).

Ethyl hydroxyethylcellulose, EHEC, and hydroxypropyl methylcellulose, HPMC, can be considered as homopolymers with a sugar chain, cellulose, as the backbone, substituted with short grafts consisting of ethyl-, hydroxyethyl-, hydroxypropyl- or methyl-groups, fig 1.2.

O

OO

OO

O

O

O

O

OO

O OO O

OOH

n

O

OO

OOO

O

O

OO O O

O O

nO H

The degree of substitution as well as the type and size of the substituents influences the solution properties of the cellulose ether. DSalkyl and MSao define the degree of substitution, where DSalkyl is the average number of alkyl substituents per anhydroglucose unit of the polymer and is thus a measure of the hydrophobic properties of the polymer. MSao, on the other hand is the average number of alkylene oxide substituents per anhydroglucose unit, and is a measure of the hydrophilicity. In table 1.1 the values for the cellulose ether fractions used are shown. If the substituents are distributed randomly or in blocks on the backbone chain is also of importance.

DSalkyl MSao Mw Mn

EHEC 1.5 0.7 1.9·105 0.9·105

HPMC 2 0.4 3.0·105 1.4·105

Values from Nilsson et al (Nilsson et al. 1995).

Fig 1.2. The molecular structure of some typical units of EHEC (left) and HPMC (right).

Table 1.1. Degrees of substitution and molecular weights for the cellulose ether fractions used.

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When polymers are synthesised either in nature or by man it is difficult to make all molecules alike. Some degree of polydispersity, distribution in molecular weight, is generally present. The PEG´s used in this thesis are however considered as having an almost monodisperse distribution as their polydispersity indexes are close to 1.1. The cellulose ethers on the other hand are polydisperse, with indexes at 2 (Nilsson et al. 1995).

1.2 AmphiphilesAmphiphiles are molecules containing two parts with different properties concerning lyophilicity, preference for a solvent. Usually molecules consisting of one water preferring part, hydrophilic, and one oil preferring or water fearing part, referred to as lipophilic or hydrophobic, are called amphiphiles. The hydrophobic part of the surfactant molecules usually consists of one or two hydrocarbon chains whereas the hydrophilic part can be ionic or contain uncharged polar groups.

Lipids are molecules that prefer oil to water. One classification of lipids is fatty acids and their derivatives, as well as substances related to them biosynthetically or functionally (Christie 1987). Lipids can be of the amphiphilic sort and are then called polar lipids, or they can be non-polar, which do not mix with water. The lipids participating in biological membranes are usually of the polar type.

The expression surfactant is used in this thesis for amphiphilic substances that interact with water and form micelles. They can then be visualised as having a conical shape as discussed below. The term amphiphiles, on the other hand is used for the ensemble of surfactants, amphiphilic drug molecules and polar lipids.

Sodium dodecyl sulphate, SDS, is one of the most well studied surfactants and it is used abundantly in detergents but also in pharmaceutical products e.g. as emulsifying agent, wetting agent and lubricant (Wade and Weller 1994).

Drug molecules from several groups, e.g. fenotiazines, local anaesthetics and non-steroid anti-inflammatory

Polydispersity index,

n

w

MMPD

11

drugs, NSAIDs, display amphiphilic behaviour (Attwood and Florence 1983). In this thesis chlorpromazine belonging to the fenotiazine group was used as well as ibuprofen that is a NSAID. Amphiphilic drug molecules are chemically more diverse than surfactants.

Monoolein, MO, is the polar lipid investigated in this thesis, fig 1.3. It belongs to the group of monoglycerides and is formed in the degradation of natural oils and fats in the gastrointestinal tract. Monoolein is considered as non-toxic for oral use and is biodegradable and included in the FDA inactive ingredients guide (Wade and Weller 1994). Monoolein has been used in several pharmaceutical applications that were recently reviewed (Ganem-Quintanar et al. 2000; Shah et al. 2001).

OO

OHOH

Fig 1.3. The molecular structure of MO. Another name for MO is glyceryl monooleate.

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2 In aqueous systems

Compared to most other solvents water is very polar and interacts primarily through intermolecular hydrogen bonds. These bonds are strong and unfavourable to break. However it is possible to break these interactions if the contributions from the entropy gain in distributing a solute in the water are larger. Dissolution of other molecules in water is thus primarily an effect of entropy gain. A substance that can form comparably more favourable interactions with water in the dissolved state is however more easily solubilised than an unpolar one.

2.1 Polymers in aqueous solution Polymer properties and interactions depend to a large degree on the chemical properties of the monomers. Polyelectrolytes for example are due to their charges usually more soluble in water than a corresponding nonionic polymer. PEG is very water-soluble compared to polymers such as polyethylene owing to the hydrophilic heteroatoms enabling hydrogen bonds with water. The cellulose backbone of the celluloseethers is not soluble in water due to very strong inter- and intramolecular hydrogen bonding between hydroxyl groups but as it is modified with hydroxyethyl and hydroxypropyl groups it can become soluble depending on the degree of grafting.

However, monomer-solvent (water) interactions are not exclusively determining polymer solubility. For example entropy gain is one of the most important parameter for dissolution. Entropy effects are also different in a polymer solution compared to a solution of monomers as the monomers are attached to each other in the polymer. Therefore, polymers are less soluble than its monomers

Fig 2.1. Three idealised forms of a polymer chain. From the left a random coil, =0.5, a completely extended chain,

=1 and a contracted chain with a close to 0.

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and solubility decreases with increasing polymer molecular weight.

The conformation of the polymer in solution is determined by the polymer-solvent interactions and the gain in conformational entropy on dissolution. A polymer with equal interactions with water as to itself will display a random conformational appearance in solution. This conformation is called a random coil and can be described by the random walk model. If the monomer-solvent interaction is more favourable than the polymer-polymer ones, the polymer chain is more extended than the random coil whereas if the polymer-solvent interaction is unfavourable the polymer chain is contracted, fig 2.1. The latter, however, is to some extent balanced by excluded volume effects, and equal balance rendered at so-called -conditions, where the polymer once more behaves as a random coil. The exponent, , in the Mark-Houwink equation

MKdescribes the appearance of the polymer chain. [ ] is

the intrinsic viscosity and can be determined by viscometry, K a constant and M the molecular weight of the polymer. If is 0.5 the polymer behaves like a random coil and if it is larger or smaller it is extended or contracted respectively. of the polyethylene glycols was determined to be equal to 0.59 indicating on a slightly extended chain formation in water, fig 2.2.

5 6 7 8 9 10 11-4

-3

-2

-1

ln[ ]=-6.98+0.59 lnM

ln M

ln[

]

Fig 2.2. Mark-Houwink plot determined by capillary viscometry for PEGs with molecular weights between 300 and 20000 (Ridell 2003).

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The intrinsic viscosity is a measure of the aqueous volume that a given polymer mass fills up. If a container is imagined as full of polymers not entangling with each other the inverse of [ ] thus gives the maximum concentration of polymer that can be kept in solution without intermolecular entanglement. This concentration is usually referred to as the critical overlap concentration or c*, fig 2.3. In the presently investigated systems c*varies between 31% for PEG 300 and 3% for PEG 20000 (Ridell 2003). The cellulose ethers EHEC and HPMC, on the other hand are larger and have c* at 0.22 and 0.13%, respectively (Nilsson et al. 1995).

0 < Cp < c* Cp=c* Cp > c*

A fascinating phase phenomenon displayed by some nonionic polymers in aqueous solution is clouding. Clouding results from a phase separation into a “cloudy” polymer rich phase and a clear solution that occurs as the temperature is raised and the so-called lower critical solution temperature, LCST, or cloud point is passed. The LCST is not always clear-cut for polydisperse samples, as the clouding is molecular weight dependent. Also the concentration of the polymer is of importance (Sarkar 1979). In fig 2.4 a clouded sample can be compared with an unclouded one. This reduced solubility at higher temperatures has been discussed as an effect of hydrogen bonding and conformational changes. The possibility for PEG to have less polar conformations increases with temperature thus decreasing the water solubility (Jönsson et al. 1998).

Fig 2.3. Polymer coils in aqueous solutions with increasing polymer concentration, Cp.

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The clouding of cellulose ethers also depends on the type and degree of substitution (Sarkar 1979) and more hydrophobic cellulose ethers display lower LCST than hydrophilic ones. In 0.1% solutions the LCST is 29 and 45˚C for the EHEC and HPMC fractions used respectively. The clouding phenomenon for the PEGs appears at much higher temperatures; for PEG 20000 the LCST is found at approximately 100˚C and higher for lower molecular weights (Saeki et al. 1976).

2.2 Amphiphiles in aqueous solution The polar part of an amphiphile is soluble in water whereas the solubility of the hydrocarbon part in water is low. This results in aggregate formation of the amphiphiles with the hydrophobic parts of the molecules away from the water and the hydrophilic parts towards the water. These aggregate structures display different shapes and properties and will be discussed below.

Molecular shape The degree of interaction between water and

amphiphilic molecules can be expressed by the term molecular shape. The molecular shape is a term for how large the lipophilic region is compared to the hydrophilic region of the molecule and thus not dependent on the actual atoms and the covalent bondings within the molecule. Surfactants, which form spherical micelles in water, have a conical shape in this aggregate type. Comparison can be made with a cornet of ice cream where the ice cream corresponds to the large polar region of the molecule and the cone corresponds to the thin lipophilic

Fig 2.4. A solution of 1% EHEC at a) room temperature and b) at 65°C.

Fig 2.5. Molecular shapes of amphiphiles; cone, cylinder and wedge.

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part. Cylindrically formed molecules have a polar region that is equal to the non-polar, whereas wedge-shaped molecules have a larger non-polar region thus forming for example reversed micelles. The molecular shapes are sketched in figure 2.5. Substances with one hydrocarbon chain often belong to the conical group whereas substances with two chains or one chain with unsaturations, giving kinks, belong to cylinders and wedges.

The critical packing parameter, cpp, has been derived to quantify the molecular shape and thus to predict of which structure will be formed by a given surfactant system. The cpp can be calculated from the area of the polar headgroup, a, the length of the hydrocarbon chain, l,and the volume of the molecule, v, according to;

alvcpp .

It has been shown that a conical molecule has a cpp < 1, a cylindrical a cpp of approximately 1 and a wedge-shaped a cpp > 1 (Israelachvili 1992; Jönsson et al. 1998).

Aggregated phases in water The phase behaviour of amphiphiles varies not only by the molecular shape but also by the concentration of the molecules. The molecular shape can also be changed by for example the temperature and salt addition. For example, if the temperature is raised the hydration of the polar head group decreases for non-ionic surfactants. A cylindrical molecule thus becomes more wedgeshaped, see fig 2.6. On the other hand, addition of salt to a solution of charged surfactants screens the head groups thus decreasing their size and the surfactants thus become more cylindrical. An overview of aggregated phases is given below.

Normal and reversed phases Amphiphilic aggregates can roughly be divided into three groups; normal, planar and reversed phases. The lamellar phase is the only planar phase. Micelles, hexagonal, cubic and sponge phases can all be either normal or reversed depending on the concentration and the molecular shape

Fig 2.6. Change of curvature and molecular shape for nonionic polyoxyethylene alkyl ether surfactants as the temperature is changed. Figure adapted from fig 4.9 in Jönsson et al (Jönsson et al. 1998).

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of the amphiphiles. The distinction between normal and reversed phases is whether the lipid surfaces circumvent water (reversed) or their hydrocarbon chains (normal). In normal phases water forms a continuum, while in reversed phases the water is circumvented by the amphiphiles. In bicontinuous cubic phases both oil and water are continuous so in a reversed bicontinuous cubic phase the lipid bilayer circumvents the water channels. Surfaces that circumvent oil, like the normal phases, are by convention called surfaces with positive curvature. Compare the micelles in figure 2.7 and 2.8. The cubic phases discussed in paper III and IV are of the reversed type whereas the micelles in paper I and II are normal.

Micelles The micellar phase, which is formed at low amphiphile

concentrations, is probably the best studied phase. Micelles start to form as a critical micelle concentration, cmc, is reached. The surfactants arrange themselves into spherical aggregates with the hydrophobic tails inside and the polar regions towards the water. The repulsive forces between the polar head groups are overcome together with the entropy loss by the gain in enthalpy in excluding the hydrophobic tails from the water region. There is, however, a dynamic exchange of monomers, single surfactant molecules, in and out of the micelles, but the number of monomers incorporated in a micelle is usually relatively constant. This number is often referred to as average aggregation number, Nav.

The chain length of the surfactant or the charge of the head group can influence the cmc. The cmc for example becomes higher as the chain length is decreased for the same type of surfactant (Jönsson et al. 1998). Uncharged surfactants can form micelles at lower concentrations than charged ones since the repulsive forces between head groups are more short-range (Jönsson et al. 1998). Adding of salt to systems of charged surfactants decreases the cmc as the entropic gain for the monomers is reduced together with the repulsive forces as the charges of the head groups are better shielded.

It is known that cmc as well as the size of the micelles are affected by the counterion of the monomer (Attwood

Fig 2.7. Micelle with the molecular shape of the amphiphile indicated.

Fig 2.8. Reversed micelle with the molecular shape of the amphiphile indicated. The surface that cirvumvents the water has a negative curvature.

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and Florence 1983; Missel et al. 1989). In paper I the effect of counterion on the cmc for dodecyl sulfates was studied. The hydrated ions of Li and Na where used as models as they differ in hydrated radius. The bare ionic radius of Li+ is smaller than for Na+ but for the hydrated ones it is the inverse due to the higher charge to surface ratio for Li+.

Ion Ion radius (Å) a

Hydrated radius (Å) a

Cmc (mM) b

Navc

Li+ 0.68 3.8 9.2 46 Na+ 0.95 3.6 8.3 59 K+ 1.33 3.3 4.3 - a Values compiled by Israelachvili (Israelachvili 1992). b Determined by micropolarity measurements at 37°C and c by fluorescence quenching at 25°C as counterions to dodecyl sulfates (paper I).

Due to the smaller size of the hydrated Na+ they can come closer to the micellar surface and thus better screen the charges of the surfactants than Li+. Consequently the micelles will be formed at lower DS- concentration and contain more monomers for Na+ than for Li+, see table 2.1. Since anions have a smaller hydration effect the effect of counterions will probably not be as large for cationic surfactants as for anionic surfactants.

The dodecyl sulfates have with their long flexible hydrocarbon chain and the ionic head group a typical conical shape, suitable for micelles. In paper II micelles built up by molecules with a more complicated shape than dodecyl sulfates are discussed. Many drug molecules are surface active and complex aromatic, or heterocyclic molecules and do thus not fit into the general picture of a conical molecule (Attwood and Florence 1983). However their amphiphilic character makes them aggregate in aqueous solution in a similar way. For the sodium salt of ibuprofen, IbuNa in fig 2.10, which was studied in paper II, an aggregation is observed from a specific concentration. The cmc was found at 180 mM and the average aggregation number for the micelles was determined to be 40. This can be compared with the much lower cmc for SDS and LiDS at 8 and 9 mM respectively.

Fig 2.9. Micellar surfaces with either Na+ or Li+as counterions.

Table 2.1. Radii and cmc for the counterions and dodecyl sulfates studied in paper I.

COO-Na

+

Fig 2.10. Molecular structure of IbuNa.

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Microviscosity data for IbuNa micelles show that they are slightly more rigid (IM/IE 1.4) than SDS micelles (IM/IE1.0) probably due to the stiffer molecular structure of IbuNa compared to the flexible hydrocarbon chain of SDS, fig 2.11 (Evertsson and Nilsson 1997; Ridell et al. 1999).

0 1 2 30.0

0.5

1.0

1.5

[amphiphile]/cmc

I M/I E

Further discussion on drug aggregation can be found in several references, see for example the review of Attwood et al. (Attwood and Florence 1983).

Lyotropic liquid crystalline phases As the concentration of amphiphiles is further increased the appearance of the bulk is no longer liquid like as the micellar solution. Instead, lyotropic liquid crystals are formed. Liquid crystals have similarities both with liquids and crystals as liquid crystalline phases display a short-range disorder (like in liquids) but some distinct order over larger distances (Jönsson et al. 1998). In lipid or amphiphile aggregation literature the expression liquid crystals usually contains all aggregated forms except the micellar phases, that is for example hexagonal, lamellar and cubic phases.

In this thesis lyotropic liquid crystals, normally composed of two or multicomponent systems, are discussed. In the liquid crystals used in for example computer screens and cellphone displays the structure change depending on temperature or electric field pulses. These thermotropic liquid crystals are usually composed of only one stiff elongated component.

Fig 2.11. Microviscosity data of IbuNa ( ) and SDS ( )in water as a function of the concentration related to the cmc. Cmc was taken as 180 mM and 8 mM respectively.Data from paper II and Evertsson et al (Evertsson and Nilsson 1997). See appendix for explanation.

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Hexagonal phase Hexagonal phases are often formed as the micelles become abundant in solution. The micelles first become elongated and finally form tubes packed in a hexagonal arrangement. Hexagonal phases are usually macroscopically quite stiff and gel like. In this thesis hexagonal phases were not studied. A reversed hexagonal phase is shown in figure 2.12.

Lamellar phase The lamellar phase is a planar structure where the monomers are packed in bilayer sheets with a water region in between. The ideal molecular shape of an amphiphile in this phase is a cylinder. Lamellar phases are fluid but frequently quite viscous. In binary monoglyceride/water-systems a liquid crystalline lamellar phase is formed at low water content (5-15%) at room temperature (Hyde et al. 1984; Qiu and Caffrey 2000). Lamellar phases in the MO/PEG/water system are discussed in paper III and IV.

Cubic and sponge phases Cubic phases can be divided into two main groups; the cubic closely packed aggregates and the bicontinuous cubic phases. The cubic packed aggregates consist of micelles or reversed micelles that are packed in a cubic symmetry. These lipid cubic phases are not so common in polar lipid systems and will not be considered more in this text.

The bicontinuous cubic phases are more spectacular since they consist of only one lipid double layer membrane that spans the whole phase, at least when the phase ideally consists of only one crystal. In the reversed bicontinuous cubic phases the membrane circumvent tubes of water with large dimensions. The water tubes are connected into two interconnected three-dimensional networks. There are different symmetries for cubic phases constituted by polar lipids, see figure 2.14, where the crossings of the water channels consists of six, four or three channels. The surface on which the lipid bilayer is centred is a so called infinite periodical minimal surface, IPMS. On this surface every point is a saddle point, i.e., it has a mean curvature equal to zero. (Lindblom et al. 1979;

Fig 2.12. Reversed hexagonal phase.

Fig 2.13. Lamellar phase with the molecular shape of the amphiphile indicated.

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Longley and McIntosh 1983; Andersson et al. 1988). In the monoglyceride monoolein/water-system at water

contents between 20 and 40% cubic phases, with two different space groups, are formed. The cubic phase, which contains the least water, belongs to the space group Ia3d. At higher water content the space group Pn3m is formed. As the water content is further increased a large two-phase region will form, cubic phase (Pn3m) and an aqueous solution of MO. The fully swollen cubic phase of the space group Pn3m has a characteristic spacing of 100Å consisting of a lipid double layer and a diameter of a water pore. Since the MO molecules are about 17 Å long (Seddon 1990; Chung and Caffrey 1994) the radius of water channels in the Pn3m cubic phase above is about 33 Å. Cubic phases are isotropic and macroscopically stiff. Cubic phases are discussed in paper III and IV.

Another phase, which has some similarities with the cubic phase, is the sponge phase, also referred to as the L3-phase. The L3-phase is a liquid but it is believed to be constituted by one lipid double layer membrane. The water channels of this phase are believed to be larger than those of the corresponding cubic phase. The IPMS of the L3-phase is believed to have the same topology as the cubic phase but since the water channels are larger the lipid bilayer is flatter. L3-phases are not formed in the binary monoolein/water-system but when solvents or other additives of different types are added an L3-phase can be formed (Ekelund 2000). L3-phases will be discussed in paper III and IV.

Cubic phases have been proposed as vehicles for various drug delivery systems (Engström 1990; Wyatt and Dorschel 1992) for example parenteral (Ericsson et al.

Fig 2.14. Units of three space groups of cubic phase; Im3m, Pn3m and Ia3d.

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1991), vaginal (Geraghty et al. 1996) and periodontal (Norling et al. 1992) drug delivery systems. The systems can also be used to incorporate proteins and maintain them in their native conformation thus protecting them from denaturation This property is also used in crystallisation of membrane proteins (Caffrey 2003) and in a enzyme-based biosensor (Razumas et al. 1994). The cubic phase may provide an environment similar to biomembranes that can be of importance for the activity of many proteins (Shah et al. 2001).

2.3 Interaction between polymers and surfactants in aqueous solution

Polymer-surfactant interaction studies are vast and stretch from pharmaceutical and cosmetic formulations to oil recovery. The research field has its origin in protein-surfactant binding studies in the 1950´s. There have been extensive studies over the last decades on various aspects of how the association of uncharged polymers and surfactant in dilute aqueous solution occur. Several reviews of the field exist (Breuer and Robb 1972; Goddard 1986; Brackman and Engberts 1993; Goddard and Ananthapadmanabhan 1993). The most studied system is the PEO/SDS-system. However cellulose ethers together with anionic and cationic surfactants have also been the subject of several studies during the last two decades (Carlsson 1989; Holmberg 1995; Nilsson 1995; Lindell 1996; Thuresson 1996; Rosén 1997; Evertsson 1999; Sjöström 2002).

Systems with EHEC, a surfactant and different drug molecules have been used as drug delivery vehicles. For example, Lindell et al investigated the release of timolol from EHEC/surfactant/water thermogelling systems, intended for ocular delivery (Lindell and Engström 1993). Moreover, Pereswetoff-Morath and collegues used the EHEC/SDS/water system to deliver insuline via the nasal route (Pereswetoff-Morath and Edman 1995; Pereswetoff-Morath et al. 1996) and the system has also been tested as a local anaesthetical delivery vehicle in the periodontal pocket (Scherlund et al. 2000).

23

As amphiphiles and polymers are mixed in solution an aggregation of amphiphilic micelles on the polymer chains is found when the amphiphilic concentration is higher than a limiting concentration, usually denoted as the critical aggregation concentration, cac. The cac is always lower or equal to the cmc, which means that the polymer induces the aggregation by a strong cooperative binding of the surfactant to the polymer or acts as sites for micellisation of the surfactant on or in the vicinity of the polymer chain (Jönsson et al. 1998).

The interactions between uncharged polymers and surfactants are weak and the primary driving force is hydrophobic attraction but other factors such as the polymer shielding of surfactant charges are also important. It is for example known that the interaction between uncharged polymers and anionic surfactants is stronger than with cationic or non-ionic surfactants (Goddard and Ananthapadmanabhan 1993). An explanation to this can be that the size of the surfactant head group is much bulkier for cationic/non-ionic than for anionic ones thus making the micellar headgroup regions less favourable to penetrate for the polymer coils.

In paper I and II, cellulose ethers together with either surfactants or amphiphilic drug molecules have been studied.

Effect of polymer In the first two papers of this thesis two cellulose ethers have been compared. They differ by their hydrophobicity, as monitored by their LCST, which has an impact on the interaction. The more hydrophobic cellulose ethers have been found to be more suitable as aggregation sites than the homogeneous and quite hydrophilic PEO chain. This is also seen in the lower cac-values for cellulose ethers than for PEO (Evertsson et al. 1998).

24

The HPMC sample investigated in the present work was more hydrophilic than the EHEC used due to higher hydrophobicity of substituted ethyl groups of EHEC compared to methyl groups of HPMC with relatively similar degree of substitution. Thus the EHEC-solution phase separated at a lower temperature (cloud point at 29°C for a 0.1% solution) than the HPMC (45°C). The onset of the interaction with amphiphiles also occurred at a lower surfactant concentration for EHEC than for HPMC. This was seen in lower cac-values for the amphiphiles together with EHEC, which is exemplified in figure 2.15.

0 5 10 15 201.0

1.2

1.4

1.6

1.8

2.0

LiDS EHECLiDS HPMC

cac EHECcac HPMC[LiDS] (mM)

I 1/I 3

Variations in polymer concentration up to 1% in the mixtures did not influence the cac whereas the saturation of the polymer was displaced to higher amphiphile concentrations.

The cooperativity against these two cellulose ethers did not differ significantly for the same amphiphile. This can be seen for example in steep decreases of the micropolarity index in figure 2.15. The effects of interaction with the less hydrophobic HPMC were however smaller than for EHEC seen both with macroscopic and microscopic methods. Macroscopically the increasing effects on the viscosity of the solutions were larger for the EHEC solutions.

Fig 2.15. I1/I3,Micropolarity index, as a function of LiDS concentration with 0.2% EHEC and HPMC. See appendix for method explanation.

25

Effect of amphiphile As mentioned earlier the amphiphilic drug molecule, IbuNa has a structure less favourable for micelle formation than many ordinary surfactants. This will obviously influence also its aggregation with the cellulose ethers. The association of IbuNa to cellulose ethers was found to be weaker than the corresponding association of dodecyl sulfates. In a simplified way the dodecyl sulfates are found to form micelles onto the cellulose ether chain below the cmc whereas IbuNa first forms micelles into which the cellulose ethers distribute. This description is however not entirely true as association effects can be seen below cmc for the drug molecule, see the increase in IM/IE in figure 2.16.

0 1 20

5

10

[konc]/cmc

I M/I E

One important factor to consider when comparing data of IbuNa with those of SDS is the much higher concentrations needed with IbuNa, due to the high cmc at 180mM. As the drug molecule is a fully dissociated salt the ionic strength of the solutions is considerable. The minimum in cloud point seen in the EHEC/IbuNa system is most likely a salting out effect resulting in conformational changes of the polymer, fig 2.17. The salting out effect is further illustrated by the addition of NaCl which lowers the cloud point over the whole drug concentration span, exemplified for EHEC in the figure.

Fig 2.16. IM/IE,microviscosity index, for IbuNa (filled symbols) and SDS (unfilled) in EHEC (squares) or HPMC (diamonds), as a function of the concentration related to the cmc. Cmc was taken as 180 mM and 8 mM respectively.

26

0 100 200 300 4000

20

40

60

80

0.5% EHEC0.5% HPMC

0.5% EHEC+NaCl

[IbuNa] mM

Clo

ud p

oint

(C

)

Effect of surfactant counterion As with ordinary micelles, the counterion of the surfactant is important for the start of interaction as well as the structure of the aggregates. The interaction was studied with both EHEC and HPMC. The onset of interaction, according to surfactant concentration, was found to be in the order KDS<NaDS<LiDS for both polymers. The same as that was found for micelle formation in aqueous solution. The explanation for this is the better shielding of the charges of the aggregates with the smaller K+ hydrated ions. Consistent with this is also the finding that the aggregates with LiDS are more fluid than those of NaDS and KDS.

Structure of the aggregates Measurement of microviscosity is a versatile tool for understanding the structure of the polymer-amphiphile aggregates formed as it gives an estimate of the fluidity in the aggregates. All systems, with both amphiphiles as well as both cellulose ethers showed the same general behaviour. The microviscosity increased up to a maximum, which was followed by a slowly asymptotically declining region as the amphiphile concentration increased, see figure 2.18.

Fig 2.17. The salting out effect seen as a minimum in cloud point. The cloud points are plotted as a function of the concentration of IbuNa.

27

0 100 200 300 400 5000.0

2.5

5.0

7.5

10.0

[IbuNa] (mM)

I M/I E

0 5 10 15 200

5

10

[XDS] (mM)

I M/I E

The aggregates first formed on the polymer chains are smaller and contain a higher degree of polymer chains since the average probe environment does not favour the excimer state of the probe. As more amphiphiles enter the aggregates, the environment for the probe becomes more fluid, so the probe can form excimers, and the aggregates become larger. This can further be seen in figure 2.19 where the average aggregation number, Nav, increases for the polymer-ibuprofen systems as the ibuprofen concentration increases.

As the total ibuprofen concentration is increased in the polymer solution, Nav strives towards 40 as of the binary ibuprofen/water system. The aggregation numbers of the polymer-bound micelles are lower that that of the unbound, also shown in the cellulose ether/SDS-system (Nilsson et al. 1995; Evertsson et al. 1996), which can be seen in the following calculation. In the 300 mM IbuNa/1% EHEC solution the EHEC-bound IbuNa concentration is 30 mM, with y=3 from figure 3 in paper II. This gives, with cmc at 180 mM, 90 mM of IbuNa incorporated into ordinary micelles, with an aggregation number of 40. There are thus 2.25 mM ordinary micelles in the solution. In table 1 in paper II the total micelle concentration for this solution is found to be 4.7 mM, giving a bound-micelle concentration of 2.45 mM and an average aggregation number for the polymer-bound micelles of 12.

In figure 2.19 it can also be seen that Nav is generally higher in the HPMC/IbuNa/water-system than in the corresponding EHEC system. This is a result of the stronger binding of IbuNa to EHEC than to HPMC, see figure 3 in paper II. The number of amphiphiles in the polymer bound aggregates were approximately the same

Fig 2.18. Microviscosity data for IbuNa interaction with 0.5% EHEC or HPMC (to the left) and for LiDS ( ), NaDS ( )and KDS ( )with 0.2% EHEC or HPMC (to the right). Filled symbols show EHEC samples and unfilled are HPMC.The lower IM/IEfor the samples with HPMC have earlier been shown to be an effect of the polymer hydrophobicity (Evertsson and Nilsson 1998).

28

for EHEC and HPMC.

200 300 400 5000

10

20

30

40

50

[IbuNa] (mM)

Nav

The variations in Nav in the SDS/EHEC/water-system have earlier been studied by fluorescence quenching (Nilsson et al. 1995; Evertsson et al. 1996). For LiDS it was unfortunately not possible to calculate Nav in that system due to method problems determining adsorption isotherms. Micelle concentrations could however be calculated from the fluorescence quenching measurements and these show that LiDS forms more micelles, unbound and polymer bound, than NaDS. This suggests that the LiDS aggregates are smaller with respect to the aggregation number taken that the total amount of LiDS or NaDS adsorbed to the cellulose ether is the same at a given total surfactant concentration.

2.4 Interaction of polymers and lipids in aqueous environment

The interactions of polymers and amphiphiles discussed in the previous section were characterised in solutions of more than 98% water. In the following discussion of polymer-polar lipid systems, the water content is decreased. Other important structures, i.e., lyotropic liquid crystals, can thus be formed.

Fig 2.19. Average aggregation numbers for ibuprofen micelles in water ( ) and in aqueous solutions of 1% EHEC ( ) and HPMC ( ) as a function of the total ibuprofen concentration.

29

The ternary PEG 400/MO/water-system As a background to the studies in paper III and IV the phase diagram of PEG 400/MO/H2O (Engström et al. 1998) can be considered. In this system PEG 400 can be described as a solvent for the water/MO mixtures as PEG 400 is completely miscible with both water and MO. A lamellar phase (L ), between 5 and 15% water, is maintained in the system up to 45% of PEG 400. The cubic phase remain up to 25% of PEG 400. As the MO concentration is further decreased, the cubic phase “melts” and forms a fluid phase, the so-called sponge phase (L3-phase). The phase diagram is shown in figure 2.20.

PEG 400

H2O MOQ

L

L3

The cubic phases The same phase behaviour as with PEG 400 with lamellar, cubic and sponge phases was found for PEGs with molecular weights up to 1500. In the phase diagrams with PEG 900 and larger, regions with solid phases were also found. The cubic phases where found to change dramatically as PEGs of even larger molecular weights were added. See examples of phase diagrams in figure 2.21.

Fig 2.20. The phase diagram of the PEG400/MO /water system. The corners of the triangle represent a sample with 100 w-% of the component. The dots indicate the samples made and the full drawn lines one-phase regions.

30

PEG 1500

H2O MOQ

L

L3

PEG 4000

H2O MOQ1

Q2L

L3

PEG 8000

H2O MOQ1

Q2

L3

L

PEG 20000

H2O MOQ1

Q2

L

L3

Polymer molecular weight Due to the total miscibility of PEG 400 both with water and MO the PEG can be present in both domains as well as on the interface. As it is quite hydrophilic it is however expected to primarily be present in the water domain of the cubic phase. The gain in conformational entropy for PEG will also be larger in water than in the lipid bilayer.

PEG molecules are easily soluble in water even at quite high molecular weights and can be used for osmotic stress experiments where the PEG extracts water from colloidal solutions and lipid bilayers (Parsegian et al. 1986).

The osmotic stress extraction process is thus the reason why the lattice parameter of the cubic phase decreases as the PEG content increases in a sample, figure 2.22. This is seen for all PEG molecular weights but at different concentrations regimes. This shrinking is also seen in the shape of the left border of the cubic region in the phase diagrams. At one point of increasing polymer concentration the lattice parameter starts to increase for PEGs up to 1500 in molecular weight whereas the cubic phase, denoted Q1 in the phase diagrams in figure 2.21,

Fig 2.21. Phase diagrams of PEG/MO/Water-systems. The molecular weight of PEG is altered.

31

goes through a phase transition for even larger PEGs. If the PEG content is further increased after the phase transition another cubic phase, denoted Q2, is found in the phase diagrams. This is seen for PEGs up to at least 20000 in molecular weight. Preliminary results indicate that this cubic phase can be found in systems with PEG 43000 and 78000 as well (Ridell 2003). For this new cubic phase it has been hard to characterise the definite space group by SAXS but we have indications that it is very swollen with large lattice spacing.

0.0 0.1 0.2 0.3 0.480

100

120

140

160

180

weight-% of PEG in sample

a (Å

)

This general behaviour of first a shrinking followed by a swelling of the cubic phase is thus seen for all molecular weights. However the shrinking of the larger PEGs is so abundant that the cubic phase finally cannot exist.

Although the hydrodynamic radius of a larger PEG (41Å for PEG 8000) is larger than the water channels (about 33Å) of the MO/water cubic phase the PEG is present in the phase. The preferred conformation of the PEG in water must thus be altered and it looses thus some of its conformational entropy. It is thus possible that the PEG also is situated at the water-lipid interface and it thus flattens the bilayer by adsorption to it. The loss in conformational entropy will be gained in adsorption. This adsorption effect is similar to the adsorption effects of polymers in microemulsions (Kabalnov et al. 1996) or the lowering of cmc for surfactants as a polymer is added (Goddard and Ananthapadmanabhan 1993). Usually more hydrophobic polymers are known to adsorb at lipid

Fig 2.22. The lattice parameter determined by SAXS of fully swollen cubic phases in the PEG/MO/water-system. The water content was kept constant at 40 % thus varying the PEG and MO content. shows the sample of a fully swollen binary MO/water-sample. ,andcorrespond to PEG 400, PEG 900 and PEG 1500 respectively. All samples belonged to the Pn3m space group except PEG 1500, which displayed an Ia3d structure.

32

interfaces but as it seems from these results also hydrophilic ones can show this behaviour.

Diffusion experiments on the swollen cubic phase (Q2),composed of 40% aqueous solution of PEG added to MO, indicate that the polymer is in the water domain to a large extent as it diffuses as if in a 50% aqueous solution. Obstruction either by adsorption to the lipid bilayer or by the network of bicontinuous water channels is however important for the decrease in diffusion coefficient.

The minor amount of PEG that can enter the shrunk cubic phase (Q1) is probably situated on the lipid bilayer as well. The sizes of the water channels, as determined by SAXS, in these cubic phases are too small for the PEG to be in its preferred coil state. It is thus probable that the PEG is forced to interact with the bilayer, evidenced by a decrease in diffusion coefficient compared to a binary aqueous solution. In Figure 2.23 the shrinking and the swelling processes are visualised. Figure 2.23. The

two opposing processes in the PEG/MO/water-system. a) The shrinking of the cubic phase results from PEGs osmotic stress on the lipid membrane. The water is extracted from the cubic phase. b) The Pn3m cubic phase with no PEG added. c) The swelling results from the curvature changing effects of PEG adsorbing to the lipid membrane. The adsorption of the polymer on the lipid surface decreases the curvature around the water.

33

The sponge phase In the PEG 400/MO/water system the L3-phase was found at fairly constant water content of 30%, with a broad variation in PEG 400 content (between 30 and 65%), see figure 2.20.

Sponge phases have also been found in MO/water systems with various types of solvents or additives such as N-methyl- -pyrrolidone, propylene glycol, dimethyl sulfoxide, 2-methyl-2,4-pentanediol, acetonitrile and ethanol (Engström et al. 1998; Ekelund 2000; Imberg and Engström 2003; Imberg et al. 2003). One striking effect was that the sponge phase appeared at approximately constant water content for a given solvent. This water content could be correlated to the lipophilicity of the solvent. A solvent with an octanol/water partition coefficient below one induces a sponge phase at a specific water content in the solvent/MO/water-system. A lower partition coefficient gives a sponge phase at low water content (Engström et al. 1998; Ekelund 2000).

Polymer molecular weight All molecules found so far inducing a sponge phase in the MO/water system are small, 50-400 in molecular weight. In the case of PEG it was found that the MW was unimportant for the sponge phase formation in the molecular range investigated. One finding was namely that polyethylene glycols gave rise to sponge phases although the molecular weight was increased considerably. The L3-phase was found for PEGs with molecular weights from 300 (Evertsson et al. 2002) up to at least 20000 (paper III or IV). The finding that the sponge phase was formed at approximately the same water content, 30 wt-%, for all PEGs implies that the degree of polymerisation is not of importance for the sponge phase formation. In the work of Evertsson and coworkers (Evertsson et al. 2002) it was inferred that the PEG (300-600), was in the water domain. The PEG had however a higher relative partition to the lipid headgroups than water.

A critical number of monomers of PEG are thus more important than the exact size of the molecule. Although the water content is constant in the sponge phase the proportions of PEG monomers and MO vary over the phase region. The amount of lipid is thus decreased in the sponge phase at higher PEG content. As MO constitutes the bilayer also the proportions of bilayers compared to surrounding PEG solution is decreased. One way to keep the L3-bilayer would then be to increase the radii of the water channels by flattening the curvature of the IPMS-surface. The object for the highly concentrated PEG monomers will thus be to flatten the membrane.

The distribution of sponge phase in the phase maps of PEG with varying molecular weight was also found to be fairly similar, from 5 to 40% MO. The area was slightly diminished to 10-40% MO, for PEG 20000, when the

34

molecular weight was increased. This decrease in size can be explained by the solubility decrease of PEG in water with increasing molecular weight.

The sponge phase, as mentioned earlier, is a quite fluid liquid. The increasing molecular weight of PEG had, however, an effect on the viscosity of the sponge phase as it increased. As the PEG mainly is in the water domain of the sponge phase the viscosity of the sponge phase will be infuenced by the corresponding high viscous binary PEG/water-system.

Amphiphilic additives The properties of the sponge phase, such as the water content, can be changed by the solvent or by adding an amphiphilic component to the phase. The curvature of the sponge phase membrane will thus be influenced and can be compensated by a larger water content. A thorough study was performed with amphiphiles of different properties.

Fig 2.24. A schematic sketch of the proposed behaviour of the sponge phase bilayer as conically shaped surfactants are added to it.

35

As mentioned earlier the curvature of the sponge phase membrane is negative, i.e., it bends around the water. A sketch of the membrane can be seen in figure 2.24a. The MO, which is the chief ingredient in the membrane, has thus a slight wedge shape. Introducing a conical molecule in the L3-membrane will then flatten the membrane and eventually induce a phase transition into a lamellar phase, figure 2.24b. However, if more water is introduced to the sample, larger water channels can be formed and an L3-phase can be stabilised, figure 2.24c. With more water added both the amphiphile and the PEG could be redistributed to the water domain decreasing their flattening effects. The membrane is thus regiven the optimal curvature for the sponge phase. This was found with for example SDS as seen in figure 2.25.

H2O MO

PEG 400

*0% SDS

1% SDS

L3

L

Q

The amount of SDS added is of course of importance for the amount of water needed to compensate the flattening effect. Since SDS is a charged amphiphile the water addition has to compensate both for the flattening of the membrane and also for the charge repulsions that exist between the monomer molecules. This effect of charge repulsion can be verified with the addition of an uncharged amphiphile in the place of SDS. Adding equal amounts of Brij 58 or -octyl glucopyranoside to the MO/PEG 400/water sponge phase did not require as much water as was the case for SDS.

Fig 2.25. The phase diagram of PEG400/MO/water at roomtemperature showing a projection of the sponge phase position as SDS is added.

36

Substance Max added

(w/w-%)a

Maxadded(mM)b

Max water content (%)c

SDS 1.0 0.036 50 IbuNa 2.8 0.123 54 ChloHCl 2.6 0.073 41 CTAB 1.5 0.040 42 Brij 58 5.9 0.052 32 Brij 700 9.1d 0.019 38 Cholesterol 4.2d 0.109 24 a The maximum amount added of the substance to a sponge phase of PEG 400/MO/water with a G/P-ratio of 1. b The same as a but in mM. c The maximum amount of water that can be incorporated into a

sponge phase of substance/PEG 400/MO/water. d At least these amounts can be added.

The results in c and a/b do not necessary come from the same samples.

Compared to SDS IbuNa could be added to the sponge phase to a much larger extent, 3% versus 1%, which probably is the effect of the stiffer and bulkier molecule and thus a lower flattening effect. This implies that IbuNa may have a more cylindrical shape than SDS or that it is incorporated in the membrane to a lower degree. The sponge phase was found at lower water contents when cholesterol was added to it than without. It seems from these results that cholesterol promotes a slightly negative curvature thus decreasing the size of the water channels.

A tempting analysis to make from these results is thus to try and find a correlation between the water swelling and the critical packing parameter of the added substances. However, such an analysis is not obvious to do since not only the molecular shape is responsible for the amount of water in the water channels but also the solubility of the substance in water and the membranes respectively as well as shape of the surfactant head group. A substance that has higher water solubility will not partition into the membrane to the same extent as a low-solubility substance.

The small increase in water content but large amount of added substance for the polyoxyethylene alkyl ethers (Brij 58 and 700) can be explained by their chemical structure. These substances can be viewed as a hydrocarbon chain coupled with a PEG. Adding them to the sponge phase can thus be compared with an increase in equal amounts of MO and PEG.

Table 2.2. Amount of different substances that can be added to a sponge phase. The maximum amount of water in a modified sponge phase is also included.

37

3 In solid state

3.1 Polymers in solid state Polymers in solid state can contain both crystal and amorphous regions. Some polymers are mainly crystalline whereas others are completely amorphous (Young and Lovell 1991). The general crystallisation behaviour will be discussed briefly here, as it is important for the results of paper V. In figure 3.2 the hierarchical structure of semicrystalline polymers are illustrated. The largest structural units, which can be seen by eye or a microscope are the spherulites. Their size is on the mm-scale (Price and Kilb 1962). Spherulites consist of fibrils growing from the center. The fibrils are bundles of twisted lamellar stacks, while the lamellar stacks are sequences of crystalline and amorphous polymer in repeating layers. The crystalline parts are often helically twisted polymer chains that sometimes are folded (Buckley and Kovacs 1976).

In the case of PEG the crystalline part has been shown to consist of helices with a repeating unit of 19.3Å which is built up of seven ethylene oxide units in two turns of the helix (Takahashi and Tadokoro 1973). The chains in the crystalline PEG can be either completely extended or folded. PEG 8000 can for example fold 1, 2 or 3 times thus creating crystalline regions of three different length (245, 164 and 122Å) (Buckley and Kovacs 1976), see table 3.1. Not the entire polymer is crystalline in solid state but there are also amorphous regions both in the lamellar stacks and in regions between the fibrils and the spherulites.

Mw N L (Å)

8000 1 245 8000 2 164 8000 3 122 4000 1 123 4000 2 82

Table 3.1. Length of the repeating part, L, of the lamellar stacks for N times folded PEG. See figure 3.1. below. Data from Buckley (Buckley and Kovacs 1976).

Fig 3.1.

38

The crystallinity of PEG varies depending on the molecular weight. It has been shown that PEG between 2000 and 10000 is quite crystalline with 6000 being most crystalline (Faucher et al. 1966). Larger PEGs will have comparably larger problems folding ideally into the lamellar stacks thus leading to lower crystallinity.

The number of folds has an impact on the thermodynamic stability of the crystals. The extended PEG is more stable than the once folded one, which in its turn is more stable than multifolded PEG of the same molecular weight.

In this thesis the behaviour of one polymer has been studied in the solid state, i.e., PEG 4000. Ordinary PEG 4000 is usually present in one form, the extended and more stable form, that is seen as the fulldrawn line in figure 3.3. However, if PEG 4000 is studied only hours

Fig 3.2. The hierarchy of structures within a solid state polymer. From the spherulite to the atoms in the helix.

39

after the crystallisation two forms of PEG 4000 is found. Also a folded form can be seen from the double peak in the thermograms.

40 50 60 70 80

temperature ( C)

dsc

(W

)

When a diluent is added to a solid semicrystalline polymer it can arrange itself in different regions. It can be enriched in the interlamellar, the interfibrillar or the interspherulitic regions (Stein et al. 1978; Chen et al. 1998). In this thesis MO has been used as a diluent with respect to the semicrystalline PEG.

3.2 Lipids in solid state Lipids in solid state are polymorphic, which means that they can be in several physical forms. The temperature history of the sample determines the crystal arrangements, the so called polymorphs. They are close-packed lipid molecules in different ways. Some main polymorphs exist for all lipids. These are the -form, ´-form and the most stable -form (Larsson 1994).

When a melted fat is cooled it organises into a membrane structure, which also can be seen to some extent in the liquid state, where the polar headgroups are packed together in lamellar sheets. Within the lamellar sheets the lipids are arranged in a hexagonal order and the individual lipids are free to rotate on their place. This polymorph is called the -form.

Fig 3.3. Thermogram of PEG 4000 obtained 10 minutes (- - -) and 10 days ( )aftercrystallisation.

Fig 3.4. The upper sketch symbolises the lamellar organisation of the -polymorph and the lower -or the ´-polymorph. The indicated distances ( ) are equal in both polymorphs as monitored by SAXS.

40

When the -form is further cooled the hydrocarbon chains are fixed in an orthorhombic arrangement as they cannot rotate any more and the polymorph ´ is formed. The hydrocarbon chains are fixed with every second chain perpendicular to the other. The most stable polymorph is the -form, which is formed upon storage or further cooling. In this polymorph the hydrocarbon chains are packed into a triclinic subcell all parallel to each other.

All three polymorphs show the same diffraction in small-angle x-ray diffraction, since the lamellar spacing, in figure 3.4, is intact throughout the stabilisation process, making the method incomplete to distinguish between the polymorphs. However, in wide-angle x-ray diffraction it is possible to distinguish between the polymorphs as these have different characteristic lattice spacings. The -form has a strong diffraction line at 4.15 Å, the ´-form has two lines at 4.2 and 3.8 Å and the -form has one strong diffraction at 4.6 Å (Larsson 1994).

This is the general behaviour for all lipids but depending on the complexity of the lipid molecules the -form, ´-form and the -form do not always exist for all lipids. Some lipids form more polymorphs and others fewer.

3.3 Mixtures of polymers and lipids Co-melting of polymers and amphiphilic compounds constitutes a way of creating solid dispersions of these systems since they can mix easily in the melted state and solidify rapidly at fairly similar temperatures. The research area of characterisation of solid dispersions of polar lipids and water-soluble polymers is however not vast. Melting behaviour of PEG and fatty acids has been studied recently (Pielichowski and Flejtuch 2003) as well

Figure 3.5. Three polymorphs seen along the hydrocarbon chains. The circles symbolise the polar head group of the lipid and the black lines indicate the direction of the hydrocarbon chains.

41

as the inclusion of amorphous polymers (Dreezen et al. 1999; Dreezen et al. 1999; Dreezen et al. 2000; Shieh et al. 2002) or surfactants (Aldén et al. 1994; Wulff and Aldén 1995; Wulff et al. 1996) into the semicrystalline PEG.

Diluents that influence or are incorporated in the lamellar structure of the polymer can be studied with good result with x-ray diffraction techniques. The correlation function enables us to study characteristic distances in the samples such as the repeating distance in the lamellae and the distances of the crystalline and amorphous parts.

The diffraction pattern of PEG 4000 without a diluent contains no peaks originating from polymer packing, fig 3.6. The absence of peaks does however not results from a 100% amorphous sample but comes from the fact that there are no detectable repetitive differences in electron density in the sample. This was confirmed by DSC where the heat of melting corresponded to the literature values of 100% crystalline PEG. The diffraction pattern of 100% MO contained one large peak and smaller peaks of the second and third order reflections of lamellar packing with 52 Å as the characteristic length. When smaller amounts of MO, 2.5% and more, are added, peaks originating from the polymer packing become detectable. These peaks become more apparent as the MO content increases and are indicative of the lamellar packing of the polymer. Examples of these peaks are seen in figure 3.6.

0.0 0.1 0.2

100% MO

100% PEG

30% MO

q (Å-1)

Inte

nsity

The MO is believed to mix with the PEG lamellar

Fig 3.6. SAXS diffraction patterns of PEG 4000, MO and a mixture of them at 25°C.

42

phase in an organised way. MO up to 5% is not detectable by DSC or SAXS and organises itself into the amorphous regions of PEG. The PEG is however mostly in its extended form at this stage. At higher MO concentrations the monoglyceride starts to form two different separate regions. The double melting peak of MO seen in samples with 15-30% MO is indicative of this. In figure 3.7 it is found one melting peak for MO at 34ºC at lower MO content and another at 32ºC at higher MO content. At intermediate compositions both peaks are found.

25 30 35 40

25%

30%

8%

15%

0%

Temperature ( C)

dsc

(W

)

The most striking result is that the lipid regions formed at higher MO contents belong to the less stable ´-formwhereas the smaller regions formed at lower lipid content belong to the -form. A model of the believed structure of the regions formed is sketched in figure 3.8. Regions of extended PEG are here mixed with regions of folded PEG in lamellar stacks of crystalline and amorphous PEG. The smaller lipid regions symbolises the -form and the larger the ´-form.

Fig 3.7. DSC thermograms of mixtures of PEG 4000 and MO showing the MO melting peak. The MO content in the samples is indicated.

43

Since the lipid regions first formed are not detected by SAXS it is probable that the regions are very small and possibly randomly distributed. A plausible reason for this is that regions with only few repeating units will show only a very weak diffraction pattern. Another possibility is that these lipid regions mainly are incorporated in the amorphous parts of the PEG lamellar structure. From the SAXS data together with the correlation function (for details see appendix), it was possible to determine dimensions of crystalline and amorphous parts in the polymer lamellar stacks, denoted lc and la in figure 3.8.

0 10 20 300

40

80

120

160

200

X MO (%)

dist

ance

(Å)

In figure 3.9 the dimensions of these parts are shown. The mean amorphous region is found to increase from about 30Å to approximately 50Å as the MO content is

Fig 3.8. A schematic sketch of how the polymer and lipid regions are distributed in the mixture.

Fig 3.9. The length dimensions of the crystalline ( ) and amorphous ( )as well as the total length ( ) of a repetitive unit in the PEG lamellar stack.

44

increased. It is thus possible for MO molecules to be incorporated there as well as in separate regions. The larger lipid regions proposed in figure 3.8 are probably of the less stable ´-polymorph, as they were detected in DSC.

Several attempts have been made within our research group to verify these regions by a microscopy method, i.e., atomic force microscopy, but due to the apparent stickiness of the sample no reproducible images have yet been achieved.

45

4 Conclusions

Physico-chemical characterisation has been the main theme in this work and several interesting aspects on the interaction of amphiphilic substances and polymers have been found.

The association of cellulose ethers, of varying hydrophobicity, and amphiphilic substances, both hydrophilic drugs and classical surfactants, has been characterised. The hydrophobicity of the polymer was found to have an impact on the interaction scheme. A more hydrophobic polymer gives an earlier onset of the interaction with for the surfactants.

The choice of counterion to the amphiphile has a small but significant effect on the interaction and the structure of the aggregates. Also amphiphilic drug molecules can interact with nonionic polymers in a way similar to that of surfactants in aqueous solution. Due to the higher cmc of ibuprofen, the interaction is largely influenced by the ionic strength of the solution. The type of amphiphile also influences the cooperativity of the amphiphile-polymer binding.

In more concentrated systems liquid crystals are formed into which the polymer interact with the amphiphiles. Both cubic and sponge phases were found with relatively large polymers interacting with polar lipids. These phases were found to swell and shrink mainly controlled by the amount of polymer inside them. Also membrane interacting substances added to the sponge phase could influence the size of the water channels in the phase.

In water free systems polymers and polar lipids were found to interact as well as forming solid dispersions. The behaviour of the phase separation between polymer and lipid depended on the concentration of the dispersed phase. The polar lipid was found to be distributed in the lamellar part of the semicrystalline polymer influencing the polymer folding.

4.1 ProspectsThe research area of polymer-amphilphile interaction has been in focus for several years due to the vast possible applications in pharmaceutics among others. However there are still problems to be solved. Characterisation of

46

these systems is always needed. With this thesis as a background, several characterisation studies and other aspects can be proposed:

The interaction of a more hydrophobic polymer, such as EHEC and more hydrophobically modified types, with the liquid crystals of polar lipids. How will it partition into the lipid domain and how will that influence the phase behaviour? An adsorption effect would probably be seen also probably with even larger flattening effects.

The polymer molecular weight dependence on the solid dispersions of PEG and MO. A larger PEG would have larger amorphous regions and might thus better mix with MO. The larger regions with MO will probably form at a larger amount of MO added. The dimensions of the polar lipid regions in the solid PEG could also be interesting to study.

The behaviour of different types of drug substances in a solid dispersion of PEG and MO. Lipophilic drug substances would probably mix in the MO regions. A distribution to the smaller lipid regions would be preferable if a fast dissolution of the drug is preferred. An amhiphilic drug substance would probably mix in the amorphous regions of PEG or with the lipid. A distribution into the lipid regions could give a slower release compared to the release from a solid dispersion with only the polymer. Studies on the fate of the solid dispersions of PEG and MO with and without the solubilised drug in water or simulated gastro-intestinal fluids could give information on this.

47

5 Acknowledgements

The studies in this thesis were performed at the former department of Pharmaceutical Chemistry and the department of Pharmacy, Uppsala University. I wish to express my sincere gratitude to:

Prof Em. Lars-Olof Sundelöf, min första handledare och framför allt lärare i fysikalisk kemi för att du fick upp mitt intresse för ämnet och inspirerade mig att börja doktorera.Prof. Sven Engström, min handledare för din smittande entusiasm för flytande kristallina faser och kemiundervisning samt ditt stöd, och för ditt tänjande på alla möjliga deadlines... Prof. Martin Malmsten, min nyaste handledare, främst på pappret, för att du kommer med många idéer till institutionen och framförallt för många bra kommentarer på avhandlingsmanuskriptet. Prof. Göran Alderborn, institutionens prefekt, för möjligheten att genomföra min forskarutbildning vid Institutionen för Farmaci och för att du varit hjälpsam i samband med disputationen samt för den trevliga stämningen på institutionen.Dr. Stefan Nilsson, min handledare för att du introducerade mig i EHEC/SDS-systemet och för din effektivitet vid pekskrivning samt för din trevliga familj som alltid är kul att stöta på i skogen. Dr. Hans Evertsson, medförfattare för att du är en entusiastisk idéspruta och för att du visade hur lugn man kan vara vid avhandlingsskrivande och för att du visar på andra värden i livet viktigare än jobbet. Dr. Christina Holmberg, my exjobbshandledare för att du introducerade mig i EHEC/SDS-systemet och din vilja att utveckla det. Denny Mahlin, medförfattare för givande samarbete med många intressanta vinklingar. Nu hoppas jag ha tid att tänka på fortsättningen! Tänk om AFM kunde funka... Dr. Göran Frenning, medförfattare för ditt intresse att förstå vårt ”system” och stimulerande samarbete. Dr. Magnus Nydén, medförfattare, för givande samarbete och hjälp att förstå NMR.Dr. Katarina Ekelund, medförfattare, för effektiv pekskrivning.

48

Dr. Satish Singh, för hjälp vid mikrokalorimetrimätningarna för länge sedan. Nils-Olov Ersson för stor hjälp med WAXS-mätningarna för inte så länge sedan.Nuvarande och f.d kollegorna i ”fysikalen” för många trevliga aktiviteter både på och utanför BMC: Adam, Dr. Anna, Anna, Dr. Anders, Dr. Andreas, Birgitta, Carina, Denny, Dr. Emma, Gunilla, Dr. Hans, Dr. Kiomars, Lotta, Prof.Em. Maggie, Dr. Marie, Nadia, Dr. Ninus, Dr. Per, Dr. Per, Peter, Dr. Tomas och Tobias. Josefina för att det varit väldigt kul att dela rum med dig. Markus Lundgren, Fia Thelin, Frida Karlsson, Johan Unga för allt ni bidragit med till det här arbetet under era exjobb och sommarjobb. Alla andra doktorander och övriga på Institutionen för Farmaci för den trevliga stämningen och hjälp på olika sätt. Göteborgsdoktoranderna (Jenny, Johanna, Johan, Pia) för att det alltid är trevligt att hälsa på i Göteborg de få gånger det händer. WC-tjejerna för god mat, trevliga stunder, skratt och whiskey. Dorothe, Sanna, Christian mfl. för att ni visar att det går att kombinera ett effektivt post-doc liv med ett intensivt orienterande.Övriga medlemmar i IF Thor, Sveriges mest PhD-frekventa klubb?, för att vi har det så roligt ihop.Övriga vänner och familj (både min och Petters) för trevlig samvaro de få gånger jag inte orienterar på fritiden! Karin och Petter för att ni orkade läsa igenom mitt manuskript trots att ni inte fattade något och ändå kunde ge vettiga kommentarer! Petter, min bästa vän för din kärlek.

49

Appendix – Physico-chemical characterisation methods

In order to determine the characteristics of a sample several techniques are available for the physical chemist in the colloid and surface field of research. The focus of this description will be on the techniques that have been used in this thesis.

Visual observations To visually observe the sample before more thorough investigations are conducted is something that should not be neglected. The samples are prepared in glass vials and usually left to equilibrate at specific temperatures before examination occurs. First the number of phases in the sample and the characteristics of them e.g. liquid, solid, gel like, clouded etc. is determined. In cross polarised light further information is achieved. For example lamellar and hexagonal phases are anisotropic whereas liquid and cubic phases are isotropic.

ViscometryViscometry is a simple but versatile method, which provides information of polymer behaviour in solution. The measurements in this thesis were made with capillary Ostwald and Ubbelodhe viscometers. The difference between the two types is that in the Ubbelodhe, which is a development of the Ostwald type, it is possible to dilute the sample between measurements thus making it easy to determine intrinsic viscosities.

According to Poiseuille’s law the viscosity, , of a liquid flowing through a capillary of length L and radius r is,

LVtPr

8

4

where P is the pressure difference across the ends of the capillary and V is the volume of the liquid flowing through. The measurements are very simple

50

as they consist of measurements of a flow time of constant volume of a solution through the capillary. This flow time, t, is compared to the flow time of a reference solution, often the solvent used, t0, giving the specific viscosity as

sp. sp is defined by,

oosp t

tt 00 .

This is true if both t and t0 are measured in the same viscometer, thus making it unnecessary to know the exact dimensions of the viscometer. The reduced viscosity,

sp/c, is usually followed as a function of polymer concentration and extrapolated to zero concentration where the intrinsic viscosity, , is given as the intercept. The intrinsic viscosity is then a measure of the hydrodynamic volume per mass unit of the polymer.

If a polymer chain is assumed to form a spherical coil in water its radius, r, can be determined from the intrinsic viscosity. Together with Stoke-Einstein’s equation an assumed diffusion coefficient, D, in water can be determined:

31

43

A

w

NMr and

rkTD

6where Mw is the molecular weight of the polymer and

NA is Avogadro´s constant. T is the absolute temperature, kBoltzmann´s constant and the viscosity of water. This approach was used in paper III.

Dye solubilisation Many of the systems studied in this thesis contain lipophilic domains. One possibility to quantify these domains or to determine the start of aggregation is to use lipophilic dyes. A dye with almost no water solubility will dissolve in these domains giving colour to the solution. The degree of colour of the sample, easily measured in an UV/Vis-spectrometer, is proportional to the amount of lipophilic domains. This is a fast and easy method to get an estimate of the critical micellar concentration of surfactants. One disadvantage of the method is that the

Fig A.1. Viscometer.

51

dye can influence the micellar aggregates and act as sites of micellisation thus giving a value lower than with other methods.

Equilibrium dialysis Equilibrium dialysis experiments can be performed in order to determine the adsorption isotherm of a substance onto the polymer. The dialysis cell used consists of two compartments of 2 mL volume each, separated by a dialysis membrane (Spectra/Por with a Mw-cut-off of 12000-14000) according to a principle developed by Fischman and Eirich (Fishman and Eirich 1971).

The polymer solution was placed on one side of the dialysis membrane, with a water solution on the other side, both sides with equal surfactant concentrations at start. The cells were placed to equilibrate and the surfactant concentrations on both sides of the membrane were determined. The equilibrium surfactant concentration not bound to the polymer ([surf]eq), the amount of surfactant bound to the polymer (y, in mmoles/gram polymer) and the total surfactant concentration ([surf]tot)can then be calculated. The mass balance equation then becomes

ycsurfsurf peqtot

where cp is the polymer concentration in grams per litre.

Thermal methods Methods involving thermal effects can roughly be divided into two groups; either the temperature is varied or it is kept constant. In this thesis one method from each group has been used and these are discussed below.

Titration microcalorimetry Microcalorimetry is a method where the temperature is kept constant over time and variations in heat added or withdrawn from the sample is recorded in order to keep it

Start:

Equilibrium:

Fig A.2. Principles of equilibrium dialysis. At start the amphiphile concentrations are equal on both sides. Water and amphiphiles are allowed to pass the membrane and at equilibrium the resulting concentrations are determined.

Fig A.3. Sketch of a titration microcalorimeter.

52

on the same temperature as a reference. Usually samples are studied over time in for example controlled humidity, constant or varied.

In titration microcalorimetry, which was used here, a solution, for example of a surfactant, is titrated in small portions into the sample compartment, containing a polymer solution or water, and the resulting heat effects recorded.

Differential Scanning Calorimetry, DSC Differential scanning calorimetry is a thermal method where the temperature of a sample and a reference is varied according to a programmed scheme. The heat recorded is the difference in added or withdrawn heat to keep the temperature of the sample the same as for the reference. The results of DSC are usually presented as transferred heat versus temperature in a thermogram. A thermogram of melting is shown in figure 3.3 and the area under the peak corresponds to the enthalpy of melting. For solid materials DSC can be used to determine the degree of crystallinity in a mixed sample by comparing the enthalpy of melting a 100% crystalline material with the enthalpy of melting of the sample.

X-ray diffraction X-ray diffraction is a technique where x-rays are used to determine distances within repetitive materials. Crystalline materials, both solid and liquid crystals, can be characterised. One approach to describe x-ray diffraction of crystals is the approach proposed by Bragg, where crystals are regarded as built up in layers acting as semitransparent mirrors. The x-rays encountering atoms on the mirror plane are reflected off the plane with the angle of reflection equal to the angle of incidence. The rest of the x-rays are transmitted to the succeeding planes. From the distances and angles in figure X Bragg´s law,

nd sin2 , can be derived. Bragg’s law is satisfied when the reflected beams are in phase and interfere constructively.

53

In the x-ray diffraction equipments used in this thesis a copper source is used to generate the x-rays. A nickel filter is used to separate off low and high energy x-rays giving a monochromatic beam of 1.54 Å. The crystals are taken to be arranged in all possible directions, as they should be in the powder method so when the x-rays hits the sample at least some of the planes are oriented at the Bragg angle, , giving rise to diffracted beams to be detected.

Small angle x-ray diffraction, SAXS Depending on the position of the detector different diffracting angles and thus distances can be examined. In classical x-ray diffraction distances between atoms and molecules in solid crystals and their unit cells are measured. The angles to achieve these measurements are quite wide, 10-50° and the technique is thus called wide angle x-ray scattering, WAXS. However if the detector is placed closer to the incident beam, diffraction at smaller angles, 0-10° can be detected. From Bragg’s law it is apparent that longer distances such as thicknesses of lamellar sheets can be estimated with SAXS.

In this thesis SAXS has been used to determine dimensions and space groups of cubic phases. It has been

Fig A.4. Principles of Braggs law.

Fig A.5. Principle of the x-ray diffraction experiment.

54

shown that the intensity peaks of a space group are arranged at specific positions in the diffraction pattern. For the Pn3m cubic phase peaks are found at scattering vectors, q,

dnq 2

,

where d is the lattice spacing of the cubic phase and ,...10,9,8,6,4,3,2n . For the Ia3d and the Im3m cubic

phases ,...11,10,8,7,4,3n , and ,...12,10,8,6,4,2n respectively.

SAXS can also be used for analysis of solid materials such as semicrystalline polymers. The one-dimensional correlation function (Vonk and Kortleve 1967) 1(x) can be used in order to obtain structural information about lamellar two-phase systems with differences in electron density, . For intensity data collected by using a slit of finite length, the one-dimensional correlation function reads (Ruland 1977)

dqqxqxJqxJIqIqQ

x b 1001 21

,

where I(q) is the slit-smeared intensity, 2sin4q is the magnitude of the scattering vector, Ib is the background (assumed to be constant in the region of interest), and J0 and J1 are zero- and first-order Bessel functions (of the first kind). Moreover, the scattering invariant Q is defined as

dqIqIqQ b021

.

From the correlation function properties such as thicknesses of repetitive crystalline and amorphous regions have been determined in paper V.

L 2L

d

x

(x)

x

(x)

x

(x)

0

1

L 2L-y

x

1(x)

0

1

L-y x

1(x)

0

1

L

x

1(x)

-y

a b c

Fig A.6. In the upper row electron density distribution, (x) with different variations both in thicknesses of the high and low density regions. In the lower row the corresponding one dimensional correlation functions, (x), with the main parameters used in the analysis; L and y, indicated. The figure is adapted from Strobl and Schneider (Strobl and Schneider 1980).

55

In a) in figure A.6 a perfectly periodical two-phase system is shown. In b) variations in thicknesses of the low density regions result in the bunt transition from the low plateau at –y to the first maximum in the correlation function (-). Finally in c) variations in both low and high density regions results in further bunt transitions as seen in the figure.

Fluorescence spectroscopy In fluorescence spectroscopy a substance can be excited by light of a specific wavelength thus emitting light of a longer wavelength due to energy loss as it returns to its ground state. Naturally fluorescent compounds, fluorescent labeled compounds and fluorescent probes, can emit fluorescence. In this thesis fluorescent probes have been used to probe different environments in the systems. Depending on the physicochemical and fluorescence emitting properties of the probes different emission spectra are achieved. As in the case of dye solubilisation a drawback with fluorescence spectroscopy is that extra molecules are added to the samples, thus risking influencing the systems. To avoid this the probe concentrations are kept very low.

There are two main fluorescence spectroscopy techniques; the time-resolved compared to the steady-state approach. Steady state measurements are the most common experiments and are performed with a constant illumination of the sample. In the time-resolved measurements the fluorescent decay is observed over time after a very short exposure of light, usually achieved with a laser pulse. The steady state measurements are in fact average of time-resolved measurements. A benefit of using time-resolved techniques is that much molecular information is not averaged out which can be the case in steady state measurements. The instrumentation is however more complex and more expensive. The work in this thesis has mainly been performed on steady state equipment.

Fig A.7. The micropolarity probe pyrene.

56

MicropolarityThe probe pyrene, fig A.7, is sensitive to the polarity of the environment where it is partitioned. Pyrene has a low water solubility of about 10-6 M. If lipophilic regions are present in the solution pyrene will partition to these regions to a higher degree than to the aqueous phase. The vibration emission spectra of pyrene in figure A.8 (recorded between 350 and 400 nm after excitation at 334 nm) change due to the environment and the peak height ratio of the first (I1, = 374 nm) and the third peak (I3, = 388 nm) is found to be a measure of the micropolarity of the probe site.

370 380 390

0.5mM LiDS20mM LiDS

wavelength (nm)

Inte

nsiti

es

In hydrophobic solvents the peaks are equally high, giving an I1/I3 index of 1.0. On the other hand, in pure water, which is the most hydrophilic solvent, the index is 2.0. A drop in I1/I3 indicates that hydrophobic regions are present in a solution and this can be used to determine the critical micellar concentration of a surfactant since the probe is believed to partition into the micellar aggregates.

MicroviscosityThe probe 1,3-di(1-pyrenyl)propane, P3P, consists of two pyrene molecules connected by a propane chain, fig A.9, making it capable of forming intramolecular excimers. An excimer is an aggregate of two or more pyrene molecules where the benzene rings of the molecules overlap. The

Fig A.8. Spectra of pyrene in a hydrophobic (20mM LiDS) and a hydrophilic (0.5mM LiDS) environment resulting in high and low micropolarity indices.

Fig A.9. The microviscosity probe P3P.

57

tendency of the probe to form excimers is believed to be a measure of the viscosity of the microenvironment of the probe, which should not be confused with the bulk property expression viscosity. Since the probe is extremely insoluble in water it is believed to partition into micelles or other hydrophobic regions as soon as these are present in the solution. If the probe is free to move in this environment it can form excimers to larger extent than if it is hindered, for example due to very small aggregates.

350 400 450 500

20mM LiDS3mM LiDS

Wavelength (nm)

Inte

nsiti

es

The degree of excimer formation can be recorded in the emission spectra of P3P, figure A.10, (recorded between 350 and 500 nm after excitation at 348 nm) as the peak ratio, IM/IE, between a characteristic monomer peak (IM,= 377 nm) and a broad excimer peak (IE, = 485 nm). A high IM/IE-ratio indicates that the probability to find a probe in the monomer state is higher than with a low IM/IE-ratio. The probability for intramolecular excimer formation is thus lower and the microviscosity relatively higher.

Aggregation number determination The average number of monomers in a micellar aggregate can be determined by both steady state and time-resolved fluorescence spectroscopy. In this thesis steady state measurements have been used in this respect whereas time resolved have been used to verify the data obtained.

Fig A.10. Spectra of p3p in a fluid (20 mM LiDS) and a stiff (3 mM LiDS)environment resulting in low and high microviscosity indices.

58

In steady state measurements a probe is used of which the fluorescence can be quenched by another molecule, a quencher. In this thesis the well studied probe-quencher pair of Ru(bipy)3

2+ and 9-MA has been used. The micellar concentration, [micelles] can be obtained from the relationship presented by Turro (Turro and Yekta 1978)

micellesQ

IIln , (1)

where I° and I are the probe fluorescence intensities in absence and presence of quencher, respectively. The mean aggregation number, N, can then be calculated by

micellescmcsurfN tot , (2)

where [surf]-cmc represent the molar concentration of surfactant monomers constituting the aggregates, taken that all surfactants exceeding cmc are incorporated in micelles.

0.0000 0.0001 0.00020.00

0.25

0.50

0.75 ln(I/Io)=0.00369+3203[Q][micelles]=1/3203M=0.31225mM

[9-MA] (M)

ln(I/

Io )

It is sufficient to use steady state measurements if there is no migration between probe and quencher and if the quenching is efficient which is the case with an efficient probe-quencher pair that distributes quantitatively to the aggregates. Time-resolved fluorescence was used to show that there were no signs of migration between probe and quencher and to monitor the presence of micelle-shaped clusters due to a large quenching rate constant.

Fig A.11. The quenching of Ru(bipy)32+ by 9-MA in 15mM LiDS and 0.2% EHEC at 37°C.

59

Nuclear Magnetic Resonance, NMR NMR is a method that is widespread in chemistry and physics with a variety of applications (Canet 1996). The most common application of the technique is structure determination in organic chemistry and on proteins. It is also used in medicine in so called “magnetic cameras” or Magnetic Resonance Imaging (MRI) machines to create images of the inside of the body. In physical chemistry different experiments are designed to determine anisotropy of samples or different dynamical aspects of molecules, for example diffusion.

Self-diffusion NMR The basic NMR experiment is performed by putting a sample in a very strong magnetic field, B0. In addition to this static magnetic field an ascillating radio frequency pulse sequence is used in order to perturb the spin systems. The response is given by an oscillating signal in the time-domain, which with a mathematical effort, i.e., a Fourier Transform, is transformed to the characteristic NMR-spectrum.

In this thesis NMR is used to determine diffusion coefficients of a polymer and a polar lipid in a cubic phase. The NMR experiments were designed according to figure A.12.

=4ms

g

=100ms

90 180

2echo

Fig A.12.The pulse sequence used in the PGSEexperiments. Adapted from Lindman et al. (Lindman et al. 1987).

60

The pulse sequence consisted of two radio frequency pulses (90 and 180 )separated by a time , grey bars in the figure. This sequence produced an echo at time 2 . This echo is the response of the sample. By introducing two magnetic field gradients of duration , indicated by white bars, and by successively varying the intensities 16 times during the experiment, the self-diffusion coefficients of the molecules in the sample can be measured as the echo is decaying with increasing gradients strengths. The distance between the magnetic field gradients is , i.e. approximately equal to .

The resulting NMR spectrum for the system investigated in the present work contains several broad peaks partially overlapping. In order to determine diffusion coefficients of the three components in the sample it is important to separate the peaks. An evaluation program developed by Stilbs (Stilbs et al. 1996) called CORE (COmponent REsolved spectroscopy) has been used for this purpose. The program takes into account the echo-decay of the complete NMR-spectrum rather than the peak area of single peaks in calculating the diffusion coefficients of the components.

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Acta Universitatis UpsaliensisComprehensive Summaries of Uppsala Dissertations

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A doctoral dissertation from the Faculty of Pharmacy, Uppsala University,is usually a summary of a number of papers. A few copies of the completedissertation are kept at major Swedish research libraries, while the sum-mary alone is distributed internationally through the series Comprehen-sive Summaries of Uppsala Dissertations from the Faculty of Pharmacy.(Prior to July, 1985, the series was published under the title “Abstracts ofUppsala Dissertations from the Faculty of Pharmacy”.)