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Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 826

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Liposomes for Drug Delivery

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from Physico-chemical Studies to Applications BY

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NILL BERGSTRAND

ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2003

Dissertation in Physical Chemistry to be publicly examined in B41, BMC, Uppsala University, on May 25, 2003, at 10.15 for the Degree of Doctor of Philosophy. The examination will be conducted in English.

ABSTRACT

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Bergstrand, N. 2003. Liposomes for Drug Delivery: from Physico-chemical Studies to Applications. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertation form the Faculty of Science and Technology 826. 71pp. Uppsala. ISBN 91-554-5592-1.

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Physico-chemical characterisation of structure and stability of liposomes intended for drug delivery is the central issue in this thesis. In addition, targeted liposomes to be used in boron neutron capture therapy (BNCT) were developed. Lysolipids and fatty acids are products formed upon hydrolysis of PC-lipids. The aggregate structure formed upon mixing lysolipids, fatty acids and EPC were characterised by means of cryo-TEM. A relatively monodisperse population of unilamellar liposomes was detected in mixtures containing equimolar concentration of the three components. The interactions between alternative steric stabilisers (PEO-PPO-PEO copolymers) and conventional PC-and pH-sensitive PE-liposomes were investigated. Whereas the PE-liposomes could be stabilised by the PEO-PPO-PEO copolymers, the PC-liposomes showed an enhanced permeability concomitant with the PEO-PPO-PEO adsorption. Permeability effects induced by different PEG-stabilisers on EPC liposomes were shown to be dependent on the length of the PEG chain but also on the linkage used to connect the PEG polymer with the hydrophobic membrane anchor. An efficient drug delivery requires, in most cases, an accumulation of the drug in the cell cytoplasm. The mechanism behind cytosolic drug delivery from pH-sensitive liposomes was investigated. The results suggest that a destabilisation of the endosome membrane, due to an incorporation of non-lamellar forming lipids, may allow the drug to be released. Furthermore, sterically stabilised liposomes intended for targeted BNCT have been characterised and optimised concerning loading and retention of boronated drugs. Key words: Liposome, steric stabilisation, BNCT, cryo-TEM, EGF, targeting, stability, permeability, pH-sensitive liposomes, triggered release. Nill Bergstrand, Department of Physical Chemistry, Uppsala University, Box 579, SE-751 23 Uppsala, Sweden

¹ Nill Bergstrand 2003 ISSN 1104-232X ISBN 91-554-5592-1 Printed in Sweden by Akademitryck AB, Edsbruk 2003

List of Papers

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This thesis is based on the papers listed below. They are referred to by their Roman numerals (I-VII) in the summary.

Aggregate structure in dilute aqueous dispersions of phospholipids, fatty acids and lysophospholipids. Nill Bergstrand and Katarina Edwards Langmuir, 2001, 17(11), 3245-3253.

II

Adsorption of a PEO-PPO-PEO triblock copolymer on small unilamellar vesicles: equilibrium and kinetic properties and correlation with membrane permeability. Markus Johnsson, Nill Bergstrand, Johan JR Stålgren and Katarina Edwards Langmuir, 2001, 17(13), 3902-3911.

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III Effects caused by PEO-PPO-PEO triblock copolymers on structure and stability of liposomal DOPE dispersions. Nill Bergstrand and Katarina Edwards submitted IV

Linkage identity is a major factor determining the effect of PEGylated surfactants, on permeability in phosphatidylcholine liposomes. Mats Silvander, Nill Bergstrand and Katarina Edwards submitted

Interaction between pH-sensitive liposomes and model membranes. Nill Bergstrand, Maria C. Afrvidsson, Jong-Mok Kim, David H. Thompson and Katarina Edwards Biophysical Chemistry, 2003, in press

VI

Optimization of drug loading procedures and characterization of liposomal formulations of two novel agents intended for Boron Neutron Capture Therapy (BNCT). Markus Johnsson, Nill Bergstrand and Katarina Edwards Journal of Liposome research, 1999, 9(1), 53-79.

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VII Development of EGF-conjugated liposomes for targeted delivery of boronated DNA-binding agents. Erika Bohl Kullberg, Nill Bergstrand, Jörgen Carlsson, Katarina Edwards, Markus Johnsson, Stefan Sjöberg and Lars Gedda Bioconjugate Chem.,2002, 13(4), 737-743.

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Reprints were made with permission from the publishers.

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Contents General Introduction of Liposomes 1.1 Amphiphiles 1.1.1 Self-Assembly 1.1.2 Aggregate structure 1.2 Lipid bilayers and Lamellar Phases 1.2.1 Phospholipids 1.2.2 Lamellar phase transitions 1.2.3 Comments on phase behaviour 1.3 Liposome formation 1.4 Applications

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Stability & Stabilisation 2.1 Chemical stabilisation 2.1.1 Oxidation 2.1.2 Hydrolysis 2.1.3 Aggregate structures induced by degradation products 2.2 Sterical stabilisation 2.2.1 Stealth liposomes 2.2.2 Alternative stabilisers 2.2.3 Stabilisation of PE-liposomes

15 15 15 16

Sustained Release 3.1 Membrane permeability… 3.1.1 …and phospholipids 3.1.2 …and PEG-ylated lipids or polymers

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Triggered Release 4.1 pH-triggered release 4.1.1 Release mechanism

38 38 41

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Loading & Targeting 5.1 Principle of BNCT 5.2 Drug loading 5.2.1 Remote loading 5.2.2 Remote loading of boronated agents 5.2.3 Comments on the lipid composition 5.3 Site specific targeting 5.3.1 Receptor mediated targeting 5.3.2 EGF-labelled liposomes 5.3.3 BNCT with EGF-labelled liposomes 5.3.4 Biodistribution of EGF-labelled liposomes 5.3.5 Antibody-labelled liposomes

46 46 48 48 49 51 51 52 52 53 54 55

6

Experimental Techniques 6.1 Cryo-TEM 6.1.1 Limitations and artefacts 6.2 Light scattering 6.2.1 Dynamic light scattering 6.3 Fluorescence assays 6.3.1 Leakage 6.3.2 Lipid mixing 6.3.3 Anisotropy 6.3.4 Time-resolved fluorescence quenching 6.4 QCM

56 56 58 59 59 60 60 61 61 61 62

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4

Acknowledgements

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References

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1

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General Introduction to Liposomes

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Lipids, along with proteins and nucleic acids, are essential biomolecules for the structure and function of living matter. Most lipids are fats and waxes, but this thesis focuses on so-called amphiphilic lipids. This type of lipid is the predominant building block of biological membranes, as well as liposomes. Liposomes are spherical self-closed structures, composed of curved lipid bilayers, which enclose part of the surrounding solvent into their interior. The size of a liposome ranges from some 20 nm up to several micrometers and they may be composed of one or several concentric membranes, each with a thickness of about 4 nm. Liposomes possess unique properties owing to the amphiphilic character of the lipids, which make them suitable for drug delivery. A schematic picture of a liposome is shown in Figure 1.1. In order to understand the behaviour of liposomes some general features of amphiphiles and their behaviour in aqueous solutions will be presented below.

Figure 1.1 A schematic representation of a liposome. ¹Göran Karlsson

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1 GENERAL INTRODUCTION TO LIPOSOMES

1.1 Amphiphiles Amphiphiles can be found in a wide rage of applications, as diverse as detergents, paints, paper coating, food and pharmaceutical products. It is their special power to aggregate spontaneously (i.e. self-assemble) into a variety of structures that enable them to be useful in a large number of areas.

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1.1.1 Self-assembly The reason why amphiphiles spontaneously aggregate, to form a variety of microstructures, is their dual preference for solvent. All amphiphiles consist of one part that is soluble in nonpolar solvents, and a second part that is soluble in polar ones. This means that solvents that are either very nonpolar or very polar, like water, promote the self-assembly. In biological applications, the solvent is water and then one usually talks about the hydrophilic and the hydrophobic part, respectively, which will be the case throughout this thesis. In most amphiphiles the hydrophobic part consists of hydrocarbon chains, while the hydrophilic part consists of what is called a polar headgroup. In water solution, the amphiphiles dissolve as monomers at first, but above a certain concentration, to minimise unfavourable hydrophobic (or solvent-hating) interactions, they spontaneously aggregate. This selforganisation is usually accompanied by increased entropy of the system [1,2]. The increased entropy originates from the water-hydrocarbon interactions that force the water molecules into an ordered structure around the hydrophobic part when the amphiphiles are freely suspended as monomers. Release of the ordered water can be achieved by driving the hydrophobic parts out of the aqueous solution and sequestering them within the interior of the aggregate. Thus the increased entropy gained by the water molecules may lead to an overall gain in free energy so that aggregation occurs spontaneously. It should be mentioned, however, that there is still debate about the existence of such locally ordered water structure [3,4]. 1.1.2 Aggregate structure Spontaneous aggregation is, however, not only determined by the hydrophobic contribution mentioned above, but it is also related to the molecular parameters of the amphiphile. The so-called surfactant

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1 GENERAL INTRODUCTION TO LIPOSOMES

parameter [5], which takes into account parameters such as the hydrophobic volume, chain length and head group area, is a useful guide for predicting the optimal aggregate structure. An effective head group area might however be difficult to estimate since it can be strongly dependent on the solution conditions. The surfactant parameter, S, is defined by v l a0

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S=

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where v stands for the volume of the hydrophobic portion of the amphiphile, l is the length of the hydrocarbon chains and a 0 is the effective area per head group. These parameters contain information about the geometrical shape of the molecule and the surfactant parameter can be considered to use geometrical packing constraints to restrict the number of forms available to the aggregate. The value of the surfactant parameter relates the properties of the molecule to the mean curvature of the formed aggregates. By convention the curvature of an aggregate is positive if the aggregate is curved around the hydrophobic part and negative if it is curved towards the polar part. The former is also said to form normal aggregates and phases, while the latter forms reversed ones. For example, small values of S imply highly curved aggregates, micelles, while for S 5 1 planar bilayers are formed. The relationship between the value of the surfactant parameter and the optimal aggregate structure is shown in Figure 1.2. Although the surfactant parameter can only be considered to be a crude and approximate model for predicting self-assembly, it provides valuable insight into how changes of molecular structure affect the shape of the formed aggregate.

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1 GENERAL INTRODUCTION TO LIPOSOMES

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Figure 1.2 The geometrical packing concept: the packing parameter of amphiphilic molecules, preferred aggregate structures and corresponding phases. L: micellar solution, H: hexagonal phase, L_: lamellar phase. Subscripts I and II denote normal and inverted phases, respectively.

1.2 Lipid bilayers and lamellar phases Throughout this thesis the most frequently encountered aggregate structure is the lipid bilayer. Typical bilayer-forming lipids consist of two hydrocarbon chains attached to a headgroup, which can either be charged (positively or negatively), zwitterionic or neutral. The molecular geometry of most lipids can be approximated as cylinders and according to the geometrical packing concept, lipids prefer to self-assemble into bilayers. At higher lipid concentrations these molecules usually form

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1 GENERAL INTRODUCTION TO LIPOSOMES

lamellar phases where two-dimensional planar lipid bilayers alternate with water layers. The class of lipids that will be considered within the present thesis is glycerophospholipids, often just called phospholipids.

R1

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DSPC

R1 = R2 = C18:0

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EPC

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Phophatidylethanolamine (PE)

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DOPE R1 = R2 = C18:1

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1.2.1 Phospholipids A phospholipid has two acyl chains linked to a headgroup by means of a glycerol-backbone. Figure 1.3 shows the structural formula of a phospholipid, where R1 and R2 are saturated or unsaturated acyl chains and R3 is the polar head group. The polar head groups are used for classification, i.e. to distinguish between different phospholipids. Phosphatidylcholines and phosphatidylethanolamines are the two groups of lipids used throughout this thesis.

H2 C

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Phosphatidylchiline (PC) R3

CH2 CH2 N(CH3 )3

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CH2 CH2 NH3

Figure 1.3 The general structure of a phospholipid and the structure of EPC, DSPC and DOPE.

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Phosphatidylcholines or PC-lipids are the most widely used lipids in liposome work. PC-lipids are zwitterionic at all relevant pH1 and can therefore form lamellar structures independently of the pH in the solution. Egg-yolk lecithin (EPC) and distearoly PC (DSPC) are the PClipids used in the present thesis and the structures are shown in Figure 1.3. DSPC is a synthetic lipid with only saturated chains, while EPC is a natural PC-lipid with both saturated and unsaturated fatty acids. Phosphatidylethanolamines have a pH dependent phase behaviour [6]. At physiological pH where the PE-lipids have zwitterionic headgroups they are not capable of forming lamellar structures. However, above pH ~ 9 the headgroup becomes charged and lamellar structures can be formed. A more detailed description of this behaviour and the advantage of using such lipids will be presented in the following sections.

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However, protonation of the phosphate group is expected at very low pH.

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1 GENERAL INTRODUCTION TO LIPOSOMES

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1.2.2 Lamellar phase transitions Phospholipid lamellar phases may exist in different physical states [1,7,8,] since the character of the bilayer changes with, for instance, lipid composition and temperature. Low temperatures or a high degree of saturation forces the bilayer into a gel state, in which hydrocarbon chains exhibit close packing and a more or less frozen conformation. Increasing the temperature or introducing unsaturated acyl chains results in a bilayer of a liquid crystalline (or fluid) state, where the chains are disordered and have high mobility. The temperature (Tm ) where the gel-to-liquid crystalline phase transition occurs is a function of the chemical composition of the bilayer, and especially of the acyl chains. Comparing an unsaturated PC-lipid with its saturated analogue, the Tm for the unsaturated lipid will be significantly lower since the double bond introduces kinks in the chain that do not allow for close packing.

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1.2.3 Comments on phase behaviour Liquid crystalline phases are build up by various microstructures [1,9]. Some phases and their corresponding microstructures are shown in Figure 1.2. Upon assembly of amphiphilic molecules in dilute solutions, the preferred aggregate shape is determined by the packing parameter. However, a change of the conditions in the solution might alter the interand intra-aggregate interactions. If, for instance, the concentration is increased, the interaction between the aggregates becomes more important. This, in turn, can either lead to ordering of aggregates relative to one another or to a change of aggregate shape, if the interactions are strong enough. Together, these effects lead to a rich phase behaviour, where a transition from one phase to another is an advanced interplay between inter- and intra-aggregate interactions. An ideal phase sequence induced by an increased water concentration is LII A I II A H II A QII A L_ A QI A H I A I I A LI

where L is the micellar solution, I the micellar cubic phase, H is the hexagonal phase, Q the biocontinuous cubic phase and L_ is the lamellar phase and the subscripts I and II denote normal and reversed phases, respectively. Many of these phases are interesting from a pharmaceutical point of view [10-13] but the work presented in this thesis focuses on lamellar

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1 GENERAL INTRODUCTION TO LIPOSOMES

phases and, especially on the dispersed particles of the lamellar phase called liposomes [14-16].

1.3 Liposome formation

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Many potential mechanisms have been suggested for the formation of liposomes, and some of these are more complex than others [17 and references therein]. One approach is to consider the self-closing of a bilayer into a liposome to be a competition between two effects, the bending or curvature energy and the edge energy of a bilayer [18-20]. For a flat lamellar fragment, in a hydrophilic surrounding, there will be a high surface tension at the rim of the lamellar sheet. Bending can reduce this edge energy but bending also implies an energy penalty due to the induced curvature. To further minimise the edge energy, a higher curvature is required and finally a closed sphere will be formed, where the edge energy is reduced to zero. The bending energy, on the other hand, has now reached its maximum and the excess free energy per liposome, regardless of the radius, is then 8/K, where K is the bending rigidity. Thus, larger liposomes are energetically favoured, while entropy would favour many small ones. However, liposomes are usually stable due to the high cost of pore formation. This means that a very long time is required before they collapse into a lamellar phase.

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1.4 Liposomes as a drug delivery system The applicability of drugs is always a compromise between their therapeutic effect and side effects. Liposomal drug delivery systems not only enable the delivery of higher drug concentrations [15], but also a possible targeting of specific cells or organs [21-24]. Harmful side effects can therefore be reduced owing to minimised distribution of the drug to non-targeted tissues. Like all other carrier systems, the use of liposomes in drug delivery has advantages and disadvantages. The amphiphilic character of the liposomes, with the hydrophobic bilayer and the hydrophilic inner core,

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1 GENERAL INTRODUCTION TO LIPOSOMES

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enables solubilization or encapsulation of both hydrophobic and hydrophilic drugs. Along with their good solubilization power, a relatively easy preparation and a rich selection of physicochemical properties have made liposomes attractive drug carrier systems. However, a complete saturation of the immune system [25] and interactions with lipoproteins [26,27] are some examples of potentially toxic and adverse effects. Efficient drug delivery systems based on liposomes need to possess a large number of special qualities [28]. First, good colloidal, chemical and biological stability is required. The fact that liposomes are nonequilibrium structures does not necessarily mean that they are unsuitable for drug delivery. On the contrary, a colloidally stable nonequilibrium structure is less sensitive to external changes than equilibrium structures, such as micelles. Hence, colloidally stable liposomes often work well in pharmaceutical applications. Biological stability includes control over the rate of clearance of liposomes from the circulatory system or compartments of the body, if the drug has been administered locally. The rate of clearance is dose dependent and varies according to the size and surface charge of the liposomes [29,30]. Early studies using conventional liposomes revealed that the clearance was too rapid for an effective in vivo drug delivery. However, circulation times that were sufficiently long were achieved by the development of the so-called sterically stabilised liposomes [30-32]. In addition, biological stability also comprises retention of the drug by the carrier en route to its destination (a phenomenon known as sustained release). For example, blood proteins were found to remove phospholipid molecules rapidly from the bilayer, leading to a disruption of the liposomes and hence drug loss before the carriers reached their target destination. In contrast to a sustained release, liposomes also have to be able to release the encapsulated drug, which might not be as easy as it sounds, and, they should preferably also be targeted. This is discussed further in section 5. Furthermore, it is important that the drug can be encapsulated in such a way that the amount required for an efficient treatment is achieved. In the following sections, the above requirements, which must be fulfilled by an efficient drug delivery system, will be presented further and discussed in the light of the results of Papers I to VII.

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Stability & Stabilisation

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Liposome stability, which can be divided into colloidal, chemical and biological stability, is one of the most important issues in liposome applications. First, the chemical stability of liposome constituents will be discussed, followed by colloidal and biological stability of liposomal dispersions.

2.1 Chemical stability

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Lipids, like most biomolecules, undergo different degradation processes and the most common degradation pathways are oxidation and hydrolysis. First, these processes will be discussed with the focus on phosphatidylcholine since it is the most commonly used lipid in pharmaceutical applications. Second, the relationship between chemical stability and structural changes of the liposomes are presented in Paper I. 2.1.1 Oxidation In the case of PC-lipids it is the hydrocarbon chains and especially the unsaturated ones that are subject to oxidation [33,34]. Saturated chains can, however, be oxidised at high temperatures [33]. The oxidation is a radical reaction, which finally results in the cleavage of the hydrocarbon chains or in the case of two adjacent double bonds, the formation of cyclic peroxides. The initiation step, abstraction of a hydrogen atom from the lipid chain, occurs most commonly as a result of exposure to light or trace amounts of metal ion contamination. The most susceptible lipids to

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this initial step are lipids containing double bonds, since the unsaturation permits delocalization of the remaining unpaired electron, which lowers the energy of this state. Polyunsaturated lipids are thus particularly prone to oxidative degradation. In the presence of oxygen, the process develops further into formation of peroxides and cleavage of the hydrocarbon chain. The use of lipids with high purity can minimise oxidation of PC-lipids in liposomes, as can storage at low temperatures and protection from light and oxygen [35]. To further enhance the protection, antioxidants and substances forming a complex with metal ions, like EDTS, can be added. A comparison of the oxidation kinetics between three different polyunsaturated fatty acids [16], at 36°C, is shown in Table 2.1. Table 2.1

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(chain length:no. of double bonds)

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* Addition of the antioxidant BHT, (butyl hydroxyl toluene).

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2.1.2 Hydrolysis The four ester bonds present in a phospholipid may all be subject to hydrolysis in water but the carboxyl esters are hydrolysed faster than the phosphate esters [36]. During the hydrolysis, the hydrocarbon chains are pinched from the lipid backbone, producing fatty acids and lysophospholipids. The lysophospholipid can be further hydrolysed into a glycerophospho-compound and ultimately the hydrolysis produces glycerophosphoric acid. The hydrolysis of the remaining ester bond appears to be negligible under pharmaceutically relevant conditions. A schematic representation of the hydrolysis reactions of PC-lipids in aqueous suspension is shown in Figure 2.1. The hydrolysis rate of PC-lipids is both pH and temperature dependent. In general, the rate of the hydrolysis has a ”V-shaped” pH dependence, with a minimum at pH 6.5 and thus an increased rate at both higher and lower pH [37]. As expected, the ester hydrolysis is both acid and base catalysed. The effect of the temperature can be described by the Arrhenius relation [37]

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(

k = A exp < Ea / RT

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where k is the hydrolysis rate, A is a frequency factor, Ea is the activation energy and RT is the thermal energy. This means that the rate is significantly slower at low temperatures. By selecting the temperature and pH, the hydrolysis can be largely avoided. However, if no special care is taken, the rate of lysolipid formation in aqueous dispersions, stored at 4°C, might be as high as ¾20% per month [16].

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Figure 2.1 The hydrolysis reaction of PC-lipids.

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Hydrolysis and oxidation of phospholipid liposomes occur, in vivo, concomitant with their interaction with serum components [38]. In addition, hydrolysis of phospholipids can be catalysed by enzymes, phospholipases. Phospholipase A2 (or PLA2) belongs to this ubiquitous family of enzymes and it hydrolyses a phospholipid at its carboxyl ester in the second position, giving 1-acyl-lyso-phospholipid and free fatty acid [39,40]. This enzyme has, for instance, an important role in the degradation of damaged or aged cell membranes and is found at elevated levels in diseased tissue [41]. Clarification of the function of PLA2 and other lipases might benefit from a detailed physico-chemical characterisation of the effects of degradation products on structure and stability of lipid membranes. In addition, a novel principle for liposomal drug release, triggered by PLA2, has been proposed [42].

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2.1.3 Aggregate structures induced by degradation products The aggregate structures formed upon mixing EPC with potential hydrolysis products, the lysolipid MOPC and oleic acid (OA) were investigated in Paper I. These three constituents have different molecular geometrical shape and thus they prefer to self-assemble into different structures, see section 1.1.2. EPC is a bilayer forming molecule and at pH 7.4, a pure EPC dispersion displays a polydisperse population of large and multilamellar liposomes. MOPC, with only one hydrocarbon chain, prefers aggregate structures with higher curvature, and thus micelles form in water solutions. Dispersions of OA have a much more complex behaviour since its headgroup size changes with the pH of the solution. At 25°C and in the presence of 150 mM NaCl, an apparent pKa value of between 7.2 and 8 may be expected for fatty acids situated in a lipid bilayer [43]. Protonation of the carboxyl group decreases the fatty acid headgroup area. The propensity for HII phase formation thus increases with decreasing pH. At high pH, where the fatty acid is essentially deprotonated, cylindrical micelles are formed. Lowering the pH to values around 9 gives rise to the formation of lamellar structures. At pH 7.4, however, aggregated lamellar and nonlamellar structures coexist [44]. In Paper I, it was shown that the phase behaviour and aggregate structure of a mixture of EPC, MOPC and/or OA, could be rationalised in terms of simple considerations of geometrical molecular shape.

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50

80 F

K G

L C

20

20 M

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H

MOPC

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50

80

Figure 2.2 A schematic diagram presenting all the compositions that were investigated by means of cryo-TEM. The concentrations are given in mol% of the total lipid concentration. The water content was above 99 wt% for all samples.

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The general trends, shown in the triangle diagram in Figure 2.2, can satisfactorily be explained by the intrinsic properties of the components. Moving along the solid line, starting at point A, it is clearly shown that the presence of OA prevents the formation of structures with high positive curvature. The structures found along this line are shown in Figures 2.3a, 2.4a, b and c. It is worth noting that at point C, where the molar amount of the three components is the same, large unilamellar liposomes are found. Furthermore, it is interesting to compare the structures found in D and E, suggested to be particles of dispersed inverted cubic (Figure 2.4c) [45,46] and hexagonal phase (Figure 2.3c) [47], respectively. A structural change towards aggregates with higher net curvature, such as dispersed particles of cubic phase, takes place upon addition of MOPC.

Figure 2.3 Cryo-TEM pictures of structures formed in the EPC/MOPC/OA system, dispersed by vortexing, at 25°C: (a) EPC/MOPC 1:1 (mol/mol), (b) OA/MOPC 1:1 (mol/mol), (c) EPC/OA 1:4 (mol/mol), (d) EPC/OA 1:1 (mol/mol). Note the threadlike micelles (marked with an arrow) in (a), (d), the open bilayer fragment (marked with an arrowhead) in (d) and the nonlamellar structures (marked with an arrow) in (c). Bar = 100nm.

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The strong influence of MOPC on the preferred aggregate structure is further demonstrated by moving along the dashed line in Figure 2.2, starting at point F. A comparison of Figures 2.3d, 2.4d, b and e shows how invaginated liposomes and complex spongelike particles transform into bi- and unilamellar liposomes and, finally, threadlike micelles are formed.

Figure 2.4 Cryo-TEM pictures of structures formed in the EPC/MOPC/OA system, dispersed by vortexing, at 25°C: (a) EPC/MOPC/OA 4:4:1 (mol/mol/mol), (b) EPC/MOPC/OA 1:1:1 (mol/mol/mol), (c) EPC/MOPC/OA 1:1:4 (mol/mol/mol), (d) EPC/MOPC/OA 4:1:4 (mol/mol/mol), (e) EPC/MOPC/OA 1:4:1 (mol/mol/mol), (f) EPC/MOPC/OA 1:4:4 (mol/mol/mol). Note the open liposomes and bilayer fragments (marked with arrowheads) in (a) and (f), and the threadlike micelles (marked with an arrow) in (f). Bar = 100nm.

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The aggregate structures along the dotted line, all containing equimolar amounts of OA and MOPC, are of special interest since this corresponds to the situation of a spontaneous or enzyme-mediated hydrolysis of EPC. Starting at point K, open and closed liposomes were detected in coexistence with threadlike micelles, Figure 2.3b. Moving via the structures in Figure 2.4f and b, towards pure EPC, it becomes clear that an addition of EPC stabilises bilayer arrangements. According to these results only a minor effect on the aggregate structure would be observed at an early stage of the hydrolysis process. When 50 mol% of the phospholipid has been hydrolysed, unilamellar liposomes are formed and a severe lamellar disruption is only observed near a complete hydrolysis. Nevertheless, the spontaneous hydrolysis at 25°C for EPC dispersions, when no special care has been taken, can proceed quite rapidly. After 23 days, cylindrical micelles appeared in pure EPC samples, Figure 2.5, which indicates formation of hydrolysis products.

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Figure 2.5 Cryo-TEM pictures of pure EPC liposomes after 23 days of incubation at 25°C. Note the threadlike micelles (marked with an arrow). Bar = 100nm.

Despite the fact that the overall structure of the liposomes is not affected at an early stage of the hydrolysis process, some other characteristics, most notably the permeability of the membrane may be seriously altered [48-50]. Similar results have also been shown for enzymatic PLA2 mediated hydrolysis of DPPC liposomes [51], which induced the formation of bilayer fragments and micelles. However, a significant size reduction of the liposomes was also found, at an early stage of the hydrolysis. But in this case the PLA2 was added to already preformed liposomes, and thus the enzyme primarily acted on the lipids in the outer layer. External

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addition of MOPC has shown similar effects in other reports and this is also in accordance with our investigation in Paper I.

2.2 Steric stabilisation

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The colloidal stability of a liposomal dispersion is determined by the inter-liposome interactions, which depend on the balance between attractive and repulsive forces [52]. An increased repulsive contribution gives rise to an enhanced colloidal stability. Steric repulsion is often used for stabilising liposomes both in vitro and in vivo. Polymer-coated liposomes are often used to create sterically stabilised liposomes. Stabilisation can be produced in two different ways, by grafting or by adsorption of the polymer to the liposomal surface [53-58]. The grafting method is the most commonly used and normally the stabilisation is achieved by incorporation of so-called PEG2-lipids, poly(ethylene glycol)-phospholipids, Figure 2.6 [53-55]. The hydrophilic PEG chains are decorating the surface of the liposome, as shown in Figure 2.6. When two polymer-covered surfaces approach each other they experience a repulsive force, as soon as the outer polymer segments start to overlap. This repulsive force is due to the unfavourable entropy associated with compressing (the loss of conformational freedom) the polymer chains between the two surfaces [52]. In addition, the difference in chemical potential between the water in the bulk and in the interaction region induces an osmotic repulsive force [59].

Figure 2.6 Schematic representation of a sterically stabilised liposome. The molecular structure of the PEG-lipid, DSPE-PEG, where n typically ranges from 17 to 114. ¹Göran Karlsson

Poly(ethyleneglycol), PEG is only one of many name for the same polymer, also the names poly(ethyleneoxid), PEO and poly(oxy ethylene), POE, are used. 2

22

2 STABILITY & STABILISATION

To describe the repulsive interactions between polymer-coated surfaces two limiting cases have to be distinguished. At a low surface coverage of the polymer, that is, no overlapping of neighbouring chains, each chain can interact with the opposite surface independently of the other chains. The interaction potential between two plates, or large liposomes, scales in this case according to

(

W ( d ) | exp
)

TE

C

H

where d is the distance between the surfaces and L is the thickness of the polymer layer [52]. In the case of a low coverage L § Rg, where Rg is the radius of gyration of the polymer. Going from low to high coverage, the polymers come so close to each other that they are forced to adopt extended configurations. Thereby the thickness of the polymer layer increases (L > Rg) and hence, within this extended region the steric stabilisation is more efficient.

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2.2.1 Stealth· liposomes3 The use of sterically stabilised liposomes does not merely increase the colloidal stability of a dispersion but it also promotes its biological stability [31,32]. Lipsomal drug delivery formulations can be administered in many different ways but here the focus will be on an intravenous administration. In the blood stream the liposomes will interact with lipoproteins and opsonins. The former interaction involves lipid exchange, which eventually leads to breakdown of the liposome [26]. Opsonisation, or adsorption of marker macromolecules, such as immunoglobulins, is a part of the body’s own defence mechanism. The marked invaders are taken up by macrophages specialised in eliminating foreign particles from the circulation. These macrophages belong to the reticuloendothelial system, RES. Thus, the majority of conventional liposomes will have a circulation time of only a few minutes [31,32]. To prolong their circulation time, markers must be prevented from reaching the liposomal surface. Sterically stabilised liposomes with their barrier of long polymer chains will protect the surfaces from interaction with both lipoproteins and RES marker molecules, thus prolonging the circulation time from minutes to days [31,32]. The mechanism behind the effective defence of the polymer chains against 3

Stealth liposome is a registered trade name from Liposome Technology, Inc.

23

2 STABILITY & STABILISATION

surface interactions has the same origin as the colloidal stability. Because of their ability to evade detection by the RES these “second generation” liposomes are also called Stealth· liposomes. The development of sterically stabilised liposomes was a major breakthrough for the use of liposomes in pharmaceutical formulations. A long circulation time is necessary for an efficient site-specific in vivo drug delivery.

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2.2.2 Alternative stabilisers PEG-lipids are the most commonly used stabiliser for liposomal drug delivery systems. The lifetime of the PEG-lipid in the membrane, which is crucial for the circulation time, depends on the length of the lipid hydrocarbon chains [60,61]. To minimise the loss of polymer from the lipid membrane, it is necessary to use lipids with long hydrocarbon chains. Triblock copolymers, adsorbed or incorporated, constitute an interesting alternative to PEG-lipids as steric stabilisers of liposomes [56-58,62-64]. Pluronics is a collective name for a large group of triblock copolymers with a hydrophobic middle block (poly propylene oxide, PPO) and hydrophilic end blocks (poly ethylene oxide, PEO). Imagine a triblock copolymer with an ability to be incorporated with its hydrophobic middle block in a membrane-spanning configuration, leaving the hydrophilic end-blocks on different sides of the membrane. This would provide a steric stabilisation that would suffer less from depletion. It has been observed, however, that adsorption of PEO-PPOPEO polymers on liquid crystalline membranes dramatically increases the membrane permeability [56,58,66-68]. In addition, PEO-PPO-PEO triblock copolymers induce significant structural perturbations when incorporated into PC-liposomes [58] and there were no indications of steric stabilisation of the liposomes. On the contrary, aggregation was observed by means of cyro-TEM. Nevertheless, a small increase of the circulation time in vivo has been observed for PEO-PPO-PEO treated liposomes, in comparison to conventional liposomes [57]. With these seemingly contradictive results in mind a more detailed investigation was performed of the adsorption behaviour of PEO-PPOPEO triblock copolymers on liquid crystalline bilayers. In Paper II the adsorption was investigated by means of a quartz crystal microbalance,

24

2 STABILITY & STABILISATION

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which allowed us to record the adsorbed amount in real time and in addition gain information about the kinetics of the adsorption process. In the first step, small unilamellar vesicles (SUVs) of EPC were adsorbed onto the gold electrode. The frequency shift, 6fSUV, originating from the adsorption of the vesicles was translated into their corresponding masses by the Sauerbrey equation (see section 6). This mass was compared to the theoretically predicted mass of a monolayer of close-packed spheres. Since a good agreement was found between the experimental and theoretical mass, a close-packed monolayer of vesicles was assumed to have formed on the gold surface, in accordance with other studies [69]. In the second step, the triblock copolymer F127 was introduced into the measuring cell. Immediately after the addition of F127, the frequency dropped, indicating an adsorption of polymers onto the EPC vesicles, as shown in Figure 2.7. Furthermore, there was a simultaneous increase of the dissipation, D , which implies that the viscoelastic properties of the adsorbed layer were changed.

PEO98-PPO67-PEO98

-260

28

-300

lp he

24 20

6f

F127

16

-340

-6 Dissipation (10 )

Frequency / Hz

-280

-320

F127

12

-360 4

1.2 10

4

1.6 10 Time / s

4

2 10

Figure 2.7 Changes in QCM resonance frequency (…) and dissipation ({) versus time for the adsorption of F127 onto the EPC SUV monolayer. Black arrows denote the introduction of the polymer solution (0.077 mg/ml) and the white arrows denote rinsing with Hepes buffer. The frequency change caused by the adsorbing polymers, 6 fF127, was extracted from the frequency difference between the solid lines.

25

2 STABILITY & STABILISATION

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In conclusion, F127 does adsorb onto EPC vesicles and the adsorption is a relatively rapid process, taking place within 10-20 s. The increased dissipation indicates an adsorbed layer reaching further out in the bulk solution. From the frequency shift, 6fF127, the mass of the adsorbed polymer was calculated. Finally, the polymer solution was exchanged for a pure buffer solution and as shown in Figure 2.7 the frequency rapidly increased, while the dissipation decreased and they both levelled off at the same value as before the addition of F127. This indicates that the polymers easily desorb from the vesicle surface and thus that the polymer/vesicle interaction is weak. In conclusion, the polymers are only weakly adsorbed to the vesicles and hence they are not able to function as steric stabilisers of liposomes, in accordance with previously discussed results [58,65,67,68]. A schematic representation of the adsorption/desorption processes described above is shown in Figure 2.7. To quantitatively determine the maximum adsorbed amount of F127 on EPC SUVs, a number of experiments were performed with different F127 concentrations. The data obtained from these measurements were used to construct an adsorption isotherm, Figure 2.8, and were fitted to the Freundlich equation, which gave a fairly good description of the isotherm at low polymer concentrations.

0.15

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mF127 / mSUV

0.2

0.1

0.05

0 0

0.2

0.4

0.6

0.8

CF127 / mg mL-1

1

1.2

1.4

Figure 2.8 Adsorption isotherm for F127. The adsorbed amount was calculated as the adsorbed mass of F127 divided by the adsorbed mass of EPC SUV´s, mF127/m SUV and C is the concentration of F127. The dashed line represents the best fit of the Freundlich equation and the full draw line indicates the plateau adsorption value.

26

2 STABILITY & STABILISATION

The best fit to the data in Figure 2.8 was obtained using the following equation T=Tp*1.61*C0.862

10-2

k / s-1

he

lp

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H

where T is the adsorbed amount, C is the F127 concentration and Tp is the maximum adsorption value, 0.187 g F127/g SUVs or 0.307 g F127/g lipid. However, this value was recalculated to the maximum adsorption value of freely suspended vesicles since in the QCM measurements, only about 50% of the total vesicle area is available for polymer binding. To recalculate Tp the adsorption of F127 on freely suspended vesicles was assumed to follow the same binding isotherm as the polymer adsorption onto the immobilised SUVs. The maximum adsorption value for freely suspended vesicles, Tp,fs, thus becomes 0.614 g F127/g lipid or 0.0382 mole F127/mole lipid. According to DLS measurements, the average radius of the SUVs is 15 nm and, thus, ~240 F127 molecules are adsorbed per vesicle. This value is very close to the plateau values obtained for F127 adsorption on hydrophobic surfaces [70] and on polystyrene latex spheres [71]. The above results were supported by fluorescence anisotropy measurements, where the anisotropy of EPC SUVs was measured as a function of F127 concentration. The anisotropy decreased monotonically, with increasing polymer concentration, until a plateau value was reached close to the concentration of F127 yielding the maximum adsorption value. A decreased anisotropy indicates an increased disorder in the membrane, possibly induced by penetration of the PPO block (or part of the PPO block) into the hydrophobic interior of the membrane.

0.01

10

-3

0.008 0.006 0.004 0.002 0 0

10

0.002

0.004

0.006

0.008

0.01

-4

0

0.01

0.02

0.03

0.04

K / mol F127 / mol lipid

Figure 2.9 CF leakage rate versus the adsorbed amount of F127. The inset figure shows an expansion of the low surface coverage regime and the solid line represents a linear fit to the data.

27

2 STABILITY & STABILISATION

C

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From the binding isotherm data and the estimated Tp,fs, the leakage rate of CF was correlated with the adsorbed amount of F127, as shown in Figure 2.9. As expected, when the polymer concentration increased, the magnitude of the leakage rate constant approached a plateau value. The plateau appears near to the F127 concentration corresponding to the maximum adsorption value. In summary, PEO-PPO-PEO triblock copolymers do adsorb onto the EPC membrane. However, the interaction between the PPO block and the lipid membrane seems to be weak. This weak interaction is most likely the explanation for the poor in vivo performance of PEO-PPO-PEO treated liposomes.

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2.2.3 Stabilisation of PE-liposomes Although PC-based liposomes are the most commonly used for pharmaceutical applications, PE-liposomes, and in particular so-called pH-sensitive PE-liposomes have been proposed as a promising alternative. The rationale for developing such liposomes is the failure of the conventional PC-liposomes to release all their entrapped substances rapidly at a specific site. pH-sensitive liposomes are believed to be promising alternatives to achieve an efficient drug release. This will be further discussed in section 4. Dioleoylphosphatidyl-ethanolamine (DOPE), one of the most studied PE-lipids, forms an inverted hexagonal phase (HII) above 10-15°C at near neutral or acidic pH [6]. However, at high pH (pH¾9) the preferred phase is the lamellar phase, which can be dispersed as liposomes. The reason for this pH-dependent phase behaviour can be explained by changes in the effective headgroup area upon acidification. An important property of the headgroup in regulating the lipid phase behaviour is its hydrophilic character, which determines the strength of the headgroupwater interactions. If attractive headgroup-headgroup interactions, such as hydrogen bonding, are present, the hydrophilicity will be reduced since hydration of the headgroups decreases. The primary amine in the PE headgroup is deprotonated at high pH and the lipid acquires a negative charge. This favours hydration and, in addition, the headgroup area increases due to electrostatic interactions. At near neutral or acidic pH, the DOPE molecule becomes net neutral (or zwitterionic) and a tight headgroup packing becomes favourable, possibly due to formation of

28

2 STABILITY & STABILISATION

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phosphate-ammonium hydrogen bonds. This corresponds to a decreased effective headgroup area. The preferred aggregate structures can thereby be related to the molecular shape of the DOPE according to the geometrical shape concept. Thus at high pH, liposomes are formed, whereas upon acidification, rapid aggregation and a transition into the HII phase occur. The stability of DOPE liposomes can be significantly improved by the incorporation of molecules that increase the spontaneous curvature of the lipid film [61,72-77]. In this way, DOPE liposomes are stabilised by addition of PEG-lipids and the amount of PEG-lipid needed in the membrane, for an effective steric stabilisation depends on the size of the PEG headgroup [75-77]. Other types of stabilisers are block copolymers, as mentioned earlier. However, no investigations concerning the interactions between HII forming PE-lipids and triblock copolymers have so far been published. In Paper III, we investigated the structure and stability of DOPE/PEO-PPO-PEO liposomal systems, at both basic and acidic pH, to evaluate the use of Pluronics as steric stabilisers of pH-sensitive liposomes. The compositions of the Pluronics used in this investigation are shown in Table I.

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Table I Pluronics (EOn-POx-EOn) –composition and molar mass Pluronic composition molar mass F127 EO 100-PO 65-EO 100 12600 F108 EO 132-PO 50-EO 132 14600 P105 EO 37-PO 56-EO 37 6500 F88 EO 103-PO 39-EO 103 11400 F87 EO 61-PO 40-EO 61 7700 P85 EO 26-PO 40-EO 26 4600

The structural effects caused by P85 and F127 when they were mixed with DOPE before the liposome formation at pH 9.5, are shown in Figure 2.10. P85 has relatively short PEO and PPO blocks and when this polymer was added in low concentrations the sample structure remained essentially the same as in the absence of polymer. The fact that the DOPE dispersion could not form a pure dispersed lamellar phase implies that the pH was not high enough. This is in accordance with other results, showing that only about 25% of the DOPE molecules are negatively charged at pH 9.5 and 150mM NaCl [78]. Single and

29

2 STABILITY & STABILISATION

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aggregated liposomes were observed in coexistence with intermediate structures4 (Figure 2.10a). When the amount of P85 was increased the intermediate structures vanished and the tendency for liposome aggregation was markedly decreased. In addition, the average size of the liposomes decreased and micelles could be detected, as shown in Figure 2.10b.

he

Figure 2.10 C-TEM micrographs showing the structure in DOPE/PEO-PPOPEO triblock copolymer samples at pH 9.5. The samples contained 3mM DOPE and (a) 10 mol% P85, (b) 50 mol% P85, (c) 10 mol% F127, and (d) 50 mol% F127. In all cases the block copolymer was present during the liposome preparation step. Arrows in (b) and (d) denote micelles. Bar = 100 nm.

Intermediates are used as a collective name for a group of structures appearing between regions occupied by lamellar and hexagonal phases. This does not include the cubic phases, which appear in the same region (see section 1.2.3). The structures of intermediates are similar to one of its neighbouring phases. 4

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2 STABILITY & STABILISATION

1.2

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Inclusion of F127, which has, in contrast to P85, comparably long PEO and PPO blocks, prevented liposome aggregation and the formation of non-lamellar structures even at low concentrations (Figure 2.10c). Furthermore, the average size of the liposomes is smaller than in the presence of P85. At higher concentrations, micelles were again revealed, in coexistence with liposomes (Figure 5.10d). Thus, inclusion of Pluronics facilitates the formation of liposomes and prevents aggregation. The effect of inclusion of Pluronics on the pH-induced L_ to HII transition was investigated and the stabilising capacity was observed to depend critically on the molecular composition of the Pluronics. Block copolymers with comparably long PPO and PEO segment lengths, such as F127 and F108, were most effective in protecting DOPE liposomes from aggregation and subsequent structural rearrangements, as shown in Figure 2.11a. A sufficiently long PPO block was found to be the most decisive parameter in order to obtain an adequate coverage of the liposome surface at low Pluronic concentrations. However, upon increasing the copolymer concentration, Pluronics with comparably short PPO and PEO blocks, such as F87 and P85, could also be used to stabilise the DOPE dispersion (Figure 2.11b). F127 F108 F88 F87 P85 P105

a

turbidity / a.u.

turbidity / a.u.

1 0.8 0.6

lp

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30

1

b

0.8 0.6 0.4

0

0

10

F127 F108 F88 F87 P85

0.2

0.2

0

1.2

40

50

60

0

10

20

30

40

50

Pluronic / mol %

time / min

Figure 2.11 Effect of PEO-PPO-PEO copolymers on sample turbidity. (a) Turbidity recorded as a function of time after acidification to pH5. The liposomes were prepared in the presence of 5 mol% Pluronic. (b) Turbidity at pH 5 as a function of Pluronic concentration. The measurements were carried out after 60 minutes incubation at pH 5.

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2 STABILITY & STABILISATION

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The aggregate structures of the DOPE/Pluronic samples at pH 5 were in agreement with the turbidity measurements. As can be seen when comparing Figure 2.12a with 2.10c, samples containing 10 mol% of F127 display essentially the same aggregate structures at pH 5 and pH 9.5. Thus, 10 mol% of F127 was sufficient to stabilise the mixture in a lamellar arrangement even at low pH. When F127 was exchanged for P85 the majority of the lipids were instead, as expected, found in transition structures and particles of dispersed HII phase, Figure 2.12b.

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Figure 2.12 Aggregate structure as observed by c-TEM 20 min after acidification to pH 5. The samples contained 3 mM DOPE and the liposomes were prepared in the presence of (a) 10 mol% F127, or (b) 10 mol% P85. Bar = 100 nm

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In order to verify the stabilising effect offered by the PEO-PPO-PEO triblock copolymers, leakage measurements were performed at pH 5. As shown in Figure 2.13, the leakage could be completely prevented by inclusion of an appropriate amount of Pluronic. It thus appears that the investigated PEO-PPO-PEO polymers are able to prevent not only aggregation of the DOPE liposomes but also, aggregation-independent, proton-induced structural rearrangements that may lead to the release of encapsulated hydrophilic compounds. The results of this study show that PEO-PPO-PEO block copolymers may be used to stabilise DOPE liposomes in a lamellar arrangement at acidic pH. While it is generally accepted that the block copolymers are anchored to the bilayer via the PPO moiety, it is not known for certain

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2 STABILITY & STABILISATION

100 F127 F108 F88 F87 P85

leakage / %

80

60

40

20

0 0

10

20

30

40

50

H

Pluronic / mol%

C

Figure 2.13 Proton induced leakage as a function of Pluronic concentration for liposome samples prepared in the presence of the Pluronics. All samples had a 0.5 mM DOPE concentration and measurements were carried out after 60 minutes incubation at pH 5.

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whether the two PEO chains protrude on the same or on the opposite side of the bilayer. The latter alternative, i.e. where the polymer adapts a membrane-spanning configuration seems less likely since it has been found that PPO is essentially immiscible with hexadecane [79]. This also implies that the PPO-block does not penetrate deep into the hydrophobic region of the bilayer. In addition, only a minor difference in the amount of released material was observed upon prolonged incubation of DOPE liposomes containing 50 mol% F127, compared to pure DOPE liposomes, at pH 9.5. This finding speaks against a deep penetration since a disturbance in the bilayer packing due to penetration is excepted to increase the bilayer permeability. It is thus plausible that the PPO unit mainly resides within the outer part of the bilayer, close to the headgroup region of the lipids. The fact that stabilised non-leaky DOPE liposomes may be produced via inclusion of Pluronics could prove useful for drug delivery applications and especially in connection with applications requiring a rapid release (see section 4). However, before Pluronic-stabilised DOPE liposomes can be evaluated for this or other types of in vivo application, it has to be established whether the Pluronic/DOPE interactions are strong enough.

33

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A sustained release of encapsulated drugs, i.e. a retention of the drug en route to its destination, in combination with a long circulation time, makes the liposomes useful as a targeted drug delivery system. Controlling the permeability of the liposome membrane, and thus avoiding drug release, will minimise the negative side effects caused by freely circulating drug molecules. The permeability of a bilayer is strongly influenced by its constituents and in Paper IV the goal was to ascertain how the permeability of the EPC-membrane was affected by different phospholipids and potential steric stabilisers.

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3.1. Membrane permeability…

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Liposome membranes are semi-permeable in that the rate of diffusion of molecules and ions across the membrane varies considerably. For molecules with high solubility in both organic and aqueous media, a phospholipid membrane clearly constitutes a very tenuous barrier, while polar solutes and high molecular weight compounds pass across the membrane very slowly. The generally accepted leakage mechanism, for polar solutes, is via defects or temporary openings (pores) in the membrane [80-82]. 3.1.1 … and phospholipids The frequency of pore formation in a membrane is mainly determined by the state of the membrane. Comparing the permeability of liposomal membranes in a liquid crystalline state with a more ordered state, the

34

3 SUSTAINED RELEASE

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former display a higher leakage rate of encapsulated hydrophilic substances [83]. In pharmaceutical application, liposomes usually contain about 40 mol% cholesterol, since cholesterol is known to increase the bilayer packing order [84]. The result is a lipid membrane with reduced permeability [8,85,86]. In Paper IV a reduced permeability was recorded when 5 mol% of the saturated DSPC was incorporated into EPC liposomes, se Figure 3.1a. DSPC is expected to create a more ordered membrane and hence fewer defects are formed. Exchanging DSPC for DSPE, and thereby introducing an amine group in the headgroup region, further reduces the permeability. The presence of the amine might promote hydrogen bond formation with the phosphate group of EPC, which could give rise to a membrane less prone to pore formation. Similar results, which support this explanation, have been found in other studies [87,88]. 0.4

0.3

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a

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0.1

0

0

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400 600 time/min 0.4

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C PEG5000 16

0.25 0.2 0.15 0.1

0.1

0.05

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0 0

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600

800

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time/min

0

200

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600

800

1000

time/min

Figure 3.1 Leakage of pure EPC liposomes { versus time, at 25°C and pH 7.4 and of EPC liposomes incorporated with 5 mol% of a) DSPC, DSPE, DSPEPEG2000 and DSPE-PEG750, b) C1 8 E 8 , Brij 700 and Myrj 59, c) DSPEPEG2000, C16-amide-PEG5000 and C18-amide-PEG5000.

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3 SUSTAINED RELEASE

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3.1.2 …and PEG-ylated lipids or polymers The PEG-ylated lipids and surfactants, shown in Figure 3.2, are micelle forming molecules and thus prefer structures with high curvature. Despite this common feature they affect the membrane permeability to a varying degree, as shown in Figure 3.1b and c. The more conventional surfactant C18E8 ((EO)8-stearyl ether) with a single hydrocarbon chain and a relatively small headgroup induced an increased permeability. The same behaviour was observed for the PEG-ylated polymers Brij 700 ((EO)100 -stearyl ether) and Myrj 59 ((EO)100 -stearate), which have a much bulkier headgroup, see Figure 3.1b. The size of the headgroup, however, seems to determine the quantity of the leakage, where a bulky headgroup leads to a lower leakage. In contrast to the PEG-ylated single-chained surfactants, PEG-lipids had a leakage reducing effect (Figure 3.1a). The reducing effect was present for both DSPE-PEG(750) and DSPE-PEG(2000), and followed the same trend as above where a lower leakage was displayed when the size of the PEG chain was increased. For a PEG chain with a high molecular weight and/or a high enough grafting density, the PEG moiety will become extended from the bilayer surface. Pore formation then demands either compression of the PEG chains or demixing of the PC and PEG-lipids in the bilayer so as to exclude the latter from the edge of the pore. These processes are energetically unfavourable and thus the probability of pore formation is expected to decrease. O

HO

lp

(A)

O

17

n

C

O

O

(B )

HO

he

n

O

17

C

O O

(C)

O

O N H

n

15 ,17

C O

O

17

C

O

O

(D)

O O

n

O

N H

O -

P O

O

17

C

O

Figure 3.2 Formula structures of A) Brij 700 (n~100), B) Myrj 59 (n~100), C) PEG-amide-surfactant (n~114), D) PEG-lipid (n~17, 45), where n is the average number of PEG units.

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3 SUSTAINED RELEASE

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The fact that the Myrj 59 and Brij 700, despite their long PEG chains, have an opposite effect on the leakage compared to the PEG-lipids, indicates that the former surfactants induce a packing disturbance in the membrane that cannot be compensated for by a reduced probability of pore formation. Since the ability to form hydrogen bonds in the headgroup region appears to obstruct pore formation, it was interesting to investigate PEG-surfactants with a capacity to form hydrogen bonds. PEG-ylated surfactants with an amide linkage were synthesised and their effect on the leakage was compared to Brij 700 (with an ether linkage) and Myrj 59 (with an ester linkage). All surfactants have the same molecular weight of the PEG chain. In contrast to ether- and ester-linked PEG polymers, the PEG-amide polymer reduced the permeability significantly as shown in Figure 3.1c. The nature of the covalent link between the hydrocarbon chain and the PEG chain, thus seems crucial for the obtained membrane properties and the reason might be their ability to form hydrogen bonds.

37

4

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Triggered Release

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Considering the multitude of factors that might cause biological destabilisation of liposomes it may seem to be a simple task to obtain drug release from liposomes. As will be shown later (in section 5) this issue represents, however, one of the more difficult challenges.

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4.1 pH-triggered release

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In many applications the liposome-encapsulated drug needs to be delivered to a specific site (section 5.3) but as long as the drug remains trapped inside the liposomes it stays inactive. A slow drug release is, in most cases, not sufficient for an efficient treatment. Different types of liposomes, such as temperature- and pH-sensitive liposomes, have been developed for this purpose [89-92]. The basic idea is that an environmental change will trigger the liposomal membrane to structural rearrangements that induce a leakage of the encapsulated substance.

Figure 4.1 A schematic representation of a pH-triggered release. ©Göran Karlsson

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4 TRIGGERED RELEASE

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In Paper V the aim was to increase the understanding of triggered release from pH-sensitive liposome systems, and more specifically to discriminate between the different release mechanisms possible for a cytosolic drug delivery. The use of PEG-lipids as stabilisers of DOPE liposomes serves dual purposes: liposome formation is facilitated and at the same time the PEG-lipids provide steric stabilisation [61,72,74,77]. However, even small amounts of PEG-lipids in the DOPE membrane prevent L_-HII transition at low pH, i.e. no triggered release is achieved [61,72,74,77]. If the PEG is attached to the lipid by an acid-labile linkage, the cleavage and loss of the PEG moieties accompanying a pH reduction, restore the pH-sensitivity of the liposomes and an L_-HII transition is made possible. A schematic representation of a pH-triggered release is shown in Figure 4.1. Mildly acidic amphiphiles, such as oleic acid (OA) and cholesteryl hemisuccinate (CHEMS), are other stabilisers that are commonly used in triggered release systems of DOPE liposomes [93-95]. In Paper V we used a novel lipid, DHCho-MPEG5000, composed of a hydrogenated cholesterol linked to a methoxy-PEG chain (Mw 5000) by means of an acid-sensitive vinyl ether bond. Upon acidification, DHChoMPEG5000 is hydrolysed to give DHCho and a MPEG5000 derivative as shown in Figure 4.2. Inclusion of DHCho-MPEG5000 was shown to have a stabilising effect on DOPE dispersions at pH 9.5, see Figure 4.3a. Wellformed, predominately spherical and non-aggregated, liposomes were formed upon incorporation of 5 mol% DHCho-MPEG5000.

O

O

O

H + / H2O

O

D HC ho-MPEG5000

n

O

O

OH

+

O

O O

H

n O

DHC ho

MPEG5000

Figure 4.2 Acid-catalyzed hydrolysis reaction of DHCho-MPEG5000. The number of PEG units, n 5 112.

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4 TRIGGERED RELEASE

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Comparison with pure DOPE liposomes in Figure 4.3b reveals that the presence of the long PEG chain protects the liposomes from aggregation. Upon lowering the content of DHCho-MPEG5000 to 1 mol%, these protective properties were notably reduced. As shown in Figure 4.3c, structures that are likely to represent intermediate structures were now quite frequently observed in the sample.

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Figure 4.3 Cryo-TEM micrographs of DOPE and DHCho-MPEG5000:DOPE liposomes at 37°C, pH 9.5, 3 mM total lipid concentration. A. 5:95 DHChoMPEG5000:DOPE. B . Pure DOPE liposomes. C . 1:99 DHChoMPEG5000:DOPE. Bar = 100nm.

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Thin layer chromatography (TLC) indicated that the hydrolysis of the acid labile linkage was a slow process at pH 4.5. This was further confirmed by the slow leakage and lipid mixing of DOPE liposomes containing 1 mol% of DHCho-MPEG5000, at pH 4.5 (Figure 4.4). The appearance of a lag phase in the lipid mixing data indicates that the membrane-membrane contact, which must precede membrane fusion (measured as the lipid mixing), is blocked. Hence, the majority of the PEG chains are presumably still attached to the liposomes during the lag phase and a rapid pH-induced release is thereby inhibited. The cleavage rate is probably too slow at pH 4.5 to make DHCho-MPEG5000 ideal for a rapid destabilisation of liposomes intended for in vivo use.

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Figure 4.4 Leakage O and lipid mixing O as a function of time for 1:99 DHChoMPEG5000:DOPE at 37°C and pH 4.5.

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4.1.1 Release mechanism The use of pH–sensitive liposomal systems requires targeting of liposomes to specific cells that are capable of internalising substance filled liposomes by means of endocytosis. Liposomes internalised via endocytosis will experience a gradual pH decrease [96-100] and this environmental change constitutes the basic idea for triggered release from pH-sensitive liposomes. Although it is well established that pH-sensitive liposomes do collapse and release their contents upon acidification, one problem still remains, that is, the active substance must also be able to cross the endosomal membrane. A number of in vitro studies indicate that internalised DOPE-based pH-sensitive liposomes are indeed able to deliver hydrophilic substances to the cytosol of target cells [90,101]. The mechanisms behind the release process are complex, however, and far from fully understood. A cytosolic delivery depends first of all on the chemical properties of the drug [102]. A drug with suitable hydrophilic/hydrophobic properties will be able to cross the endosome membrane by simple diffusion. This is not the case, however, for most liposome-encapsulated drugs and transportation into the cytosol thus requires other mechanisms [94]. One possibility is that the liposomes, upon acidification, fuse with the endosomal membrane. This would lead to a “microinjection” of the drug into the cytosol. A second alternative is that the liposomes, as a result of a lowered pH, first collapse and release their contents into the endosomal compartment. In a second step the DOPE molecules, initially

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Figure 4.5 Two possible mechanisms for a cytosolic delivery. A) The fusion or the “microinjection” mechanism and B) The destabilisation mechanism.

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situated in the liposomes, may interact with the endosome membrane, which could lead to a higher permeability and perhaps also major structural rearrangements of the endosome membrane. These two possible mechanisms are schematically represented in Figure 4.5. In order to try to distinguish between the possible mechanisms we set out to investigate the interaction between our pH-sensitive liposomes and membranes designed to mimic endosome membranes. Early endosomes are believed to contain an overall lipid composition similar to that of the plasma membranes [103]. The composition thus varies with the cell type [104,105], but normally includes PC, PE, SM, Cho and PS as major components. The so-called endosome liposomes were designed to have a standard composition of EPC:DOPE:SM:Cho 40:20:6:34 (mol%), but PS was excluded from these standard membranes to avoid complications due to the presence of a charged component. First we performed lipid mixing experiments between our endosome liposomes and liposomes composed of either OA:DOPE 40:60 (mol%) or DHCho:DOPE 3:97 (mol%). The latter corresponds to the composition of 3:97 (mol%) DHCho-MPEG5000:DOPE-liposomes, after complete cleavage of the DHCho-MPEG5000. After 2 days at pH 5.5 and 37°C, no significant lipid mixing was observed for either of the pH-sensitive liposomes (Figure 4.6a). This suggests that a spontaneous fusion with the endosome membrane is not likely to occur, even after prolonged

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incubation at low pH. Nevertheless, it does not exclude the possibility of fusion mediated by endosome specific proteins. In the second release mechanism, mentioned above, a pH reduction would lead to an escape from the endosomal compartment due to a change in lipid composition of the endosome membrane, as a result of incorporation of lipids originating from the pH-sensitive liposome. To investigate this possibility, we prepared samples with various lipid compositions corresponding to such a lipid exchange between endosome liposomes and liposomes composed of OA:DOPE 40:60 (mol%), DHCho:DOPE 3:97 (mol%), DHCho-MPEG5000:DOPE 1:99 (mol%) and DSPE-PEG2000:DOPE 3:97 (mol%), see Table I.

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Table I Lipid compositiona and size ratio of endosome:lipsome mixtures. sample EPC DOPE SM Cho OA DHCho DHCho DSPE SRb -MPEG -PEG 1 21.9 38.1 3.3 18.6 18.1 1 2 22.8 53.1 3.4 19.4 1.3 1 3 21.1 57.2 3.2 18.0 0.5 1 4 23.2 52.3 3.5 19.8 1.3 1 5 31.7 35.9 4.8 27.0 0.6 3.5 6 40 20 6 34 n/a a mol% ; b SR is the size ratio, i.e the size of the endosom/size of the lipsome

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Samples 1-4 in Table I have the compositions that would result from a 1:1 mixture between pH-sensitive and endosome liposomes. Since early endosomes have been reported to have a size of about 100 nm [106], samples 1-4 serve as models for events taking place early within the endocytotic process. Late endosomes are considerably larger [106,107] and to investigate the effect of size on liposomes leakage, the composition in sample 5 represents the mixture obtained by incorporation of the lipid from a DHCho:DOPE 3:97 (mol%) liposome into an endosome liposome having a diameter that is about four times larger. Figure 4:6b shows the leakage of the samples in Table I at 37°C and pH 5.5, as a function of time. The samples corresponding to a mixture with early endosomes, sample 1-4, displayed a moderate leakage in the case when PEG-lipids had been included, while the samples with no stabilising PEG-lipids in the membrane had a very rapid leakage. To draw this conclusion we assume that sample 3 still contains a large

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Figure 4.6 Leakage and lipid mixing with endosome liposomes, 37°C, pH 5.5, 250 µM total lipid concentration. A: Lipid mixing as a function of time between endosome liposomes and OA:DOPE or DHCho:DOPE liposomes. B: Leakage as a function of time for sample 1 - 6. The numbers refer to the sample numbers used in table I .

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amount of intact PEG-lipids because of the slow hydrolysis rate. Sample 5, which serves as a model for the mixing between late endosomes and pH-sensitive liposomes, has a very different release profile compared to sample 2, which corresponds to the same pH-sensitive liposomes mixed with early endosomes. The release profile of sample 5 reveals that the rate of the release decreases with a decreasing amount of DOPE in the membrane. The leakage experiments support the notion that incorporation of HII-phase promoters may increase the permeability of the endosome membrane. Further, it is clear that this effect is counteracted by the presence of low concentrations of PEG-lipids that stabilise the lamellar phase. The suggestion that the leakage behaviour could be explained by changes in phase propensity was confirmed by cryo-TEM investigations. Cryo-TEM micrographs of endosome liposomes, at pH 5.5 and 37°C, revealed a polydisperse collection of structures, all displaying a lamellar structure (Figure 4.7a). As expected from the leakage measurements, large aggregates displaying the complex morphology associated with the transformation from lamellar to inverted phase were frequently found in samples 1 and 2. The micrograph shown in Figure 4.7b corresponds to sample 2. The structures found in sample 4, Figure 4.7c, confirm that only a small quantity of PEG-lipids is needed to stabilise the lamellar arrangement of the lipid mixture.

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Figure 4.7 Cryo-TEM micrographs of various liposome formulations imaged pH 5.5 at 37°C (3 mM total lipid concentration). A. endosome liposomes, B. sample 2, C. sample 4. Numbers 1 to 4 refer to the sample numbers used in table I. Micrographs A and C were imaged >60 minutes and B within 15 minutes after acidification. Bar = 100nm.

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Taken together, the results suggest that the observed ability of DOPE-containing liposomes to mediate cytoplasmic delivery of hydrophilic molecules cannot be explained by a mechanism based on a direct, and non-leaky, fusion between the liposome and endosome membranes. A mechanism involving destabilisation of the endosome membrane due to incorporation of DOPE seems more plausible.

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In the present section, the development and optimisation of liposomes intended for cancer therapy by means of BNCT (Boron Neutron Capture Therapy) will be introduced. First, the basic principle of BNCT for cancer treatment and our potential drugs will be presented. This will be followed by an introduction to the concept of remote loading of drugs into liposomes, and a description of the optimisation of this procedure for two novel boronated compounds. Finally, the concept of targeted drug delivery of liposomal formulations will be presented and our development of targeted liposomes intended for BNCT will be discussed. Furthermore, some in vitro cellular investigations and in vivo biodistribution studies of these systems will be presented.

5.1 Principles of BNCT

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BNCT is a two-part therapy in which first a non-toxic compound containing the stable boron nuclide, 1 0B, has to be accumulated in tumour cells, in a concentration higher than that in the surrounding tissue. Then the cells are exposed to thermal neutrons, resulting in a nuclear fission yielding two highly lethal ions and characteristic gamma radiation [108]. In tissue, the lethal ions, 4He2+ and 7Li3+, have a range of about 9 and 5 µm, respectively, which is less than the average diameter of a cell (approximately 10µm). 10

B +1n th A

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5 LOADING & TARGETING

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The deposited energy is thereby mainly distributed within the cells that have accumulated 10B.The main advantage of BNCT is the fact that an external activation source is required to initiate a cell-toxic activity of the stable boron nuclide, since the boron itself is non-toxic. Accumulation of 10B-containing compounds can be achieved by the use of tumour-seeking (or tumour-targeting) drug vehicles and the most promising vehicles for this purpose are liposomes. Tumour-targeted liposomes will mainly deliver their cargo to tumour cells and hence the average concentration of the drug will be kept low in the body and in surrounding healthy tissues. In our investigations, receptor-mediated targeting has been used and this will be described in more detail below and in Paper VII. The boron compounds most often used in combination with liposomes are hydrophilic substances. Paper VI and VII, are focused on hydrophilic compounds consisting of a boron-rich carborane cage coupled to a moiety that is known to intercalate into, or interact with, DNA. One of the compounds, WSA (water soluble acridine), is shown in Figure 5.1. In WSA the carborane cage has been coupled to acridine and spermidine, which are both known to interact with DNA [108]. The rationale for using DNA-intercalating/interacting compounds is that the therapeutic efficiency is substantially increased when the neutron-activated fission can take place in, or in the vicinity of, the cell nucleus. Furthermore, the retention of the compound in the tumour cell should increase if the compounds are bound to the DNA. The drug loading optimisation of such compounds are presented below and in Paper VI.

Figure 5.1 Structural formula of WSA (or water soluble acridine).

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5.2 Drug loading

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Drug molecules can interact with liposomes in several different ways depending on the drug’s solubility and polarity characteristics. Watersoluble drugs are usually entrapped in to the aqueous cavity, while water-insoluble drugs can be solubilised in the hydrophobic part of the liposomes. Hydrophilic drugs are most simply encapsulated automatically upon hydration of the lipids with a corresponding drug solution. The non-encapsulated drug molecules are then removed by gel filtration. However, the efficiency of this method is quite low. There are several ways to improve the drug encapsulation of hydrophilic drugs and one of the more elegant solutions is so-called remote loading where the drug is loaded into preformed liposomes using different ion gradients [109-111].

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5.2.1 Remote loading Remote loading can be used for loading of weakly acidic or alkaline drug molecules [109-111]. The basic idea of the method is that neutral molecules are shuttled, due to the different pH between the inside and the outside of the membrane, into liposomes where they become charged. Charged drug molecules will thereby be trapped in the liposomal interior, owing to a low diffusion rate of charged molecules over the membrane. Figure 5.2 shows a representation of the remote loading of a weak base into preformed liposomes by means of the so-called pH-gradient method. The pH inside the liposome is lower than the pH outside and the weak base B, which is in equilibrium with its protonated form, passes through the membrane in its unchanged form. Inside it is, however, protonated and thereby inhibited to leave the liposome. Although the pH-gradient loading method can accumulate large amount of acids or bases in the liposomes, a decaying pH-gradient may cause problems with premature drug leakage [110]. However, some drugs have been shown to have the ability to precipitate inside the liposomes at higher concentrations and so this problem becomes negligible [109].

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A3H

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Figure 5.2 The pH-gradient method, for loading of weak hydrophilic alkaline drugs.

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5.2.2 Remote loading of boronated agents In order to optimise the encapsulation efficiency of WSA, see Figure 5.1, two types of remote loading methods were used; the pH-gradient [110], mentioned above, and the ammonium sulphate gradient [111]. The results in Figure 5.3a show clearly that by using the pH-gradient method, trapping efficiencies > 95% can be achieved even at rather large drug-to-lipid ratios. The high trapping efficiency obtained yielded concentrations of the drugs inside the liposomes that were two orders of magnitude higher than the solubility of the drug in the aqueous phase. At such high concentrations it is likely that the drug molecules precipitate inside the liposomes. This was confirmed by cryo-TEM, as shown in Figure 5.4a, where the dark globular spots in the liposome interior represent a drug precipitate. Surprisingly, the encapsulation efficiency (in Figure 5.3a) using the ammonium sulphate gradient was shown to decrease substantially at higher drug-to-lipid ratios. However, the reason for this was found using cryo-TEM where the formation of discs was observed in the samples of high drug-to-lipid ratios, shown in Figure 5.4b. The disc formation is assumed to occur due to precipitate growth. Eventually the precipitate “pierces” the lipid membrane resulting in a breakdown of the liposome structure and hence reduced trapping efficiency. The release rate of the encapsulated WSA is a very important parameter, and the stability of WSA loaded liposomes was shown to be excellent, both in buffer and in 25% human serum. For most formulations the release rate was only around 10% after 24 hours at 37°C, as shown in Figure 5.3b. When the leakage measurements were performed in media containing human serum, no elevated leakage was observed and hence the retention of WSA during blood circulation should be rather good.

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Figure 5.3 a) Trapping efficiency of WSA using the pH-gradient loading procedure (z) or the ammonium sulphate gradient ({ ). b) Effect of drug-to-lipid ratio on the release of WSA at 37 °C, when loaded by means of the pH-gradient method. The initial drug-to-lipid ratios (mol/mol) were 0.15:1 (z), 0.20:1 („), 0.25:1 (‹), 0.30:1 (V) and 0.50:1 ().

Figure 5.4 Cryo-TEM micrographs of DSPC/Cho/PEG(2000)-DSPE (55/40/5) (mol%) loaded with WSA using the a) pH-gradient, b) ammonium sulphate gradient. Arrows in b) denote bilayer discs as observed face-on (A) and edge-on (B). Bar = 100nm.

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5.2.3 Comments on the lipid composition In many pharmaceutical formulations, cholesterol is included in the lipid mixture to further increase the in vivo stability since it has been shown to reduce the leakage of liposome encapsulated hydrophilic substances [8,85,86]. Furthermore, it has been shown that liposomes with 5-10 mol% PEG(2000)-lipid exhibited the longest circulation time in vivo [31,32]. The upper limit of PEG(2000)-lipid that is possible to incorporate without a breakdown of the liposome structure, is about 10 mol% [53-55]. On the other hand, 5 mol% is the critical surface concentration of PEG needed to yield an effective steric stabilisation [31,32]. In addition, saturated phospholipids are preferred when considering the long-term stability of the liposomes. The liposomes in the studies described in this section were composed of DSPC/Cholesterol/PEG(2000)-DSPE in proportions of 55/40/5 mol%. This composition is one of the most commonly used formulations for liposomes intended for drug delivery.

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5.3 Site-specific targeting

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In an ideal therapy for cancer, the treatment should be able to completely eradicate the tumour cells without injuring normal tissues. The development of a targeted nuclide therapy originates from the fact that conventional methods like surgery, chemo- and external radiotherapy, all have limitations. Surgery and external radiotherapy are well suited for treatments of large and well-defined tumour masses, but not for spread cells. Chemotherapy is efficiently used for some types of tumours with disseminated cells, but not for all, since some cases require high doses and hence normal organs, for instance the bone marrow, are easily damaged. An ideal treatment of spread cells would imply a delivery system with a high affinity towards the tumour cells, leaving the normal and healthy tissue unaffected. In tumour tissue and at sites of inflammations the blood vessels are rather leaky [30]. This helps to bring a large fraction of small liposomes loaded with the drug to these sites. However, this passive accumulation does not mean that the liposomes are internalised into the tumour cells but rather that they slowly leak their content in the tumour area. For most tumour therapies based on a delivery of cytotoxic agents to the

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tumour cells, this non-specific leakage is not sufficient for an efficient treatment. In BNCT a specific targeting towards the tumour cells, and further into the cell cytoplasm, is necessary since the lethal ions, induced by external thermal neutrons, have a short range and the drug should preferably be able to make it all the way to the cell nucleus [112]. To increase the accumulation of the liposomes in the tumour area, as well as to increase the efficiency of cytoplasmic delivery, receptortargeted sterically stabilised liposomes have been developed.

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5.3.1 Receptor-mediated targeting A characteristic difference between normal and tumour cells is essential for targeting. This could be a structure expressed solely on tumour cells, however, there are very few known structures exclusively expressed on tumour cells. It could also be a difference in the quantity of an expressed structure. These structures, called tumour-associated antigens, are also often found in the organs from which the tumour originated. Antibodies are the most commonly used biomolecules in targeting against tumourassociated antigens. Other types of biomolecules suggested for targeting are hormones or ligands, which are directed towards normal cellular receptors that are overexpressed in certain tumour cells. An example of such a biomolecule is the epidermal growth factor, EGF, which can be directed towards the corresponding epidermal growth factor receptor, EGFR [21].

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5.3.2 EGF-labelled liposomes EGF has been suggested as a good candidate for tumour targeting in BNCT for several reasons. The EGF receptor is overexpressed in many cancer forms, including gliomas, breast, colon and prostate-cancer [113,114]. It is also known that EGF undergoes receptor-mediated endocytosis, which can bring the ligand-receptor complex inside the cell. In Paper VII, the conjugation of EGF to liposomes, loaded with WSA, was investigated. EGF was conjugated to the distal end of PEG-DSPE lipids in a micellar solution. The EGF-PEG-DSPE micelles were then mixed with preformed liposomes, either empty or loaded with WSA. By this “micelle-transfer” method the EGF-lipids became incorporated into the liposome membrane. This approach is appealing since the development of new formulations can be reduced to only redesigning the conjugation or the loading procedure.

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Optimisations of the micelle-transfer method, by changing conditions like time, temperature and concentration, were performed on both empty and WSA-loaded liposomes. For the optimised EGF-labelled liposomes ~10-15 EGF molecules/liposome was obtained and this amount is assumed to be enough to achieve a satisfactory cellular uptake of antibody-targeted liposomes [115,116]. Combining the optimisation of the WSA loading procedure and the EGF-labelled liposomes, the drug-tolipid ratio was set to 0.2, for all following experiments. This amount of WSA corresponds to about 105 to 106 boron atoms in each liposome, which is enough to make the liposomes interesting for therapy. It is known that 108 to 109 boron atoms are needed per tumour cell for therapeutic effect [117], meaning that in our case 102 to 104 receptor interactions per cell are required. The stability of WSA-loaded EGF-labelled liposomes, regarding WSA leakage and EGF conjugation degradation, was investigated in cell culture medium and at different temperatures. At 4°C the liposomes could be stored for weeks without any substantial leakage and/or degradation. The stability at 37°C was also acceptable and high enough to perform in vitro tests in cultured tumour cells. A so-called displacement test was performed to analyse the receptor specific binding of the EGF-labelled liposomes. Increasing amounts of EGF were shown to displace both empty and WSA-loaded EGF-labelled liposomes in cultured human glioma cells5. This proved that we could indeed obtain receptor-specific binding of the EGF-labelled liposomes.

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5.3.3 BNCT with EGF-labelled liposomes Two important requirements must be met for BNCT to be efficient. First, the 10B-compounds must be distributed specifically to the tumour cells and, second, a high concentration of 10B must be delivered to each cell. These two obstacles can be overcome by the use of liposomes in combination with a concept involving two-step targeting. A schematic representation of the two-step targeting mechanism is given in Figure 5.5. EGF-labelled liposomes loaded with WSA have been found to exhibit receptor-specific binding and receptor-mediated internalisation in two different cultured tumour cell lines [102]. However, the liposome encapsulated WSA was not able to reach the nucleus after the 5

The cultured human glioma cells were U-343 MGa cells.

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EGFR

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Figure 5.5 Schematic presentation of the two-step targeting. Step 1) targeting of EGF-labelled liposome, with encapsulated DNA intercalating compound, to tumour cells overexpressing the EGFR. After receptor mediated intercalating of the liposomes and liposomal degradation, step 2) the released boronated DNA intercalators bind to cellular DNA.

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internalisation [102]. Nevertheless, the retention of the boronated compound in the cytoplasm was good and hence a nuclear uptake might not be necessary. Preliminary results indicate that neutron activation of cytoplasmic WSA in cultured glioma cells, internalised by receptor mediated endocytosis of EGF-labelled liposomes, gives a therapeutic effect [118]. This effect would be further increase if the boronated compound consisted of 100% enriched 10B instead of 20%, which was used within this study. When WSA was exchanged for the conventional cytotoxic drug doxorubicin the two-step targeting concept was proven. The use of EGFlabelled liposomes loaded with doxorubicin yielded the desired delivery to the nucleus. 5.3.4 Biodistribution of EGF-labelled liposomes One problem related to the use of EGF-mediated targeting is the toxic effects on normal cells expressing EGFR, e.g. the liver [119]. However, recent biodistribution tests of EGF-labelled liposomes in mice indicate that when EGF is conjugated to stabilised liposomes with a diameter of about 100nm, the distribution to the liver is significantly reduced [120]. This implies that the possibility for the EGF-labelled liposomes to reach the tumour cells increases substantially.

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5.3.5 Antibody-labelled liposomes Antibody-labelled liposomes have also been investigated with regard to the two-step targeting concept [121]. The antibody used was herceptin, which is specific towards HER-2 receptors. HER-2 is overexpressed in several types of cancers, including breast cancer, lung cancer, gastric cancer and bladder carcinoma [122-125]. Herceptin-labelled liposomes loaded with WSA showed a receptor-specific binding and internalisation. In addition, a high retention of the boron compound was achieved but without any delivery to the nucleus. Thus, the same trends were observed for both the EGF- and the herceptin-labelled liposomes.

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In BNCT, however, the DNA is the most highly prized target for boron neutron capture damage. If the boron could accumulate in the nucleus, lower amounts would be needed to achieve therapeutic effects. So far, the boronated compound did not reach the cell nucleus, even though it was internalised via endocytosis. This indicates that the drug is inhibited from leaving the endosomal compartments. To overcome this obstacle, pH-sensitive liposomes have been suggested as a promising alternative to the conventional PC-liposomes. The gradual decrease in pH experienced by liposomes that are internalised via endocytosis [96100] constitutes a potentially very useful intrinsic stimulus. Today, several pH-sensitive liposome formulations based on this strategy have been developed and evaluated biologically [90].

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Experimental Techniques

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This section will briefly describe the techniques used to obtain the results presented in this thesis. The aim is merely to illustrate the central ideas of the techniques with particular emphasis on the cryoTEM technique.

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The Cryo-Transmission Electron Microscopy (cryo-TEM) technique is a powerful tool for studying aggregate structures of amphiphilic molecules in water solutions [126]. The uniqueness of this technique lies in the fact that no staining, drying or chemical fixation is used for the preparation of the specimen. Instead, a thin sample film (< 500nm) is quickly frozen, vitrified, and thereafter transferred directly to the microscope for examination. Owing to the vacuum in the microscope and the low vapour pressure of the frozen film, the electrons can penetrate the thin film and if the aggregates residing in the film have high enough electron density compared to water, they can be visualised. Vitrification of a sample is achieved by rapidly plunging the sample into liquid ethane held at ~ 100 K. The procedure of rapidly plunging the sample into a cold liquid of relatively good heat capacity ensures a good probability for creating a vitrified film of the sample solution, and avoiding the crystalline ice formed when water is cooled slowly. Preparation of a thin sample film requires, however, certain controlled environmental conditions and for that reason we use a socalled climate chamber. Inside the chamber the temperature and

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humidity conditions are controlled. By keeping a high humidity, evaporation of water can be avoided during the preparation procedure. A schematic view of the preparation technique is shown in Figure 6.1 along with an electron microscopy (EM) grid covered with a perforated polymer film. The first step of the film preparation takes place within the climate chamber by means of a blotting procedure. A small drop of the sample solution is placed onto an EM-grid and excess solution is removed by blotting with a filter paper. The EM-grid with the thin sample film is then plunged into the liquid ethane for vitrification. After this treatment the specimen films can be considered as a transparent glass film with a thickness between 10 to 500 nm. In the last step, the vitrified film is transferred to the grid-holder and further to the microscopy, all performed at a temperature below 108 K to prevent sample perturbation and the formation of ice crystals.

Figure 6.1 A schematic representation of the climate chamber and the EMgrid. ¹Göran Karlsson

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6 EXPERIMENTAL TECHNIQUES

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6.1.1 Limitations and Artefacts A thin film favours good vitrification and contrast of small objects. In thick films containing much material, the effects of multiple scattering and crowding become pronounced, leading to a reduced contrast of the objects. In general, the viscosity of the solution must not be too high. Thereby the cryo-TEM technique will be suitable for investigations of dilute aqueous solutions, >95 wt% water. Sample perturbation in cryo-TEM studies has mainly been focused on freezing damage but since the cooling of the aqueous film is rapid enough (<1ms), there is no reorganisation of large aggregates. The fast kinetics of micellar solutions, however, make surfactants with a strong temperature-dependent aggregation unsuitable for cryo-TEM. There is also a possibility that some films freeze insufficiently. Given some experience it is, however, generally straightforward to distinguish and avoid such perturbed films. To make correct interpretations of the cryo-TEM images, knowledge about some common “structural artefacts” is required. Liposomes under certain conditions may, for instance, appear to be invaginated. This effect is thought to be caused by an osmotic stress of the liposomes. The thin films used for vitrification have large area-to-volume ratios and are thus extremely sensitive to evaporation. If a liposome sample contains salt, osmotic stress might be created during the preparation by solvent evaporation. Another “structural artefact” is size-sorting, originating from the structure of the film, yielding a segregation of structures with different sizes. A perforated polymer film, as shown in Figure 6.1, covers the grid and the blotting of the solution on the grid will result in thin solution lenses spanning the holes. Larger aggregates will be located further away from the centre of the hole, in the thicker part of the lens. In samples containing large aggregates, it is important to remember that aggregates larger than the thickness of the film will not be able to reside in the vitrified film. Nevertheless, when the sample contains small aggregates, a reasonable agreement of the size distribution is normally found between cryo-TEM and dynamic light scattering measurements [127]. The cryo-TEM technique has been used in all the papers included in the thesis.

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6.2

Light Scattering

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Light scattering is the reason why milk is white because milk contains aggregates large enough to scatter light of all wavelengths. The same phenomenon is observed in liposome dispersions. By scattered light we mean light that has been deflected from its original direction and the turbidity is a measure quantifying the degree to which light travelling through a solution is scattered. The turbidity can be used to measure relative changes in aggregate size. The scattered light is proportional to the second power of the volume (or the sixth power of the radius) of the aggregate and thus it is dependent on the size of the aggregates. Larger aggregates result in higher scattering intensity, which is shown by a higher apparent absorbance, according to

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where I0 is the incident intensity, I is the intensity after passage through sample. Turbidity measurements have been used in Papers I, III and V.

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6.2.1 Dynamic light scattering From dynamic light scattering (DLS) measurements we get information about how the intensity varies with time. The variation of the intensity with time contains information on the random motion of the aggregates. Furthermore, this can give information about the size of the dispersed aggregates [128]. DLS can thus be used to measure the size and the size distribution of aggregates in dilute solutions, according to Rh =

kBTq2 6WdKx

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6 EXPERIMENTAL TECHNIQUES

where C 1/C2 is the concentration ratio of population 1 and 2 with different size distributions (assuming that K/q2 | M-1/3, the molecular weight) and A1/A2 is the ratio of the peak amplitudes. DLS has been used in Paper II.

6.3

Fluorescence assays

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Steady-state fluorescence assays were used to measure leakage, lipid mixing and lipid packing order of liposomes, while a time-resolved fluorescence quenching (TRFQ) assay was used for determining micelle aggregation number.

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6.3.1 Leakage Fluorescence quenching measurements have been used to detect leakage of hydrophilic fluorescent molecules from liposomes. Depending on the pH of the sample solution, two different types of probes were used. Carboxyfluorescein (CF) was chosen for measurement where just one pH was required since the fluorescence spectra is pH-dependent [129-130]. Preferably, the solution should have a pH > 7, at lower pH the CF molecule fluorescence intensity will be much lower. The fluorescence of CF is >95% self-quenched at concentration >100 mM. Concentrated solutions of CF are encapsulated in liposomes, which are separated from any remaining free dye by gel filtration. Upon leakage, dye release is accompanied by an increase in fluorescence due to dilution of the CF. For leakage assays requiring measurements at different pH and especially at low pH a mixture of the probes ANTS6 and DPX7 were used since ANTS has a pH-independent fluorescence spectra [131]. This assay is based on the collisional quenching of the polyanionic fluorophore ANTS and the cationic quencher DPX. ANTS and DPX were co-encapsulated into liposomes, using the same procedure as for CF. Upon dilution into the surrounding medium, ANTS fluorescence will increase because quenching by DPX will be diminished. Leakage measurements have been used in Papers II, III, IV and V.

6

8-aminoaphthalene-1,3,6-trisulfonic acid, disodium salt (ANTS) bromide (DPX)

2p-xylene-bis-pyridinium

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6.3.2 Lipid mixing A flourometric method was used for observing membrane fusion by determining to what extent liposomes mix their membrane lipids. The lipid mixing assay is based on NBD/Rhodamine energy transfer [132]. Membranes, labelled with a combination of fluorescence energy transfer donor and acceptor lipid probes, NBD-PE8 and Rh-PE9 respectively, are mixed with unlabelled membranes. By exciting the donor, the fluorescence energy transfer detected as the emission of the acceptor, decreases when the average spatial separation of the probes is increased upon fusion between labelled and unlabelled liposomes. It should be noted that this lipid mixing assay, used in Paper V, cannot detect aggregation, which is the first step in the fusion process.

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6.3.3 Anisotropy A fluorescent hydrophobic probe, DPH (diphenylhexatriene), has been used to investigate changes in the mobility and packing of the hydrocarbon chains in lipid membranes [133], assuming the DPH is stationary in the membrane. The magnitude of the anisotropy is dependent on the motional freedom and rotational diffusion of the probe. A decreased anisotropy indicates a decreased chain order and thus, most likely, an increase of the number of defects in the membrane. The steady-state anisotropy was calculated according to

(

)(

)

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r = IVV < GIVH / IVV + 2GIVH

he

where G = IHV / IHH is an instrumental correction factor and IVV, IVH, IHV and IH H refer to fluorescence intensity polarised in vertical and horizontal detection planes (second subscript index) upon excitation with either vertically or horizontally polarised light (first subscript index). Anisotropy measurements were used in Paper II. 6.3.4 Time-Resolved Fluorescence Quenching Time-Resolved Fluorescence Quenching (TRFQ) was used to determine micelle aggregation numbers [134]. The method requires, for best results, a probe and a quencher that can be regarded as stationary N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (NBD-PE) 9 Lissaminea rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (Rh-PE) 8

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6.4

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during the time scale of the experiment. Pyrene excimer quenching was used, utilising pyrene as both probe and quencher. The excimer is a dimer that merely exists in an excited state (*). Since the concentration of the pyrene* monomer is reduced by the excimer formation, the emission intensity from free pyrene* will be reduced. Hence excimer formation represents a form of emission quenching or concentration quenching since the extent of the quenching is directly related to the pyrene concentration. The collected time-resolved fluorescence decay curves were fitted to the Infelta equation, giving the average number of quenchers per micelle. Knowing the concentration of the surfactant and the added pyrene, the aggregation number of the micelle can be determined. The TRFQ technique was used in Paper I.

QCM

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The Quartz Crystal Microbalance (QCM) is a relatively new technique for adsorption measurements [135]. The major advantage with this technique is that it allows adsorption studies on a large variety of surfaces simply by coating the quartz crystal in the desired way. The QCM technique is based on the piezoelectric effect, which occurs naturally in some materials. Applying an electric field across a piezoelectric material subjects the material to stress. The quartz crystal disc, which is a piezoelectric material, is sandwiched between two gold electrodes. When an AC electric field is applied to the electrodes, the disc starts to oscillate along the surface. The crystal has a resonance frequency, which depends on the total oscillating mass (in a resonating material both a mass and a current are oscillating simultaneously). An adsorption of material onto the disc will thus change this frequency in proportion to the added mass according to the Sauerbrey equation: 6f = < k u 6m

where 6f is the change in resonant frequency, 6m is the change in deposited mass and k is the mass sensitivity constant of the crystal. However, this equation is only valid when the deposited mass can be considered a thin rigid film, evenly distributed over the disc and much smaller than the mass of the crystal itself. The QCM technique can

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monitor frequency changes small enough to achieve a mass sensitivity of ~ 5 ng/cm2 in water. In Paper II a QCM-D™10 apparatus was used, which enables measurements of both the deposited mass and the dissipation, D. The dissipation is a measure of the viscoelastic properties of the adsorbed layer. An adsorbed layer which is viscoelastic does not follow the crystal oscillation to the full extent and consequently the oscillation is damped. Hence, the dissipation gives information about interactions between an adsorbed layer and its bulk solution.

10

The QCM-D is trademark of Q-Sense AB, Gothenburg, Sweden.

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Acknowledgement

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Först och främst vill jag tacka Katarina för att jag har fått möjligheten att göra mina forskarstudier här på Fysikalen, och för att jag har fått friheten att pula på med mina egna projekt och på mitt eget sätt. Sen vill jag även passa på att säga att jag verkligen har trivts och det har varit en kul tid. En av anledningarna till den sköna stämningen här på Fyiskalen är Mats A. och hans sköna stil. (Hoppas silverputsen kommer till andvändning.) Naturligtvis så vill jag även tack alla som jag har lärt känna här. Tack för alla långa stunder i fikarummet, för snack i uppehållsrummet, för känslan att bli slagen med 9-0 i squash, för känslan att vinna med 12-2 i badminton (mot samma motspelare) och för tillfället att få träna mina biceps (hoppandes på kryckor) efter en ”fair” tackling (öh, jag tänkte inte klämma till dig så hårt). Sen vill jag gärna minnas alla roliga partaj som vi har haft. Vad sägs om kräftskivan? Den minns vi väl alla (??). Jag vill även tacka Staffan, för att han allt som oftast ”tutar” förbi uppehållsrummet (eller som det numera heter, Skönhetsrådet). Den oumbärliga kvintetten, Göran å Göran, Gösta, Dick och Laila, utan er skulle det inte hända så mycket på det här stället. Tack! En särskilt tack till Göran K, jag vet inte hur många tack jag vill säga, men många är det. Du är otroligt bra! Sist, men absolut mest, så vill jag tack Makrus, Mato och Maria. Det har varit otroligt kul att jobb tillsammans med er och ett privilegium att få lära känna er. Markus med sin sanna forskarsjäl har hjälp mig många många gånger och andra gånger har han varit helt biiiindgalen. Ja, djävlar i havet! Med Mats, som förövigt har en förkärlek för helspeglar (speciellt framåt nattkröken), har jag har haft ett (1) väldigt långt och trevligt samarbete (flera år långt, faktiskt) och jag hoppas att det blir längre, som Geishan sa. Å till Maria vill jag säga, huhuhuhurra! Synd att vi inte hann göra mer jobb tillsammans, det hade verkligen varit roligt! Vi ses på stadshotellet i Karlstad! Hälsningar Nill

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