Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Science and Technology 340
Bilayer Discs - Fundamental
Investigations and Applications of
Nanosized Membrane Models
EMMA JOHANSSON
ACTA
UNIVERSITATIS
UPSALIENSIS
UPPSALA
2007
ISSN 1651-6214
ISBN 978-91-554-6962-7
urn:nbn:se:uu:diva-8200
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List of papers
I.
Structure of mixed micelles formed in PEG-lipid/lipid
dispersions
Maria C. Sandström, Emma Johansson, Katarina Edwards
Langmuir, 2007, Vol. 23, pp 4192-4198
II. On the formation of discoidal versus threadlike micelles in
surfactant/lipid systems
Emma Johansson, Maria C. Sandström, Magnus Bergström, Katarina Edwards
Submitted to Langmuir
III. Development and initial evaluation of PEG-stabilized disks as
novel model membranes
Emma Johansson, Caroline Engvall, Maria Arfvidsson, Per
Lundahl, Katarina Edwards
Biophysical Chemistry, 2005, Vol. 113, pp 183-192
IV. Nanosized bilayer disks: Attractive model membranes for
drug partition studies
Emma Johansson, Anna Lundquist, Shusheng Zuo, Katarina
Edwards
Biochimica et Biophysica Acta, 2007, Vol. 1768, pp 1518-1525
V. Influence of preparation path on the formation of discs
and threadlike micelles in DSPE-PEG2000/lipid systems
Maria C. Sandström, Emma Johansson, Katarina Edwards
Submitted to Biophysical Chemistry
Reprints were made with the kind permission of the publishers.
Contents
1 Introduction..................................................................................................9
1.1 Amphiphilic molecules and self-assembly.........................................10
1.1.1 Structure of amphiphilic aggregates ...........................................11
1.1.2 Lamellar phases ..........................................................................12
1.1.3 Formation of liposomes and discs ..............................................15
1.2 The cell membrane and models thereof .............................................18
1.2.1 Transport across cell membranes................................................18
1.2.2 Model membranes ......................................................................20
1.3 Aims of the present investigation.......................................................20
2 Experimental techniques............................................................................22
2.1 Preparation of liposomes and discs ....................................................22
2.2 Cryo-TEM ..........................................................................................22
2.2.1 Artefacts in cryo-TEM................................................................24
2.3 Light scattering...................................................................................25
2.3.1 Dynamic light scattering.............................................................25
2.4 Drug partition chromatography ..........................................................26
2.5 Isothermal titration calorimetry..........................................................26
2.6 Fluorescence measurements ...............................................................27
3 Results and discussion ...............................................................................28
3.1 Aggregate structure in surfactant/lipid systems .................................28
3.1.1 Aggregate structures in PEG-lipid/lipid systems........................29
3.1.2 Aggregate structures in C12E8, CTAB and SDS/lipid systems ...33
3.2 Discs as model membranes ................................................................37
3.2.1 Initial disc development..............................................................37
3.2.2 Refinement and optimization of lipid composition ....................39
3.2.3 Drug partition studies .................................................................40
3.2.4 Protein reconstitution..................................................................44
3.3 Effect of preparation path on aggregate structure ..............................45
4 Conclusions................................................................................................50
Svensk sammanfattning ................................................................................51
Acknowledgements.......................................................................................55
References.....................................................................................................56
Abbreviations
BBM
Brush border membrane
bR
Bacteriorhodopsin
C12E8
Octaethylene glycol monododecyl ether
Ceramide-PEG5000 N-palmitoyl-sphingosine-1-[succinyl (methoxy (polyethylene glycol) 5000]
CF
5(6)-carboxyfluorescein
cryo-TEM
Cryo-transmission electron microscopy
CTAB
Hexadecyltrimethylammonium bromide
DLS
Dynamic light scattering
DMPC
1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine
DPC
Drug partitioning chromatography
DPPC
1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine
DOPC
1,2-dioleoyl-sn-glycero-3-phosphatidylcholine
DOPE
1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine
DSPC
1,2-distearoyl-sn-glycero-3-phosphatidylcholine
DSPE
1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine
DSPE-PEGn
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[methoxy (polyethylene glycol)-n]
EPC
Egg phosphatidylcholine
IAM
Immobilized artificial membrane
ITC
Isothermal titration calorimetry
Liquid order phase
lo
MLV
Multilamellar liposomes
MSPC
1-stearoyl-2-hydroxy-sn-glycero-3-phosphatidylcholine
OG
Octyl glucoside
PC
Phosphatidylcholine
PE
Phosphatidylethanolamine
PEG
Polyethylene glycol
PI
Phosphatidylinositol
POPC
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine
PS
Phosphatidylserine
SDS
Sodium dodecyl sulphate
Transsition temperature
TC
ULV
Unilamellar vesicles
1 Introduction
Every living cell is enclosed by a cell membrane that separates the inner
compartment of the cell from its surroundings. This membrane not only
serves as a barrier, but is responsible for the transport of substances into and
out of the cell, and also creates an environment for the many membrane proteins. To investigate the processes occurring in the cell membrane there is a
need for models that can be used to explore, for example, the interaction of
molecules with the membrane. One area that has created a need for model
membranes is the rapid development of new drugs, where numerous drug
candidates are screened in the search for the optimal molecule. To test these
many potential drugs, the majority of which have to cross one or several
biological membranes to be active, methods are required that can quickly
screen these compounds to test their bioavailability. The study of membrane
proteins requires a suitable environment for the protein, i.e. an environment
that resembles the protein’s natural environment, but which can be prepared
in the laboratory.
Due to their structural similarity with cell membranes, liposomes are often
used as model membranes for the above mentioned purposes. However,
some difficulties in using liposomes for these purposes have been identified,
as further discussed in Section 3.2.
Another aggregate structure that can serve as a model membrane is the bilayer disc. The bilayer disc is a circular bilayer fragment composed of the
same material as the liposomes, but without an inner, water-filled compartment. These flat discs can sometimes provide a superior alternative to liposomes, and alleviate some of the problems encountered when using liposomes as model membranes.
The work described in this thesis was focused on the understanding of
systems in which discoidal aggregate structures exist. The effects of various
preparation methods on the disc aggregate structures have been investigated,
and discs intended to serve as model membranes have been developed. The
discs have also been tested as model membranes using two different drug
partitioning methods with good results. An attempt has also been made to
incorporate a protein into these discs.
9
1.1 Amphiphilic molecules and self-assembly
The building blocks of model membranes are so-called amphiphilic molecules. Amphiphilic molecules can be found around us in our everyday life,
for example, in soap. What makes these molecules special is their dual character, as they have a hydrophilic and a hydrophobic part (Figure 1). This
dualistic property will cause the molecules to self-assemble when added to
polar or non-polar medium. One part of the molecule will interact with the
surrounding medium while the other will not.
Figure 1. A schematic illustration of an amphiphilic molecule.
The solubilizing medium used throughout the work described in this thesis
was water containing a low concentration of amphiphilic molecules, and in
such a solution the molecules will accumulate at the air-water interface. This
is why these molecules are often referred to as surfactants, or as being surface active. Above a critical aggregation concentration the amphiphiles will
assemble in a way that allows the hydrophilic part to remain in contact with
the water, while the hydrophobic part is shielded from the surrounding water. The driving force behind this behaviour is called the hydrophobic effect
[1, 2], and is the result of the gain in entropy of the system when aggregation
takes place. When free monomers of amphiphilic molecules are dissolved in
water the adjacent water molecules are forced to arrange themselves around
the amphiphilic molecules. The formation of amphiphilic aggregates will
relieve the entropic stress of the water molecules, making the aggregation of
the amphiphilic molecules energetically favourable. The simplest aggregate
structure, which is responsible, for example, for the cleaning properties of
soap, is the micelle (illustrated uppermost in Figure 2). A number of other
types of surfactant aggregates can also be formed. The aggregate structure
formed will depend on the nature of the amphiphilic molecule and on the
environment surrounding it.
10
Molecular shape and
packing parameter
Predicted aggreagate
structure
Ns ≤ 1/3
Ns ~ 1
Ns > 1
Figure 2. The preferred aggregate structures and the corresponding packing parameters of the surfactants.
1.1.1 Structure of amphiphilic aggregates
The ability to predict the shape of the aggregate structures formed when
amphiphilic molecules are added to water is one of the main topics of this
thesis. One of the most frequent methods of predicting the aggregate struc-
11
ture is to consider the geometric shape of the amphiphilic molecule added
through the use of the surfactant packing parameter, Ns [3]. This is given by
Ns
v
a0lc
where v is the hydrophobic volume, a0, the effective head-group area and lc,
the critical length of the hydrophobic chains. The shape of the aggregates
depends on the value of Ns, as shown in Figure 2. An approximate value of
the three parameters can be estimated relatively easily, which makes this
theory a useful tool. The effective head-group area is somewhat more problematic to predict than the other two parameters as a0 is largely curvaturedependent and is more a property of the self-assembled aggregate structure
than the single molecule [4]. The difficulty in predicting a0 is one of the
drawbacks of this theory. Another limitation is that it is difficult to predict Ns
in mixtures of two or more amphiphilic molecules, as the energy difference
between mixing the molecules and separating them must be taken into account. This has been studied in Papers I and II where, instead of the surfactant parameter, the concept of spontaneous curvature, c0, was considered.
The spontaneous curvature is defined as the curvature adopted by an unconstrained monolayer of amphiphilic molecules. By convention, the definition
of positive curvature is when the polar interface of the aggregate is curved
towards the non-polar medium, and negative curvature is when the polar
interface is curved towards the polar medium. Accordingly, regular micelles
have a high positive curvature, whereas a flat lamellar aggregate has zero
curvature and the inverted aggregate structure have negative curvature.
Parameters such as pH, salt concentration and water concentration can
also change the packing parameter or the spontaneous curvature.
1.1.2 Lamellar phases
All cells are surrounded by a cell membrane, in which the amphiphilic molecules adopt a lamellar arrangement. The most abundant lipids in animal cell
membranes are the glycerophospholipids, often referred to as phospholipids;
these are also the main lipids studied in this work. The cell membrane constitutes an essential border between the cell interior and its surroundings, and
controls some transport into and out of the cell. The rest of the transport is
governed by membrane proteins that are anchored to, or embedded in, the
cell membrane.
12
DMPC: R1=R2=C14:0
DPPC: R1=R2=C16:0
DSPC: R1=R2=C18:0
EPC: Average R1=C16:0, R2=C18:1
MSPC: R1=C16:0 , R2=OH
DSPE: R1=R2=C18:0
DOPE: R1=R2=C18:1
POPC: R1=C16:0, R2=C18:1
phosphatidylcholine
R1 O CH2
R2 O CH2 O
H2C O P O R3
O
R3 CH2CH2N(CH3)3
phosphatidylethanolamine
R3 CH2CH2NH3
Figure 3. The molecular structure of the phospholipids used in the present work.
In a phospholipid (glycerophospholipid) the hydrophobic part, i.e. the hydrocarbon chains, and the hydrophilic part, i.e. the head group, are linked to
a glycerol backbone via ester bonds (Figure 3). The head group, length and
saturation of the hydrocarbon chain may vary, but two major kinds of phospholipids are phosphatidylcholine (PC) and phosphatidylethanolamine (PE).
Both PC and PE are zwitterionic over a broad pH interval. Normally, two
hydrocarbon chains are attached to the glycerol backbone at position R1 and
R2 (see Figure 3). Depending on the length and saturation of the hydrocarbon
chains, they form lamellar structures or inverted phase structure. Lysolipids,
used in the study described in Paper I, have only one hydrocarbon chain and
are therefore micelle forming.
Phospholipid lamellar phases may exist in a number of physical states [2,
5, 6]. At low temperatures the bilayer is in a crystalline phase, denoted Lc or
Lcc, with a dense crystalline structure. The prime indicates that the phase has
tilted hydrocarbon chains. Increasing the temperature results in a greater
rotational motion of the chains and at a specific temperature the bilayer will
adopt a lamellar gel phase, denoted L or Lc. In this phase the lipids are not
as tightly packed as in the crystalline phase, but the membrane still has a
high order. Increasing the temperature further will eventually result in another phase transition where the gel phase passes into the liquid crystalline
phase, L, in which the chains are disordered and have high mobility. In the
L phase the phospholipids exhibit rapid lateral diffusion and the interior of
the membrane is liquid-like. PC lamellar phases often adopt another phase
between the Lc and L phases, the so-called rippled phase, Pc. The main
transition from Lc to L is shown schematically in Figure 4.
13
T
C
L
β’
L
α
Figure 4. Illustration of a lamellar phase transition from Lc to L.
The transition temperature, at which the bilayer transforms from the gel
phase into the liquid crystalline phase, TC, is of special interest, and depends
on the nature of the phospholipid. Especially the length and saturation of the
hydrocarbon chains affect this temperature. Phospholipids with long and/or
saturated hydrocarbon chains have a higher transition temperature than those
with short and/or unsaturated chains. The interaction between the hydrocarbon chains increases the longer the chains are, and thus raises TC. Unsaturated lipids do not pack as densely as saturated ones due to the kink on the
hydrocarbon chains, which will lower the transition temperature.
HO
Figure 5. The molecular structure of cholesterol.
Cholesterol belongs to a lipid class called the sterols, and is one of the
lipid components in animal cell membranes, constituting between 20 and 50
mol% of the total lipid content [7]. Due to its small hydrophilic head and
large hydrophobic part (Figure 5), cholesterol has a specific effect on phospholipid lamellar phases. In the lipid bilayer the hydroxyl group will be situated at the hydrocarbon-water interface, while the stiff ring system and the
hydrocarbon tail are embedded in the hydrophobic interior of the membrane
[8]. Depending on the phase state of the lipid membrane and the amount of
cholesterol that is incorporated, cholesterol can alter the packing order in the
membrane [9]. When situated in a gel phase bilayer, the sterol will break the
lateral packing order and induce more disordered lipid acyl chains. When
situated in a liquid crystalline bilayer cholesterol will reduce the mobility of
the phospholipid molecules leading to a more ordered membrane [10, 11].
14
When the concentration of cholesterol reaches 20-30 mol% the main phase
transition is eliminated and a new phase, the liquid order phase, lo, is formed
[11, 12]. This phase has mixed properties of the liquid crystalline and gel
phases, with both relatively rapid lateral diffusion and a high lipid chain
order.
Cholesterol also has the property of reducing the membrane permeability
and increasing the membrane’s elastic bending rigidity when included in
phospholipid membranes with saturated or unsaturated chains [13-15]. These
effects were observed and are discussed in Papers I to V.
1.1.3 Formation of liposomes and discs
In this section the thermodynamics behind liposome formation will first be
discussed. The second part deals with the behaviour of disc formation, without using the theory of the packing parameter.
A liposome is an aggregate structure composed of a closed lipid bilayer
with an aqueous interior (Figure 6). The similarity with biological membranes and the hollow interior have made these aggregates interesting in
many applications, such as drug delivery vehicles, components in cosmetics
and as model membranes [16-19].
Figure 6. Illustration of a liposome. © Göran Karlsson.
Generally, liposomes are not thermodynamically stable aggregates. In line
with this energy is needed to curve the flat bilayer to form a liposome. However, what appear to be thermodynamically stable liposomes have been ob15
served in specific cases [20-24]. Because liposomes are kinetically trapped
they can be stable for a long time, from days up to years depending on the
molecules present in the bilayer and the environmental conditions.
The formation of liposomes is a result of the closure of a circular planar
bilayer, a lamella. The closure is determined by the energy balance between
the edge interaction and curvature energy. This mechanism has been discussed at various levels of complexity [25-28], and here the bilayer will be
analysed as a thin elastic film, as described by Helfrich [29]. An open lamella has an energetically unfavourable boundary interaction energy, Eedge,
arising from the adverse exposure of the hydrophobic hydrocarbon chains to
water, and can be written as:
E edge
J u 2Sr
where is the effective edge tension and r is the radius of the flat lamella. To
reduce Eedge the lamella can bend, and the edge interaction energy of the disc
decreases as the lamella is curved according to:
E edge
§ § r2
J u 2Sr ¨¨ 1 ¨¨
2
© © 4R
··
¸¸ ¸
¸
¹¹
1/ 2
Two terms, one elastic and one inelastic, can describe the energy required to
curve the bilayer. The energy penalty for the elastic deformation of a bilayer
per unit area, Ecurv, can be calculated using the Helfrich expression [29]
which becomes:
E curv
§ 2k c
k' ·
¨¨ 2 c2 ¸¸S r 2
R ¹
© R
where kc is the elastic bending rigidity and kcc the elastic saddle splay
modulus. The inelastic contribution to the curving energy is only important
when r becomes very small [26] and has been neglected here. Hence, the
resulting expression for the excess energy of a flat or a curved lamella is:
E excess
§ § r2
J u 2Sr ¨¨1 ¨¨ 2
© © 4R
··
¸¸ ¸
¸
¹¹
1/ 2
§ 2k
k'
¨¨ 2c c2
R
©R
· 2
¸Sr
¸
¹
When a liposome is formed, the first term, originating from the edge energy
will, of course vanish, and r=2R, which leads to the resulting expression:
16
E excess,liposome
8Sk c 4Sk c'
This expression indicates that large vesicles are energetically preferred over
small ones. However, small liposomes are entropically favoured.
Figure 7. Illustration of a disc. Dark grey molecules represent micelle-forming amphiphilic molecules. © Göran Karlsson
If the flat lamella does not bend and form liposomes, the aggregate structure may be called a disc (Figure 7). Discs have been both theoretically predicted and experimentally found in various lipid systems [4, 28, 30-33].
Normally, these aggregate structures are very short-lived due to the unfavourable edge interaction energies mentioned above, but stable discs have
been found in systems containing bilayer-forming lipids together with PEGlipids [34, 35]. The PEG-lipids are preferentially accumulated at the rim of
the disc protecting the hydrophobic acyl chains from the surroundings and
the discs from aggregation or closure. Hristova and Needham predicted that,
at bilayer saturation, the PEG-lipid would form cylindrical or globular micelles when combined with cylindrically shaped lipids [36]. This has been
shown to be true in some cases. To form cylindrical micelles the bilayerforming lipid, which prefers zero curvature, must be packed into a highly
curved monolayer. Hence, the formation of cylindrical micelles is accompanied by a bending energy penalty. However, the formation of discs requires
partial component segregation, with most of the cone-shaped surfactants
situated at the rim and the cylindrically shaped lipids in the flat part of the
aggregate. Aggregation into a disc will then be associated with a free energy
penalty in the form of reduced mixing entropy. This means that discs should
only form when this entropy loss is small compared with the energetic cost
of forcing all components in the lipid mixture into a highly positively curved
monolayer, as in the cylindrical micelle. Disc formation in various surfactant/lipid systems, focusing on how environmental conditions and lipid characteristics affect the energy balance, was studied and is reported in Papers I
and II.
17
1.2 The cell membrane and models thereof
The cell membrane has three main tasks. First, it separates the cell organelles
from their surroundings creating a barrier for molecular diffusion. The second task of the cell membrane is to serve as a solvent for hydrophobic membrane proteins. Thirdly, the membrane participates in many of the metabolic
processes taking place in the living cell.
The composition of cell membranes varies depending on where the cell is
in the body, but the main features of mammalian cell membranes are the
same. The main building blocks of the cell membrane are the membrane
lipids, which create a suitable environment for the many proteins present.
The total amount of protein varies between different kinds of cells, but is in
the range of 25 to 75% by weight. Proteins will, of course, affect the membrane characteristics but will not be discussed here.
There are four classes of lipids in cell membranes: glycerophospholipids,
sphingolipids, glycolipids and sterols. Glycerophospholipids or phospholipids have been described in Section 1.1.2 and are the most common membrane lipids. Apart from PC and PE, which are the most common phospholipids, the negatively charged phosphatidylinositol (PI) and phoshatidylserine (PS) are also present in mammalian membranes. There are also sphingolipids, which, instead of the glycerol backbone, have a long-chain amino
alcohol connected to the fatty acids. Glycolipids are phospholipids and
sphingolipids with a sugar added to the head-group. Normally, two fatty
acids are linked to phospholipids and sphingolipids; they are generally 16 or
18 carbons long and one of the chains is usually unsaturated. This length and
saturation would normally lead to a membrane in the liquid crystalline state,
but the situation is affected by the fact that cholesterol is present in the membrane. The cholesterol concentration normally varies between 20 and 50
mol% of the total amount of lipids and influences both the fluidity and permeability of the membrane, see Section 1.1.2. The various transport processes that can occur across the membrane will be discussed in the next section.
1.2.1 Transport across cell membranes
Transport across the cell membranes can take place via passive or active
transport. Active transport consumes energy, while passive transport does
not require any energy input but is driven by a concentration gradient. Passive transport can be divided into facilitated and simple diffusion; proteins or
carrier molecules being involved in facilitated diffusion. Simple diffusion, or
diffusion, is normally responsible for the transport of relatively small molecules like oxygen and carbon dioxide. However, the uptake of many drug
substances also occurs through diffusion.
For the sake of simplicity, a non-charged molecule will be assumed during the following arguments. Every system strives to reach equilibrium
18
where all chemical species have the same chemical potential. A system with
different concentrations of a solute on the two sides of a barrier will equilibrate by flow of the solute across the barrier according to Fick’s first law:
J
D
wc
wx
The flux (J) of molecules across the membrane is proportional to the diffusion coefficient (D) and the concentration gradient over the membrane
(c/x). Integration of this expression yields:
J
D
G
'c mem
where is the thickness of the membrane and cmem is the difference in concentration within the membrane. If the flux of molecules over a membrane is
calculated, the entrance of the molecules into and exit out of the membrane
must be considered. This is because the molecules do not dissolve equally
well in the organic environment in the membrane compartment and in the
aqueous surrounding medium. The solubility of the molecule in the membrane versus its solubility in water is determined by the difference in standard state chemical potential in the two environments (μ0j (aq) - μ0j(mem))
and is defined by the partition coefficient (K):
K
^ª P
Cmem
Caq
e¬
0
0
º
j ( aq ) P j ( mem ) ¼ / RT
`
Now the flux can be written:
DuK
J
G
'c aq
where caq is the difference in concentration over the membrane and a permeability coefficient, P [ms-1], can be defined as:
P
Du K
G
19
The diffusion coefficient in the membrane is mainly governed by the size of
the solute. In the present studies the sizes of the various drugs can be approximated to be equal, and hence D is roughly the same. Because the model
system is the same, , is constant. Measurements of the partition coefficient
will therefore reflect the permeability.
1.2.2 Model membranes
Studying the transport across natural cell membranes can be rather complicated due to the many processes simultaneously taking place in the membrane. The complexity of natural membranes has created a need for model
membranes. These models can be of a more or less sophisticated nature. One
of the more crude methods used to determine the partition of drugs into a
cell membrane is the octanol/water method [37-39]. Here, octanol is used to
model the hydrophobic part of the membrane. The apparent distribution coefficient of a molecule into the octanol phase at pH 7.4 is defined as Doct.
Due to its wide use, and the large amount of data available, the drug partition
data obtained in the present work were compared with log Doct (Paper IV).
Octanol can mimic the hydrophobic part of a biological membrane reasonably well. However, when using lipids in various aggregate structures to
measure drug partitioning, the interaction with the polar head groups will
also be taken into account. One method of measuring the partition coefficient into lipid aggregates is the use of an immobilized artificial membrane
(IAM) [40, 41]. The model consists of a monolayer of phospholipids covalently bound to a carrier surface. The binding of the lipids onto the surface
results in restricted acyl chain motion and hence a model that does not exactly mimic fluid bilayers. Examples of discrete aggregate structures that
have a fluid character used in drug partition studies are detergent micelles
[42, 43], cubic phases [44] and liposomes [45, 46]. The structural similarities
of liposomes with biological membranes have led to them being widely used
as model membranes [17, 19]. However, as further discussed in Section 2.3,
there are some drawbacks associated with the use of liposomes as a model
membrane which could be solved by using discs.
1.3 Aims of the present investigation
This thesis is focused on fundamental studies and some applications of bilayer discs formed in phospholipid systems.
Papers I and II describe the investigation of various lipid mixtures and
environmental conditions to explore the conditions under which discoidal
structures form. Paper I concerns the study of PEG-lipid/lipid systems and
Paper II surfactant/lipid systems.
The intentions of the studies described in Papers III and IV were to develop and optimize discs that could be used as model membranes and for the
20
reconstitution of proteins. The potential of the discs was evaluated by drug
partition studies. In the study described in Paper IV a membrane protein was
also incorporated into the discs.
Paper V reports on the influence of preparation path on disc formation.
Detailed descriptions of the aims can be found in the respective section.
21
2 Experimental techniques
2.1 Preparation of liposomes and discs
Four different preparation methods were used in the present work. All the
preparation protocols started with the same procedure. The appropriate lipids
were dissolved in chloroform, which was removed under a gentle stream of
N2 gas, followed by further evaporation in vacuum overnight to remove residues of chloroform. Thereafter, the preparation methods differed. Discs or
liposomes were produced by the following four methods.
1) Simple hydration, where buffer was added to the vial and the mixture
was hydrated at temperatures between 50 and 80ºC, depending on
lipid composition, for approximately 30 minutes with intermittent vortex mixing.
2) Freeze-thawing, where the sample went through 5-8 freeze-thawing
cycles: freezing in liquid nitrogen, heating to above 55ºC and thereafter vortexing.
3) Sonication, where buffer was added to the sample and then sonicated
for 10, 30 or 45 minutes depending on lipid composition.
4) Detergent depletion, where octyl glucoside (OG) micelles were added
to the sample, which was then allowed to equilibrate. The equilibrated
sample was gel filtrated followed by detergent dialysis.
The influence of preparation path on the formation of discs and threadlike
micelles is reported in Paper IV.
2.2 Cryo-TEM
Cryo-transmission electron microscopy (cryo-TEM) is a useful technique
that provides direct visualization of aggregate structures of amphiphilic
molecules. Cryo-TEM is superior to many other techniques when dealing
with systems including various aggregate structures. An important advantage
of cryo-TEM is that it does not require staining, drying or chemical fixation
of the sample. A small droplet of the sample is frozen quickly so that the
surrounding water is vitrified and arranged in an amorphous configuration.
Rapid cooling is necessary for the amorphous structure to form, but also
makes it unlikely that the larger amphiphilic aggregates will reorganize. The
aggregate structures can be visualized due to the difference in electron density between the water and the lipid aggregates.
22
Objects between 4 nm and 500 nm can be viewed in cryo-TEM; the lower
limit is due to optical limitations and the higher one to the film thickness. If
the sample is thicker than 500 nm the sample will scatter the electrons too
much and the image will appear black. Figure 8 illustrates how the 2D image
of liposomes and discs is generated from a 3D sample [47].
Figure 8. Illustration showing how the vitrified sample will appear in a 2D image.
The figure at the bottom is a cryo-TEM image showing liposomes and discs from
various angles. © Göran Karlsson
To avoid evaporation and to control the temperature, the sample is prepared in a climate chamber. The preparation set-up can be seen in Figure 9.
A small copper grid covered with a porous polymer film is used as a support
for the sample. A small droplet of the sample is placed on the copper grid
and excess solution is removed with a filter paper. The grid is rapidly
plunged into liquid ethane for vitrification, and then transferred into the mi23
croscope. The sample is cooled by liquid nitrogen during preparation, transfer and viewing. For vitrification to take place satisfactorily a thin film is
needed, hence the viscosity of the solution must not be too high. The technique is suitable for samples of dilute aqueous solutions >95 wt% water.
Figure 9. Illustration showing the preparation unit used for cryo-TEM. To the right
in the figure the grid with polymer film is displayed. © Göran Karlsson
2.2.1 Artefacts in cryo-TEM
Although the cryo-TEM technique is useful for studying amphiphilic aggregate structures, one must be aware of the possible artefacts. One such artefact is size sorting. The thin sample solution film spanning the polymer holes
is concave. Close to the centre of the sample, the film thickness is about 10
nm, while at the edges it is about 500 nm. If the aggregate size of the sample
varies within this range, size sorting may occur, such that the larger aggregate structures are only present at the edges. Satisfactory agreement has,
however, been demonstrated between aggregate sizes obtained by cryo-TEM
and dynamic light scattering for samples with relatively small aggregates
[48].
Another problem is that liposomes in the liquid crystalline state sometimes become invaginated. This phenomenon is caused by osmotic stress
[49]. When the solvent is evaporated the salt concentration increases in the
external buffer and a concentration gradient is formed over the liposome
membrane. This gradient can be equalized by water transport from the liposomes to the surroundings, causing the liposome membrane to invaginate.
24
2.3 Light scattering
Light scattering is the phenomenon that makes dust visible when the sun
shines into a room. Light scattering is also a non-invasive technique used to
measure the size of small particles.
Changes in the average aggregate size, for example when liposomes are
solubilized into micelles, can be detected with light scattering. This can be
done by monitoring the change in apparent absorbance, also called the turbidity. A high turbidity indicates large particles in the solution whereas a low
turbidity indicates small particles. Turbidity measurements were used in the
study presented in Paper II.
2.3.1 Dynamic light scattering
Dynamic light scattering (DLS) measures the intensity fluctuation of a sample over time. The variation in intensity contains information about the random motion, i.e. the diffusion, of the aggregates. Since small and large particles will diffuse differently, DLS can be used to measure the size and size
distribution of aggregates in dilute solutions. Using the Stokes-Einstein relation, the diffusion coefficient can be related to the hydrodynamic radius, Rh:
D
k BT
6SK Rh
were kB is Boltzmann’s constant, T is the absolute temperature and is the
solvent viscosity. Rh from the Stokes-Einstein relation gives the radius assuming the particles are spherical. The samples investigated in this work
contained mainly disc-shaped aggregate structures, and Rh can be converted
into the disc radius Rdisc, via the disc model suggested by Mazer et al. [50]:
Rh
3Rdisc
2
1 ª
­
½
2 1/ 2
2 1/ 2 º
® ª¬1 D º¼ ln «D ª¬1 D º¼ » D ¾
¼
D ¬
¯
¿
1
where =Ldisc/(2Rdisc) and Ldisc is the thickness of the disc. DLS was used in
the studies presented in Papers III and V to measure the size of the aggregate
structures.
DLS is a useful technique for measuring the size and size distribution of a
sample, however, without information on the aggregate structure in the sample the interpretation of the data is difficult and sometimes impossible. As
shown in several studies e.g. [35], TEM together with DLS is a powerful
combination where both experimental techniques can be exploited to their
utmost.
25
2.4 Drug partition chromatography
Drug partition chromatography (DPC), also referred to as immobilized liposome chromatography, can be used to investigate the interactions of drugs
with an immobilized membrane [51, 52]. It is a chromatographic method in
which dry gel beads are rehydrated by the lipid suspension. During the
swelling of the beads the lipid aggregates are sucked into and sterically
trapped in cavities in the gel beads. A freeze-thawing cycle is often performed to enhance the amount of trapped lipid aggregates. A freeze-thawing
cycle was used when the lipid aggregates were multilamellar liposomes (Paper III). After immobilization the beads are packed into a glass column. The
drugs are applied to the column and their elution volume is measured spectroscopically. The drug partition is evaluated from the retention volume, and
expressed as a normalized capacity factor (Ks):
Ks
VE V0
A
where VE is the elution volume of the drug, V0 is the elution volume of an
analyte, Cr2O72-, assumed to not interact with the lipids in the column, and A
is the amount of immobilized lipid. There is also a small interaction between
the drugs and the gel beads which is generally negligible [53] and has been
neglected in this work. DPC was used in the studies described in Papers III
and IV.
To compare the results of DPC with partition data obtained by other
methods a unitless partition coefficient (K) can be calculated by dividing the
determined value of Ks by the apparent lipid molar volume [54]. This was
done in the study reported in Paper IV.
2.5 Isothermal titration calorimetry
Isothermal titration calorimetry (ITC) is a technique that can be used to
measure the heat associated with the binding of a small solute, for example a
drug or surfactant, to lipid aggregates, such as liposomes or discs. The heat
produced or absorbed is monitored and compared with a reference cell. The
results are plotted against concentration allowing partition coefficients to be
calculated. Paper III describes how a modified ITC assay was used, the solvent-null method [55], to calculate the partition coefficient for the partitioning of drugs into liposomes or discs. In the solvent-null method a syringe is
filled with the lipid aggregates together with a drug and is allowed to equilibrate. Aliquots are then titrated into the sample cell containing various
amounts of the drug. When the amount of free drug in the syringe matches
the drug concentration in the sample cell no heat change is observed. Correc26
tions were made for the heat of dilution of the lipid and the heat change
when adding the free drug concentration to the sample cell containing the
various amounts of drug. The partition coefficient, Kp, was calculated according to:
Kp
>drug @bound
>lipid @>drug @ free
where [drug]bound is the concentration of drug bound to the lipid phase,
[drug]free is the free drug concentration and [lipid] the lipid concentration in
the syringe.
2.6 Fluorescence measurements
Fluorescence is a process in which a molecule discards its excitation energy
as a photon. This emission of light can be used to investigate processes such
as liposome leakage and phase changes. A fluorescence quenching method
was used to calculate the entrapped volume in order to estimate the amount
of liposomes in a preparation (Paper III). In the study reported in Paper III
the sample was prepared in 100 mM 5(6)-carboxyfluorescein (CF) buffer. At
this concentration, the fluorescence of CF is self-quenched by more than
95%. Non-encapsulated CF was removed by gel filtration. When the liposomes start to leak or collapse, CF is diluted by the external buffer and is no
longer self-quenched but fluoresces. The intensity of the fluorescence was
measured and the liposomes were thereafter lysed by the addition of Triton
X-100, and the fluorescence intensity measured again. The intensity of the
fluorescence from the sample was then compared with the intensity from the
pure liposome preparation and the amount of liposomes in the sample was
estimated.
27
3 Results and discussion
3.1 Aggregate structure in surfactant/lipid systems
Previous results from our lab have shown that discs may form as an intermediate aggregate structure between liposomes and globular micelles in PEGlipid/lipid systems. The disc structure was found in systems with lipids in the
gel state and lipids in the liquid crystalline state upon supplementation with
cholesterol [34, 35]. Threadlike micelles, on the other hand, were formed in
systems containing EPC. Papers I and II present systematic studies of the
factors promoting disc formation in phosphatidylcholine-based systems.
Schematic illustrations of a disc and a threadlike micelle are shown in
Figure 10. In the case of the disc it is quite easy to understand that the coneshaped surfactants prefer to be situated at the highly curved rim. In the case
of the cylindrical micelle the entire aggregate structure has a high spontaneous curvature and therefore the surfactant curvature preferences are met
anywhere in this aggregate.
Figure 10. A schematic representation of a cylindrical micelle (left) and a discoidal
micelle (right). Dark grey molecules represent PEG-lipids.
As already mentioned (Section 1.1.3), the discoidal aggregate structure requires partial component segregation and hence the formation of discs is
associated with an entropy loss. Discs may only form when the entropy loss
is small compared with the energetic cost of forcing all the components in
the lipid mixture into a highly curved monolayer. It is therefore to be expected that sample components and/or environmental conditions that reduce
the surfactant/lipid miscibility would increase the tendency for disc formation. Furthermore, properties of the lipid mixture, such as monolayer bending rigidity and spontaneous curvature, are expected to have a decisive influ28
ence on whether threadlike or discoidal mixed micelles form in the system.
However, the spontaneous curvature and bending rigidity are closely related
and, therefore, these two effects can not easily be separated and will thus be
discussed together.
In the study described in Paper I the micelle-forming component was represented by a PEG-lipid, while in the next study, Paper II, the PEG-lipid was
replaced by more conventional surfactants. One of the main aims of the
study presented in Paper I was to explore how various sample properties,
such as bending rigidity and spontaneous curvature, affected the structure of
the mixed PEG-lipid/lipid systems. Investigations were then carried out (Paper II) to establish whether the findings reported in Paper I could be more
generally applied to surfactant/lipid systems.
3.1.1 Aggregate structures in PEG-lipid/lipid systems
In the first study, (Paper I) all samples were simply hydrated and contained a
fixed amount of DSPE-PEG2000 (25 mol% of the total amount of lipids).
Figure 11. Cryo-TEM images of dispersions of: a) EPC:DSPE-PEG2000 (75:25
mol%), b) EPC:cholesterol:DSPE-PEG2000 (65:10:25 mol%), c)
EPC:cholesterol:DSPE-PEG2000 (55:20:25 mol%), d) EPC:cholesterol:DSPEPEG2000 (45:30:25 mol%) and e) EPC:cholesterol:DSPE-PEG2000 (35:40:25 mol%).
The arrow in a) indicates a liposome. The arrowhead in a) denotes a bilayer flake
with threadlike extensions. The arrow and arrowhead in e) indicate disks observed
face-on and edge-on, respectively. Scale bars indicate 100 nm.
29
Figure 11a shows the aggregate structures found using cryo-TEM in a sample composed of DSPE-PEG2000:EPC (25:75 mol%). Liposomes, bilayer
flakes with threadlike extensions, and cylindrical to globular micelles were
all present in the sample. As more and more cholesterol was used in the mixture the cylindrical micelles disappeared and an increasing amount of discshaped aggregates was formed. When 40 mol% cholesterol was used mainly
circular discs of various sizes were observed. The change in structure with
increasing amount of cholesterol can be seen in Figure 11. The shape of cholesterol, with its relatively small head group and bulky hydrophobic part,
means that it prefers a flat monolayer rather than a monolayer with high
positive curvature, as in the threadlike micelles. In line with this, previous
studies have shown that increasing concentrations of cholesterol progressively increase the bending rigidity of phosphocholine membranes [56] and,
as expected, decrease the phospholipid membrane curvature [57]. Forcing
cholesterol into a highly curved monolayer is thus more energetically unfavourable than separating the PEG-lipid from EPC and cholesterol and forming discs.
HO
Figure 12. The molecular structure of lanosterol.
The structure of lanosterol, an evolutionary precursor to cholesterol, resembles that of cholesterol but has three additional methyl groups on the
hydrocarbon ring system (compare Figure 12 with Figure 5). Similar to cholesterol, lanosterol increases the lipid chain order, but not to the same extent
[58], and it does not promote and stabilize the liquid ordered state [58-62].
30
Figure 13. Cryo-TEM images of samples containing: a) EPC:lanosterol:DSPEPEG2000 (64:11:25 mol%) and b) EPC:lanosterol:DSPE-PEG2000 (35:40:25 mol%).
Scale bars indicate 100 nm.
In Figure 13 samples with DSPE-PEG2000/EPC supplemented with 11 and 40
mol% lanosterol are shown. The images show that lanosterol induced disc
formation to at least the same extent as cholesterol. Inclusion of lanosterol
does increase the bending rigidity, but not as much as the inclusion of cholesterol. The effect of lanosterol on the spontaneous curvature has not been
elucidated in detail but might be even more pronounced than that of cholesterol due to the methyl groups on the ring system of lanosterol.
Figure 14. Cryo-TEM images of dispersions of: a) EPC:DSPE:DSPE-PEG2000
(46:29:25 mol%), b) EPC:DSPE:DSPE-PEG2000 (35:40:25mol%), c) DSPE:DSPEPEG2000 (75:25 mol%) and d) EPC:DSPE:DSPE-PEG2000:MSPC (40.5:26.8:22.7:10
mol%). Scale bars indicate 100 nm.
Inclusion of DSPE in DSPE-PEG2000/EPC mixtures had a similar effect
on the aggregate structure, as found upon inclusion of the sterols, although
not as pronounced (Figure 14a-b). When comparing DSPE-PEG2000/EPC
samples supplemented with either 40 mol% cholesterol or 40 mol% DSPE
(compare Figure 14b with Figure 11e) the same amount of DSPE led to the
formation of more irregular bilayers but importantly no threadlike micelles.
When all the EPC was replaced by DSPE only discs of varying sizes were
31
formed (Figure 14c). At the temperature used, 25ºC, DSPE forms a lamellar
gel phase and this probably contributes to the almost circular shape of the
discs in the pure DSPE-PEG2000/DSPE sample. Disc formation below TC, as
in DSPE-PEG2000/DSPE mixtures, is partly the result of the high bending
rigidity, which is known to be about ten times higher in the gel than in the
liquid crystalline state [63]. Furthermore, the PEG-lipid/lipid miscibility is
probably reduced in the gel phase, and the energy loss due to component
segregation is less at lower temperatures. The results obtained by imaging
DMPC-PEG2000/DMPC above and below TC support this reasoning (Figure
15). Due to the smaller head group of DSPE than EPC, inclusion of DSPE in
the PEG/EPC mixture probably also reduces the spontaneous curvature of
the mixture. Inclusion of 10 mol% MSPC in DSPE-PEG2000:EPC:DSPE
(25:45:30) induced the formation of threadlike micelles (Figure 14d). This
behaviour can be partly explained by the high positive curvature of MSPC,
which counteracts the curvature-reducing effect of DSPE. Additionally, the
inclusion of a surfactant in a bilayer will often also reduce the bending rigidity [64, 65], which further helps promote the formation of threadlike micelles.
Figure 15. Cryo-TEM images of preparations of: a) DMPC:DMPE-PEG2000 (75:25
mol%) at 30qC and b) DMPC:DMPE-PEG2000 (75:25 mol%) at 5qC. Scale bars
indicate 100 nm.
In conclusion, discoidal structures are preferred over cylindrical micelles
when the lipid mixture contains components that reduce the spontaneous
curvature and increase the monolayer bending rigidity. Examples of such
components are cholesterol, lanosterol and DSPE. Discoidal structures are
furthermore preferred at temperatures below the transition temperature of the
lipid mixture. In this case, disc formation is probably promoted by a combination of high bending rigidity and reduced PEG-lipid/lipid miscibility.
32
3.1.2 Aggregate structures in C12E8, CTAB and SDS/lipid
systems
In Paper I it was reported that DSPE-PEG2000 induced the formation of discs
when mixed with DPPC or EPC and cholesterol, whereas threadlike micelles
were formed when the PEG-lipid was mixed with EPC. Due to the large
polymer attached to the head group of DSPE, DSPE-PEG2000 is a somewhat
special surfactant. In particular, the large polymeric head groups effectively
prevent fusion or self-closure of the aggregates. The effect on aggregate
structure of replacing the PEG-lipid by other more conventional surfactants
was explored (Paper II). In this studie EPC was replaced by the synthetic
lipid POPC. However, aggregate structures found in POPC/hexadecyltrimethylammonium bromide (CTAB) systems were comparable to the aggregate structures found in EPC/CTAB systems (results not shown).
The turbidity was measured in the samples with increasing amounts of
surfactant and just before the turbidity reached its minimum, i.e. where
globular mixed micelles are formed, the aggregate structure was investigated
by cryo-TEM.
Figure 16. Cryo-TEM images of samples containing DPPC and: a) 39 mol% C12E8,
b) 49 mol% CTAB and c) 53 mol% SDS. Scale bars indicate 100 nm.
As can be seen in Figure 16, all three surfactants, octaethylene glycol
monododecyl ether (C12E8), CTAB and sodium dodecyl sulphate (SDS),
were found to induce the formation of discoidal micelles when mixed with
DPPC, as was the case with DSPE-PEG2000. The investigations were carried
out at 25qC, i.e. well below TC for DPPC, and the same explanation as for the
disc formation in DSPE-PEG2000/DPPC mixtures can be applied here. That
is, both the low bending rigidity and the reduced miscibility between the
lipid and surfactant are factors that promote disc formation in these mixtures.
33
Figure 17. Cryo-TEM images of samples containing POPC and: a) 40 mol% C12E8,
b) 90 mol% CTAB and c) 80 mol% SDS. Scale bars indicate 100 nm.
When DPPC was replace by POPC, which has a transition temperature
below room temperature (-3qC [6]), all three surfactants eventually induced
the formation of threadlike micelles (Figure 17). This was not surprising
since the comparatively low bending rigidity of POPC monolayers is expected to reduce the energetic cost of forming highly curved structures.
There were, however, some important differences between the three investigated surfactant/POPC systems.
Figure 18. Cryo-TEM images of samples containing POPC and: a) 70 mol% SDS, b)
80 mol% CTAB and c) 20 mol% C12E8. The arrow in c) indicates an ice crystal.
Scale bars indicate 100 nm.
In contrast to C12E8, the two charged surfactants induced the formation of
bilayer discs at relatively low POPC/surfactant ratios (Figure 18a-b). This
should be compared with C12E8, where a low amount of surfactant in combination with POPC resulted in mesh-like structures (Figure 18c). The observed difference could have several explanations. First, C12E8 may mix
better with POPC than the other surfactants, which is not very likely, as
POPC is in the liquid crystalline state and should mix equally well with all
three surfactants. Second, C12E8 may increase the spontaneous curvature of
the mixture more than the other two. Third, C12E8 could be more effective in
lowering the bending rigidity of the mixture than the other two surfactants.
By calculating the bending rigidity for mixtures of POPC and C12E8 or SDS
it was shown that the bending rigidity for C12E8/POPC mixtures was lower
34
than for the SDS/POPC mixture. This suggests that the bending rigidity of
the lipid mixture has a decisive influence on the structure of the mixed micelles formed.
When DSPE-PEG2000 was used as the micelle-forming surfactant, the inclusion of cholesterol changed the structure of the mixed micelles formed in
the DSPE-PEG2000/EPC system (see Section 3.1.1). The mixed micelles
formed in the SDS/POPC and CTAB/POPC systems were similarly affected
upon the inclusion of cholesterol (Figure 19a,b). The threadlike micelles,
appearing before the globular micelles are formed in the CTAB/POPC and
SDS/POPC systems, are now replaced by discs. Obviously, the curvaturereducing effect of cholesterol and the increased bending rigidity caused by
cholesterol inclusion will also result in disc formation in these systems. For
C12E8 no mixed micellar structures, except globular micelles, were observed
at any of the surfactant/POPC/cholesterol ratios (Figure 19c). This observation could be explained by very poor mixing of C12E8 into the
POPC/cholesterol environment. It is, however, not easy to see why C12E8
would differ from the other surfactants in this respect. Another explanation
could be that the chemical potential of liposomes and globular micelles is
similar enough to allow the coexistence of the two types of aggregates. Liposomes and globular micelles in coexistence as the only aggregate structures
present has also been observed in sodium taurocholate/egg yolk lecithin/cholesterol systems [66].
Figure 19. Cryo-TEM images of samples containing POPC/cholesterol and: a) 70
mol% C12E8, b) 67 mol% CTAB and c) 70 mol% C12E8. The arrow in c) indicates an
ice crystal. Scale bars indicate 100 nm.
To elucidate whether the sample structure was stable over time, a sample
containing a homogeneous population of mixed micelles was selected from
each system and stored at 4qC for one month and then reinvestigated with
cryo-TEM. DPPC, POPC and POPC/cholesterol samples containing SDS
remained stable over time. As judged from cryo-TEM images, changes in
aggregate size and structure could not be observed after 30 days of storage at
4qC. The stable structure and absence of aggregation may in part be explained by the ability of the surfactant to electrostatically stabilize the
lipid/surfactant aggregates. However, for samples containing C12E8 and
35
CTAB, changes were observed after 30 days of storage. Generally, in the
samples with C12E8 only very small changes were observed. The relatively
large head group of C12E8 can perhaps offer some steric stabilization, but not
enough to prevent the aggregates from fusing and growing larger during
storage. In Figure 20a the observed changes in the C12E8/DPPC (39 mol%
surfactant) sample are shown. The main aggregate structure is still discs in
the sample, but they have grown larger during the month of storage (compare with Figure 16a). The changes in aggregate structure upon storage are
most pronounced for CTAB/lipid systems. Figure 20b shows
CTAB/POPC/cholesterol (67 mol% surfactant) samples (compare with Figure 19b). Why CTAB, as a charged surfactant, can not stabilize the aggregates to the same extent as SDS, may be explained by the more shielded
charge on CTAB than on SDS.
Figure 20. Cryo-TEM images of stored samples containing: a) C12E8/DPPC (39
mol% surfactant) and b) CTAB/POPC/cholesterol (67 mol% surfactant). Scale bars
indicate 100 nm.
In Table 1 the aggregate structures formed in the surfactant/lipid systems
are summarized. The main conclusions were that the results in Paper I concerning the formation of discs or threadlike micelles in systems with lipids
and PEG-lipids can be ascribed more generally to the surfactants studied
here, namely C12E8, CTAB and SDS. The potential for applications of discs
formed in these systems is not as promising as for the PEG-stabilized discs.
This is due to the rather high amount of surfactant needed to create homogeneous discs and the fact that the discs formed are comparatively small. Additionally, the discs formed in samples containing C12E8 or CTAB are not stable over time. The poor stability upon dilution is due to the high monomer
aqueous solubility of, in particular, SDS and CTAB.
36
Table 1. Summary of the aggregate structures formed in the surfactant /lipid systems
studied.
DPPC
POPC
C12E8
Discs
Threadlike micelles
CTAB
Discs
SDS
Discs
Discs threadlike
micelles
Discs threadlike
micelles
POPC/cholesterol
Liposomes and
globular micelles
Discs
Discs
3.2 Discs as model membranes
As stated in Section 1.2.2, liposomes have been extensively used as model
membranes due to their structural similarity with cell membranes. However,
there are some drawbacks when using liposomes for this application. Firstly,
one problem associated with the use of liposomes as model membranes for
interaction studies is that the inner lipid layer of the liposome is not initially
accessible for interaction. If sufficient time is not allowed for equilibration
over the membranes, some proportion of lipid will be inaccessible for the
analyte. In addition, as a rule, liposome preparations contain a population of
bi- or multilamellar structures. This fact further complicates the evaluation
of the partition data. Secondly, when reconstituting membrane proteins with
one active site into liposomes, an unknown fraction of the active sites will be
oriented towards the centre of the liposome, and will therefore not be available for interactions from the outside. The unknown amount of initially
available active sites might complicate the interpretation of the interaction
data. Thirdly, the liposome structure is not stable over time.
In the present work the bilayer disc was developed for use as a model
membrane (Paper III), and further improvements were made to this model
membrane (Paper IV). Protein reconstitution into these aggregate structures
was also performed (Paper IV). The usefulness of the disc as a model membrane was investigated with drug partition studies, described in Paper III and
Paper IV.
3.2.1 Initial disc development
A mixture of DSPC, 40 mol% cholesterol and an increasing amount of the
micelle-forming component 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-5000] (DSPE-PEG5000) was studied
(Paper III). As has been discussed above, when added at concentrations ex-
37
ceeding the bilayer saturation concentration, the PEG-lipid will induce the
formation of circular, bilayer discs.
Figure 21. Cryo-TEM images of DSPC/cholesterol/PEG-DSPE5000 mixtures with
varying amount of PEG-DSPE5000: a) 0 mol%, b) 5 mol%, c) 12 mol%, d) 15 mol%
and e) 30 mol% PEG-DSPE5000. Scale bars indicate 100 nm.
A series of samples with DSPC/cholesterol and increasing amounts of
DSPE-PEG5000 is shown in Figure 21a-e. Due to the fact that the samples
were not extruded, multilamellar liposomes were formed in the absence of
PEG-lipid (Figure 21a). When only 5 mol% PEG-lipid is included, the liposomes appear unilamellar and a small number of bilayer discs coexist with
the unilamellar liposomes. As the PEG-lipid concentration is increased
above 5 mol%, the discs become more frequent and the number of liposomes
decreases. In Figure 22 the decrease in size, measured from cryo-TEM images, with higher amounts of PEG-lipid is obvious. When evaluating the
actual diameter of the discoidal structure it is important to realize that the
PEG layer can not be visualized by means of cryo-TEM. Due to the poor
contrast between the PEG and the vitrified matrix only the lipid body is visible to the electron beam.
38
80
70
% (number)
60
50
40
30
20
10
0
1-10
11-20
21-30
31-40
41-50
51-60
61-70
71-80
81-90
91-100
Size [nm]
Figure 22. Size distribution (diameter) of disks determined from cryo-TEM images
in DSPC/cholesterol/PEG-DSPE5000 samples containing 12 mol% (black), 15 mol%
(white) and 30 mol% (grey) PEG-DSPE5000. More than 680 structures counted.
3.2.2 Refinement and optimization of lipid composition
The discs used in the above study (Paper III) showed some electrostatic interactions with charged drugs (discussed in Section 3.2.3). The electrostatic
interaction seen for charged drugs with discs composed of DSPEPEG5000/DSPC/cholesterol arises from the negative charge of DSPE-PEG5000.
To overcome this problem DSPE-PEG5000 was replaced by an uncharged
PEG-lipid (Paper IV). The lipid composition was also changed to more
closely mimic that of natural membranes.
Figure 23. Cryo-TEM images of samples containing: a) DSPC:cholesterol:Ceramide-PEG5000 (48:40:12) prepared by hydration and b) DSPC:cholesterol:Ceramide-PEG5000 (45:40:15) prepared by sonication. Scale bars indicate 100 nm.
When the uncharged N-palmitoyl-sphingosine-1-[succinyl (methoxy
(polyethylene glycol) 5000] (Ceramide-PEG5000) was mixed with DSPC and
39
cholesterol a sample with heterogeneous size and aggregate distribution was
formed (Figure 23a). The variety of aggregate structures and sizes indicates
that the Ceramide-PEG5000 distribution probably differs between the various
aggregates. Ceramide-PEG5000 does not resemble DSPC as much as DSPEPEG5000 (compare the structures in Figure 24 with Figure 3), and this could
explain the poorer mixing. To overcome this problem the samples were prepared by sonication; the result is shown in Figure 23b. The sonicated sample
displayed a fairly homogeneous population of discs with satisfactory size,
but still contained a fraction of liposomes. The latter were removed by centrifugation.
a
O
CH3O(CH2CH2O)113
O
OP O
O
O
C N
H
O
O
b
O
CH3O(CH2CH2O)113 C
H O
O
H NH
C
O
O
HO H
Figure 24. The molecular structure of a) PEG-DSPE5000 and b) Ceramide-PEG5000.
In order to prepare discs that mimicked biological membranes a lipid
composition that closely resembled that of the porcine brush border membrane (BBM) [67] together with Ceramide-PEG5000 was used. After sonication most of the material was found in disc-shaped aggregates and therefore
no centrifugation step was needed to purify these samples.
3.2.3 Drug partition studies
In the study presented in Paper III, three model membranes were investigated: multilamellar liposomes, unilamellar liposomes and discs, all composed of DSPC, 40 mol% cholesterol and 0, 5 or 12 mol% DSPE-PEG5000.
ITC was used to investigate the partitioning of three drugs into unilamellar
liposomes and discs. Multilamellar liposomes could not be investigated with
this technique since the liposomes sedimented in the injection syringe. In
Table 2 the values of log Kp are given and it can be seen that the partition
behaviour is similar in unilamellar liposomes and discs. This indicates that
there is enough time for the drug to equilibrate between the lipid and water
phases and the initially inaccessible inner lipid layer of the liposomes does
not seem to pose a problem. Furthermore, this finding indicates that varying
the amount of PEG-lipid in the two different aggregates has little effect on
the drug partitioning. However, the ITC method is limited to drugs with a
40
comparatively high aqueous solubility and is therefore not suitable for many
drugs. ITC is also time consuming and not suitable for studying a large
number of drugs.
Table 2. Drug partitioning into unilamellar liposomes and disks analysed by isothermal titration calorimetry. Average log Kp values ± standard deviation for two
positive drugs and a negative, n=3.
Unilamellar
liposomes
Drugs and solutes
Alprenolol
Charge at pH 7.4
+
Discs
Log K
1.22r0.06
1.21r0.04
Lidocaine
+
1.18r0.12
1.10r0.10
Ibuprofen
–
0.85
0.98
Further comparisons of the three model membranes were made with DPC.
This method is relatively fast and can be used to scan a large series of drugs
in a short time. The results of the DPC measurements showed that higher log
Ks values were obtained for discs than for liposomes, particularly when
compared with multilamellar liposomes (Figure 25a). Higher values of log
Ks for the discs than for the liposomes indicate greater membrane partitioning into the discs than into the liposomes. This correlates with the amount of
PEG-lipid included in the various model membranes. An inherent drug/PEGlipid interaction may, however, be ruled out based on both the ITC results
and additional DPC measurements. DPC measurements made with unilamellar EPC liposomes with and without PEG-lipid resulted in the same log Ks
values. The general trend observed in Figure 25a may instead be explained
by the fact that unilamellar and, in particular, multilamellar liposomes have a
lipid fraction that is initially not available to the analyte. In the case of multilamellar liposomes this fraction might never be available to the analyte due
to long equilibration time over the consecutive bilayers. The capacity factor
is calculated by dividing the retention volume by the amount of immobilized
lipid, A (see the equation on page 26). Therefore, if the amount of accessible
lipid is overestimated, the value of log Ks will be underestimated. The extent
to which the multilamellar structure will affect the capacity factor depends
on the properties of the drug. A drug that has a low propensity to partition
into the membrane will equilibrate slowly over the membrane. A drug with a
high propensity to partition into the membrane will equilibrate slowly across
the aqueous inner compartments in a multilamellar liposome.
41
4
a
1,5
log K s
Average log K s
2,0
1,0
0,5
Discs with 12 mol% DSPE-PEG 5000
2,5
b
3
2
1
0
0,0
MLV
ULV
Discs
0
1
2
3
log K s
Multilamellar liposomes
4
Figure 25. a) Partition of drugs for uncharged (white), positively charged (grey) and
negatively charged (black) into multilamellar liposomes (MLV), unilamellar liposomes (ULV) and discs expressed as average log Ks values. b) Comparison of log Ks
values between multilamellar liposomes and discs stabilized by 12 mol% DSPEPEG5000. Positive drugs (), uncharged drugs () and negative drugs ().
An electrostatic effect on the drug retention can be observed in Figure
25b. Here, discs composed of DSPE-PEG5000:DSPC:cholesterol (12:48:40
mol%) are compared with liposomes composed of DSPC:cholesterol (60:40
mol%). The values of log Ks for the positively charged drugs are considerably higher for the discs than the liposomes. This is probably due to the negative charge on DSPE-PEG5000, which will interact positively with the positively charged drugs. The opposite is true for the negatively charged drugs.
The electrostatic effect was investigated further, as described in Paper IV.
When the negatively charged DSPE-PEG5000 was replaced by the uncharged Ceramide-PEG5000 (Paper IV) no electrostatic differences between
discs and liposomes could be observed (Figure 26a). In Figure 26b discs
composed of DSPE-PEG5000:DSPC:cholesterol (15:45:40 mol%) are compared with discs composed of Ceramide-PEG5000:DSPC:cholesterol
(15:45:40 mol%), and differences due to electrostatic interactions can be
seen.
Apart from replacing the negatively charged PEG-lipid with an uncharged PEG-lipid, (Paper IV), a model membrane with a lipid composition
that resembled a natural membrane was developed and evaluated. Figure 26c
shows a comparison between discs mimicking porcine BBM, and discs composed of DSPC/cholesterol. The charge also had an effect in this case. The
amount of charged lipids in the discs mimicking porcine BBM is around
13.5 mol%, which can be compared with 15 mol% of the negatively charged
PEG-lipid in the DSPE-PEG5000:DSPC:cholesterol discs. Electrostatic interactions have an important influence on drug-membrane interactions [68-72]
and attractive interactions between positively charged drugs and negatively
charged membranes are expected to result in increased log Ks values. Inter42
estingly, the results reveal that the partition behaviour of uncharged drugs is
very similar in systems containing Ceramide-PEG5000-stabilized
DSPC:cholesterol discs and brush border membrane discs. Given the large
variation in lipid composition, including a considerable difference in cholesterol content, between the two disc formulations, this finding was somewhat
unexpected. It is well known that the amount of cholesterol affects the membrane permeability [73, 74]. The lower cholesterol content in the BBM discs
is probably compensated for by the presence of other lipid components that
reduce the membrane permeability. Phosphaditylethanolamine is known to
have a condensing effect on the membrane and should thus reduce the drug
partitioning [13, 68].
a
b
4
Log K s
Discs with Ceramide-PEG 5000
Log K s
Discs with Ceramide-PEG 5000
4
3
2
1
3
2
1
0
0
0
1
2
3
4
Log K s
Multilamellar liposomes
Log K s
Discs with Ceramide-PEG 5000
4
0
1
2
3
Log K s
Discs with DSPE-PEG 5000
4
c
3
2
1
0
0
1
2
3
4
Log K s
Discs mimicking porcine BBM
Figure 26. Comparison of log Ks values between: a) multilamellar liposomes and
discs stabilized by Ceramid-PEG5000, b) discs stabilized by DSPE-PEG5000 and by
Ceramid-PEG5000, and c) discs mimicking porcine brush border membrane and discs
stabilized by Ceramide-PEG5000. Positive drugs (), neutral drugs () and negative
drugs ().
43
In Figure 27 log K data obtained from studies based on DSPC:cholesterol
discs stabilized by Ceramide-PEG5000 are compared with previously published log Doct data [68, 75]. For neutral drugs, the agreement and correlation
between the two partition coefficients were reasonable. However, correlations between the values of log K and log Doct determined for charged drugs
were very poor. This strongly supports the use of lipid-based model membranes, rather than octanol, as the lipophilic phase in drug partition studies.
Log K
Discs with Ceramide-PEG 5000
4
3
2
1
0
0
1
2
3
4
Log D oct
Figure 27. Comparison of log Doct data [68, 75] with log K data for discs stabilized
by Ceramide-PEG5000.
3.2.4 Protein reconstitution
To further investigate potential applications of discs, reconstitution of a
membrane protein into the disc was made (Paper IV). As mentioned in Section 3.2, there are advantages of utilizing discs for protein reconstitution.
The membrane protein bacteriorhodopsin (bR) was successfully reconstituted into DSPC/cholesterol discs stabilized by Ceramide-PEG5000 as well as
DSPE-PEG5000. Amino acid analysis was used to confirm the protein incorporation. However, more experiments must be performed to test the activity
of the protein, and other membrane proteins should also be tested to further
investigate the potential of these discs.
44
3.3 Effect of preparation path on aggregate structure
Depending on the application, discs with certain characteristics, such as a
certain lipid composition, particular size or size homogeneity, may be required. In the studies described above it was shown that the composition of
the discs can be varied and, for example, tailored to reflect that of biological
membranes (Paper IV). Furthermore, the size of the discs can be altered by
changing the PEG-lipid content (Paper III). Another way of influencing the
size and homogeneity of the discs is by altering the preparation method.
Systematic investigations of three PEG-lipid/lipid mixtures, prepared using
four commonly employed preparation techniques, were made (Paper V). The
techniques used were simple hydration, freeze-thawing, sonication and detergent depletion; the preparation methods are described in Section 2.1. The
aggregate size and structure were analysed using cryo-TEM and DLS.
Figure 28. Cryo-TEM images of DPPC:DSPE-PEG2000 (75:25 mol %) mixtures. The
samples were prepared by: a) hydration, b) freeze-thawing, c) sonication and d)
detergent depletion. Scale bars indicate 100 nm.
Regardless of the preparation method used, all DPPC/DSPE-PEG2000
samples contained almost only discs of a relatively homogeneous size (Figure 28). However, the actual size of the discs varied depending on the preparation path. Storage for one week at room temperature did not change the
aggregate size or structure in the samples noticeably, as judge from cryoTEM investigations. However, according to DLS measurements, the apparent hydrodynamic radius of the small particles increased from 9 to 12 nm in
the detergent-depleted samples (Table 3).
45
Table 3. Hydrodynamic radius determined from DLS data. (n=3 unless otherwise
stated.)
Hydrated
DPPC/DSPEPEG2000
20 r3 / 292 *
EPC/chol/ DSPE- EPC/ DSPEPEG 2000
PEG2000
162**
77 r11
Hydrated, stored 1
week
Freeze-thawed
20 r3 / 199 r72
77 r10
162**
19 r2 / 432*
52 r4
101**
Freeze-thawed,
stored 1 week
Sonicated
19 r2 / 261 r97
33 r10
90**
15 r2 / 307*
30 r3
24 r2
Sonicated, stored 1
week
Detergent-depleted
15 r1 / 161r29
30 r2
27r3
9 r1 / 144 r18
13 r2 / 181 r64
12 r1 / 121 r41
12 / 136
15 / 351
Detergent-depleted,
stored 1 week ***
* The larger aggregates were not detected in all samples.
** n=1.
*** n=2.
15 / 113
All samples based on EPC/cholesterol/DSPE-PEG2000 resulted in discs
and, depending on the preparation path, a varying amount of liposomes (Figure 29). Storage for one week at room temperature resulted in size changes
in samples prepared by hydration and freeze-thawing, while the other samples were unaltered (Table 3).
46
Figure 29. Cryo-TEM images of EPC:cholesterol:DSPE-PEG2000 (35:40:25 mol %)
mixtures. The samples were prepared by: a) hydration, b) freeze-thawing, c) sonication and d) detergent depletion. Scale bars indicate 100 nm.
The choice of preparation path was found to have the greatest effect on
the EPC/ DSPE-PEG2000 samples (Figure 30). In samples prepared by hydration, or freeze-thawing, liposomes, large bilayer flakes with cylindrical extensions and threadlike to globular micelles were present. However, the
sample prepared by freeze-thawing exhibited a considerably larger amount
of long threadlike micelles and the liposomes were generally smaller in size
(compare Figure 30a and b). Storage of the hydrated and freeze-thawed
samples did not cause any major changes (Table 3). Sonication of
EPC/DSPE-PEG2000 mixtures resulted in the formation of mainly discoidal
structures (Figure 30c). A fraction of small liposomes was also present, as
well as a few very long threadlike micelles. The number of threadlike micelles increased after one week of storage at room temperature. Detergentdepleted samples contained small aggregates, including both irregular and
relatively circular discs (Figure 30d). Furthermore, some large bilayer flakes
and a few liposomes were observed. After one week the sample appeared
unchanged based on cryo-TEM inspection, but DLS measurements indicated
that the small aggregates had increased slightly in size (Table 3).
47
Figure 30. Cryo-TEM images of EPC:DSPE-PEG2000 (75:25 mol %) mixtures. The
samples were prepared by: a) hydration, b) freeze-thawing, c) sonication and d)
detergent depletion. Scale bars indicate 100 nm.
Freeze-thawing clearly influences the aggregate structure in EPC/cholesterol/DSPE-PEG2000 and EPC/DSPE-PEG2000 mixtures. In the hydrated EPC/
DSPE-PEG2000 sample it is probable that the PEG-lipid distribution differs
between the various aggregates. Freeze-thawing can be expected to lead to a
more homogeneous component distribution and thus a structurally less
polydisperse appearance. Another effect of freeze-thawing was the decrease
in liposome size found in the EPC/DSPE-PEG2000 sample. This is a documented effect of freeze-thawing for liposomes in the liquid crystalline phase,
and has been shown also to apply to liposomes containing a relatively high
amount of PEG-lipids [76-79].
A general observation was that sample preparations using sonication or detergent depletion generated aggregates with a smaller size than with the other
preparation paths. Sonication is a high-energy-input method, which may
force the amphiphilic molecules to assemble into structures deviating from
the equilibrium state, such as very small aggregates [80]. Relaxation into
larger aggregates will probably be prevented, or at least delayed, by the high
amount of PEG-lipid used in the preparations. The most surprising finding
was that discs, rather than cylindrical micelles, were formed in EPC/DSPEPEG2000 systems prepared by sonication and detergent depletion. As discussed above, sonication is a high-energy-input method and the energy input
is apparently high enough to compensate for the partial component segregation needed to make disc formation possible. The small aggregates formed in
the detergent-depleted EPC/DSPE-PEG2000 samples are also disc-shaped
48
rather than threadlike. A rod-shaped micelle of small size would have a very
high average curvature compared with a longer threadlike micelle. When
growth from initially small globular micelles into larger assemblies is hindered by the presence of PEG-lipids it may thus be more energetically favourable to partially segregate EPC and PEG-lipid components and form
discs.
49
4 Conclusions
The results presented in Papers I, II and V show how lipid mixture, environmental conditions and preparation path can be chosen so as to promote
the formation of either discoidal or cylindrical micellar structures in surfactant/lipid mixtures.
In the study described in Paper I, where the surfactant was represented by
a micelle-forming PEG-lipid, it was shown that discoidal structures are preferred over cylindrical micelles when the lipid mixture contains components
that reduce the spontaneous curvature and increase the monolayer bending
rigidity. Furthermore, discoidal aggregate structures are preferred at temperatures below the transition temperature of the lipid mixture.
The study reported in Paper II, where the surfactant was represented by
C12E8, CTAB and SDS, showed that the preference for either discoidal or
threadlike micelles can be tuned by the choice of lipids and environmental
conditions in much the same way as that observed for the PEG-lipid/lipid
system.
In the study presented in Paper V it was found that the aggregate homogeneity and the size of the aggregates were significantly affected by the
choice of preparation path.
The discoidal micelles, which in certain lipid systems adapt sizes large
enough to be called bilayer discs, can be useful as model membranes. In the
study described in Paper III it was shown that bilayer discs could be prepared from mixtures of DSPE-PEG5000:DSPE:cholesterol (12:48:40 mol%).
The discs showed good long-term stability and the size could be varied by
changing the PEG-lipid concentration.
The disc as a model membrane was investigated in drug partition studies
with encouraging results. The study reported in Paper IV describes the development of a disc with a lipid composition resembling that of BBM and
stabilized by an uncharged PEG-lipid. Furthermore, investigations using bR
suggested that membrane proteins could be reconstituted into bilayer discs.
50
Svensk sammanfattning
Alla celler är omgivna av ett cellmembran som separerar cellens inre delar
från den omgivande miljön. Barriären som cellmembranet utgör har bland
annat till uppgift att släppa igenom små molekyler genom diffusion samt att
hindra stora makromolekyler som exempelvis proteiner att diffundera över
membranet. Membranet skapar också en miljö för alla membranproteiner
som i sin tur är delaktiga i många livsviktiga funktioner.
Att studera olika processer som sker i cellmembranet är komplicerat på
grund av cellmembranets komplexa karaktär. Ett exempel där det finns ett
behov att studera interaktioner med cellmembranet är inom läkemedelsindustrin. I dagens snabba utveckling av läkemedel tas många substanser fram i
jakten på nya läkemedel; de flesta av dessa läkemedel är tänkta att tas oralt
och måste för att komma ut i blodsystemet passera ett flertal cellmembran i
tarmväggen. Att göra fördelningsstudier, det vill säga testa hur lätt de tilltänkta läkemedlen tar sig över ett sådant membran är därför ett viktigt steg i
processen när nya läkemedel utvecklas. Istället för att använda naturliga
membraner kan modellmembran användas. Att studera en ny typ av nanometerstora (en nanometer = en miljarddels meter) modellmembran har varit
fokus i min forskning.
Hydrofil del
Hydrofob del
Figur1. Till vänster i figuren syns en schematisk bild på en membranlipid med den
hydrofila delen representerad av en rund boll och den hydrofoba delen representerad
av två vågiga svansar. I mitten i figuren illustreras det dubbellager som membranlipiderna bildar då dessa molekyler tillsätts till vatten. Till höger syns en schematisk
bild på en liposom.
De huvudsakliga komponenterna i cellmembranet är lipider. Membranlipider är amfifila molekyler, det vill säga en del av molekylen är vattenäls51
kande (hydrofil) och en del av molekylen är vattenhatande (hydrofob), se till
vänster i Figur 1. När amfifila molekyler blandas med vatten över en kritisk
koncentration kommer de, till följd av molekylernas amfifila karaktär, att slå
sig samman och bilda aggregat (Figur 1, mitten). Då aggregaten bildas kommer lipidernas hydrofila delar att vara vända mot vattnet medan de hydrofoba delarna vänder sig mot varandra och kommer på så sätt att undvika interaktion med vattnet. Beroende på faktorer såsom den omgivande miljön och
de amfifila molekylernas form kan de bilda olika typer av aggregatstrukturer
(Figur 2). Då molekylerna är cylinderformade kommer de att bilda ett dubbellager, det vill säga två monolager med de hydrofila delarna vända mot
varandra (Figur 1, mitten). Detta dubbellager kan sedan sluta sig och bilda
en liposom (Figur 1, till höger) på grund av ofördelaktiga interaktioner vid
sidorna av dubbellagret, där de hydrofoba delarna är exponerade mot vattnet.
Molekylens form
Aggregatstruktur
Figur 2. Figuren visar hur formen på den amfifila molekylen påverkar det aggregat
som de bildar.
52
Om molekylerna är konformade (som högst upp i Figur 2) passar de bäst
att sitta i ett krökt monolager, som tårtbitar i en tårta. När molekylerna sitter
på detta sätt såsom i sfäriska och cylindriska miceller (överst i Figur 2) kallas det för att de sitter i en hög positiv kurvatur. Negativ kurvatur har de
omvända micellerna (nederst i Figur 2), ett dubbellager har kurvaturen noll
(mitten i Figur 2).
Om amfifila molekyler med olika form blandas kan det vara svårt att förutse aggregatstrukturen. Exempelvis så kan det bildas två fundamentalt olika
strukturer då cylinderformade lipider blandas med en speciell typ av konformade amfifila molekyler, så kallade polyetylenglykol (PEG)-lipider. I
Figur 3 visas de två olika aggregatstrukturerna, cylindriska miceller och
diskar.
Figur 3 31. Schematisk bild på en cylindrisk micell och en disk. De mörkast gråa
lipiderna representerar PEG-lipider.
Min avhandling behandlar diskformade aggregat. Jag har undersökt i vilka typer av system som diskar uppträder och studerat om diskar kan bildas i
andra system än i de ovannämnda PEG-lipid systemen. Att ta fram diskar
som är optimerade för att användas som modellmembran och testa diskarnas
funktion i läkemedelsfördelningsstudier har också varit ett mål med avhandlingen.
För att bestämma när diskar bildades, studerades system med PEG-lipid
tillsammans med en cylindriskt formad lipid. Om alla molekylerna blandar
sig jämnt kommer en cylindrisk micell att bildas. Detta är fördelaktigt med
tanke på blandningen men den cylindriskt formade lipiden tvingas till att
sitta i ett krökt monolager trots att dess form gör att den passar bäst att sitta i
ett platt monolager. Om en disk bildas istället för en cylindrisk micell får
molekylerna sitta som deras individuella form föredrar, med de konformade
PEG-lipiderna på kanten och de cylindriska lipiderna i den platta delen. Rent
geometriskt är detta fördelaktigt för molekylerna, däremot är det inte fördelaktigt att inte blanda PEG-lipiden med lipiden. I min avhandling visas att
diskar bildas i system med PEG-lipid tillsammans med en lipidblandning
som innehåller lipider som ökar stelheten på blandningen och minskar den
53
spontana kurvaturen, det vill säga molekyler med en omvänd konisk form
som nederst i Figur 3 (delarbete I).
I delarbete II visades att PEG-lipiden inte var nödvändig för att skapa stabila diskar utan att andra mer konventionella konformade lipider också kan
användas för att diskar ska bildas. Diskarna som bildas med dessa konventionella lipider fungerar dock inte lika bra som modellmembran då de inte
har lika bra stabilitet över tid samt inte är stabila vid utspädning som PEGlipid/lipid diskarna är.
Diskarna som utvecklades med avseende på att användas som modellmembran är optimerade så att den största delen av en preparation endast
består av diskar. Diskar med ungefär samma storlek är också önskvärda. I
delarbete III visades att diskarnas storlek kunde styras genom att mer eller
mindre PEG-lipid tillsattes till blandningen. För att efterlikna naturliga
membraner så mycket som möjligt togs diskar fram med en lipidsammansättning som liknade tarmmembranet hos gris (delarbete IV). För att testa
diskarnas duglighet som modellmembran utfördes läkemedelsfördelningsstudier. I vissa fall visade det sig att diskar kan vara bättre lämpade i fördelningsstudier i jämförelsen med liposomer (som mer traditionellt används).
Diskarna var bättre lämpade som modellmembran än liposomer i de fall då
en snabb analysmetod användes för fördelningsstudierna. I dessa fall hinner
inte läkemedlet att interagera med all lipid i liposomen eftersom en del av
lipiden är ”gömd” i liposomen från början.
Förhoppningsvis kan resultaten som presenteras i denna avhandling, såväl
som förståelsen för faktorer som påverkar diskbildning och hur diskarnas
storlek och sammansättning kan förändras, att komma till nytta för utveckling av diskar för olika biokemiska, biotekniska och farmaceutiska tillämpningar.
54
Acknowledgements
Det kanske inte blev Den Bästa Avhandlingen men jag hade i alla fall…
… Den bästa handledaren, Katarina.
… Den bästa biträdande handledaren, Mats.
… De bästa medarbetarna, Anna Lunquist, Caroline Engvall, Magnus
Bergström, Maria C Sandström, Per Lundahl, och Shusheng Zuo.
… De bästa fixarna, Göran S, Gösta och Laila.
… Den bästa superfixaren, Göran K.
… Den bästa trion, Anna, Maria och PEr.
… Den bästa familjen och svärfamiljen.
… Den bästa maken, Anders!
55
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
56
C. Tanford, The Hydrophobic Effect: Formation of Micelles and
Biological Membranes, Wiley, New York, 1976.
D.F. Evans and H. Wennerström, The Colloidal Domain: Where
Physics, Chemistry, Biology and Technology Meets, VCH Publichers Inc, New York, 1994.
J.N. Israelachvili, D.J. Mitchell and B.W. Ninham, Theory of SelfAssembly of Hydrocarbon Amphiphiles into Micelles and Bilayers,
J. Chem. Soc., Faraday Trans. 2 2 (1976) 1525-1568.
L.M. Bergstrom, Bending energetics of tablet-shaped micelles: A
novel approach to rationalize micellar systems, Chemphyschem 8
(2007) 462-472.
J.F. Nagle and S. Tristram-Nagle, Structure of lipid bilayers, Biochim Biophys Acta-Reviews on Biomembranes 1469 (2000) 159195.
G. Cevc, Phospholipids handbook, Cevc, G., Marcel Dekker Inc.,
New York, 1993.
O.G. Mouritsen, Life-As a Matter of Fat. The Emerging Science of
Lipidomics, Springer-Verlag, Heidelberg, 2005.
H. Ohvo-Rekila, B. Ramstedt, P. Leppimaki and J.P. Slotte, Cholesterol interactions with phospholipids in membranes, Prog Lipid Res
41 (2002) 66-97.
R.A. Demel and B. De Kruyff, The function of sterols in membranes, Biochim Biophys Acta 457 (1976) 109-32.
T.P. McMullen and R.N. McElhaney, Differential scanning calorimetric studies of the interaction of cholesterol with distearoyl and
dielaidoyl molecular species of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine, Biochemistry 36 (1997)
4979-86.
M.R. Vist and J.H. Davis, Phase equilibria of cholesterol/dipalmitoylphosphatidylcholine mixtures: 2H nuclear magnetic
resonance and differential scanning calorimetry, Biochemistry 29
(1990) 451-64.
J.H. Ipsen, G. Karlstrom, O.G. Mouritsen, H. Wennerstrom and M.J.
Zuckermann, Phase-Equilibria in the PhosphatidylcholineCholesterol System, Biochim Biophys Acta 905 (1987) 162-172.
S.E. Wright, J.C. White and L. Huang, Partitioning of teniposide
into membranes and the role of lipid composition, Biochim Biophys
Acta 1021 (1990) 105-13.
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
D. Needham and R.S. Nunn, Elastic deformation and failure of lipid
bilayer membranes containing cholesterol, Biophys J 58 (1990) 9971009.
J.M. Smaby, M.M. Momsen, H.L. Brockman and R.E. Brown,
Phosphatidylcholine acyl unsaturation modulates the decrease in interfacial elasticity induced by cholesterol, Biophys J 73 (1997)
1492-505.
T.M. Allen and P.R. Cullis, Drug delivery systems: Entering the
mainstream, Science 303 (2004) 1818-1822.
D. Papahadjopoulos and H.K. Kimelberg, Phospholipid Vesicles
(Liposomes) as Models for Biological Membranes: Their Properties
and Interactions with Cholesterol and Proteins, Prog Surf Sci 4
(1974) 141-232.
N. Weiner, L. Lieb, S. Niemiec, C. Ramachandran, Z. Hu and K.
Egbaria, Liposomes: a novel topical delivery system for pharmaceutical and cosmetic applications, J Drug Target 2 (1994) 405-10.
D.D. Lasic. in (Lipowsky, R. and Sackmann, E., eds.) Handbook of
Biological Physics, Elsevier Science B. V. 1995, pp. 491-519.
R. Joannic, L. Auvray and D.D. Lasic, Monodisperse vesicles stabilized by grafted polymers, Phys Rev Lett 78 (1997) 3402-3405.
D.D. Lasic, R. Joannic, B.C. Keller, P.M. Frederik and L. Auvray,
Spontaneous vesiculation, Adv Colloid Interface Sci 89-90 (2001)
337-49.
E. Marques, A. Khan, M.D. Miguel and B. Lindman, Self-Assembly
in Mixtures of a Cationic and an Anionic Surfactant - the Sodium
Dodecyl-Sulfate Didodecyldimethylammonium Bromide WaterSystem, J Phys Chem 97 (1993) 4729-4736.
S.A. Safran, P. Pincus and D. Andelman, Theory of Spontaneous
Vesicle Formation in Surfactant Mixtures, Science 248 (1990) 354356.
I. Szleifer, O.V. Gerasimov and D.H. Thompson, Spontaneous liposome formation induced by grafted poly(ethylene oxide) layers:
Theoretical prediction and experimental verification, Proc Nat Acad
Sci 95 (1998) 1032-1037.
P. Fromherz, lipid-vesicle-structure: size control by edge-active
agents, Chem phys lett 94 (1983) 259-66.
D.D. Lasic, The mechanism of vesicle formation, Biochem J 256
(1988) 1-11.
M. Bergstrom and J.C. Eriksson, The energetics of forming equilibrated bilayer vesicles, Langmuir 12 (1996) 624-635.
D.D. Lasic, A molecular model for vesicle formation, Biochim Biophys Acta 692 (1982) 501-2.
W. Helfrich, Elastic properties of lipid bilayers: theory and possible
experiments, Z Naturforsch [C] 28 (1973) 693-703.
M.M. Kozlov, D. Lichtenberg and D. Andelman, Shape of phospholipid/surfactant mixed micelles: Cylinders or disks? Theoretical
analysis, J Phys Chem B 101 (1997) 6600-6606.
57
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
58
L. van Dam, G. Karlsson and K. Edwards, Direct observation and
characterization of DMPC/DHPC aggregates under conditions relevant for biological solution NMR, Biochim Biophys Acta 1664
(2004) 241-256.
A. Walter, P.K. Vinson, A. Kaplun and Y. Talmon, Intermediate
structures in the cholate-phosphatidylcholine vesicle-micelle transition, Biophys J 60 (1991) 1315-25.
M. Silvander, G. Karlsson and K. Edwards, Vesicle solubilization by
alkyl sulfate surfactants: A cryo-TEM study of the vesicle to micelle
transition, J Colloid Interface Sci 179 (1996) 104-113.
K. Edwards, M. Johnsson, G. Karlsson and M. Silvander, Effect of
polyethyleneglycol-phospholipids on aggregate structure in preparations of small unilamellar liposomes, Biophys J 73 (1997) 258-66.
M. Johnsson and K. Edwards, Liposomes, disks, and spherical micelles: aggregate structure in mixtures of gel phase phosphatidylcholines and poly(ethylene glycol)-phospholipids, Biophys J 85 (2003)
3839-47.
K. Hristova and D. Needham, Phase-Behavior of a Lipid PolymerLipid Mixture in Aqueous-Medium, Macromolecules 28 (1995) 9911002.
J.A. Rogers and A. Wong, The temperature dependence and thermodynamics of partitioning of phenols in the n-octanol-water system,
Int J Pharm 6 (1982) 339-348.
C. Hansch, R.M. Muri, T. Fujita, P.P. Maloney, F. Geiger and M.
Streich, The Correlation of Biological Activity of Plant Growth
Regulators and Chloromycetin Derivatives with Hammett Constants
and Partition Coefficients, J Am Chem Soc 85 (1963) 2817-2824.
A. Leo, C. Hansch and D. Ekins, Partition coefficients and their
uses, Chem Rev 71 (1971) 525-616.
S.W. Ong, H.L. Liu and C. Pidgeon, Immobilized-artificialmembrane chromatography: Measurements of membrane partition
coefficient and predicting drug membrane permeability, J Chromatogr A 728 (1996) 113-128.
S. Ong, H. Liu, X. Qiu, G. Bhat and C. Pidgeon, Membrane partition
coefficients chromatographically measured using immobilized artificial membrane surfaces, Anal Chem 67 (1995) 755-62.
M. Molero-Monfort, Y. Martin-Biosca, S. Sagrado, R.M.
Villanueva-Camanas and M.J. Medina-Hernandez, Micellar liquid
chromatography for prediction of drug transport, J Chromatogr A
870 (2000) 1-11.
D.W. Armstrong and F. Nome, Partition Behavior of Solutes Eluted
with Micellar Mobile Phases in Liquid Chromatography, Anal Chem
53 (1981) 1662-1666.
S. Engstrom, T.P. Norden and H. Nyquist, Cubic phases for studies
of drug partition into lipid bilayers, Eur J Pharm Sci 8 (1999) 243254.
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
G.P. van Balen, C.M. Martinet, G. Caron, G. Bouchard, M. Reist,
P.A. Carrupt, R. Fruttero, A. Gasco and B. Testa, Liposome/water
lipophilicity: methods, information content, and pharmaceutical applications, Med Res Rev 24 (2004) 299-324.
G.M. Pauletti and H. Wunderliallenspach, Partition-Coefficients inVitro - Artificial Membranes as a Standardized Distribution Model,
Eur J Pharm Sci 1 (1994) 273-282.
M. Almgren, K. Edwards and G. Karlsson, Cryo transmission electron microscopy of liposomes and related structures, Coll Surf A 174
(2000) 3-21.
S.U. Egelhaaf, E. Wehrli, M. Muller, M. Adrian and P. Schurtenberger, Determination of the size distribution of lecithin liposomes: A
comparative study using freeze fracture, cryoelectron microscopy
and dynamic light scattering, J Microscopy 184 (1996) 214-228.
J. Dubochet, M. Adrian, J.J. Chang, J.C. Homo, J. Lepault, A.W.
Mcdowall and P. Schultz, Cryo-Electron Microscopy of Vitrified
Specimens, Quart Rev Biophys 21 (1988) 129-228.
N.A. Mazer, G.B. Benedek and M.C. Carey, Quasielastic lightscattering studies of aqueous biliary lipid systems. Mixed micelle
formation in bile salt-lecithin solutions, Biochemistry 19 (1980)
601-15.
F. Beigi, Q. Yang and P. Lundahl, Immobilized-Liposome Chromatographic Analysis of Drug Partitioning into Lipid Bilayers, J Chromatogr A 704 (1995) 315-321.
P. Lundahl and F. Beigi, Immobilized liposome chromatography of
drugs for model analysis of drug-membrane interactions, Adv Drug
Deliver Rev 23 (1997) 221-227.
F. Beigi, I. Gottschalk, C.L. Hagglund, L. Haneskog, E. Brekkan,
Y.X. Zhang, T. Osterberg and P. Lundahl, Immobilized liposome
and biomembrane partitioning chromatography of drugs for prediction of drug transport, Int J Pharm 164 (1998) 129-137.
Q. Yang, X.Y. Liu, K. Umetani, N. Kamo and J. Miyake, Partitioning of triphenylalkylphosphonium homologues in gel beadimmobilized liposomes: chromatographic measurement of their
membrane partition coefficients, Biochim Biophys Acta 1417 (1999)
122-30.
F. Zhang and E.S. Rowe, Titration calorimetric and differential
scanning calorimetric studies of the interactions of n-butanol with
several phases of dipalmitoylphosphatidylcholine, Biochemistry 31
(1992) 2005-11.
J. Henriksen, A.C. Rowat and J.H. Ipsen, Vesicle fluctuation analysis of the effects of sterols on membrane bending rigidity, Eur Biophys J 33 (2004) 732-41.
Z. Chen and R.P. Rand, The influence of cholesterol on phospholipid membrane curvature and bending elasticity, Biophys J 73
(1997) 267-76.
59
[58]
[59]
[60]
[61]
[62]
[63]
[64]
[65]
[66]
[67]
[68]
[69]
[70]
[71]
60
O.G. Mouritsen and M.J. Zuckermann, What's so special about cholesterol?, Lipids 39 (2004) 1101-13.
L. Miao, M. Nielsen, J. Thewalt, J.H. Ipsen, M. Bloom, M.J. Zuckermann and O.G. Mouritsen, From lanosterol to cholesterol: structural evolution and differential effects on lipid bilayers, Biophys J 82
(2002) 1429-44.
C.E. Dahl, J.S. Dahl and K. Bloch, Effect of alkyl-substituted precursors of cholesterol on artificial and natural membranes and on the
viability of Mycoplasma capricolum, Biochemistry 19 (1980) 14627.
J.A. Urbina, S. Pekerar, H.B. Le, J. Patterson, B. Montez and E.
Oldfield, Molecular order and dynamics of phosphatidylcholine bilayer membranes in the presence of cholesterol, ergosterol and
lanosterol: a comparative study using 2H-, 13C- and 31P-NMR
spectroscopy, Biochim Biophys Acta 1238 (1995) 163-76.
X. Xu and E. London, The effect of sterol structure on membrane
lipid domains reveals how cholesterol can induce lipid domain formation, Biochemistry 39 (2000) 843-9.
K.R. Mecke, T. Charitat and F. Graner, Fluctuating lipid bilayer in
an arbitrary potential: Theory and experimental determination of
bending rigidity, Langmuir 19 (2003) 2080-2087.
I.I. Szleifer, D. Kramer, A. Ben-Shaul, D. Roux and W.M. Gelbart,
Curvature elasticity of pure and mixed surfactant films, Phys Rev
Lett 60 (1988) 1966-1969.
L.M. Bergstrom, Bending elasticity of charged surfactant layers: The
effect of mixing, Langmuir 22 (2006) 6796-6813.
A. Kaplun, F.M. Konikoff, A. Eitan, M. Rubin, A. Vilan, D.
Lichtenberg, T. Gilat and Y. Talmon, Imaging supramolecular aggregates in bile models and human bile, Micr Res Tech 39 (1997)
85-96.
K. Christiansen and J. Carlsen, Microvillus membrane vesicles from
pig small intestine. Purity and lipid composition, Biochim Biophys
Acta 647 (1981) 188-95.
T. Osterberg, M. Svensson and P. Lundahl, Chromatographic retention of drug molecules on immobilised liposomes prepared from egg
phospholipids and from chemically pure phospholipids, Eur J Pharm
Sci 12 (2001) 427-39.
S.D. Kramer, C. JakitsDeiser and H. WunderliAllenspach, Free fatty
acids cause pH-dependent changes in drug-lipid membrane interactions around physiological pH, Pharm Res 14 (1997) 827-832.
S.D. Kramer, A. Braun, C. Jakits-Deiser and H. WunderliAllenspach, Towards the predictability of drug-lipid membrane interactions: The pH-dependent affinity of propranolol to phosphatidylinositol containing liposomes, Pharm Res 15 (1998) 739-744.
C. Lagerquist, F. Beigi, A. Karlen, H. Lennernas and P. Lundahl,
Effects of cholesterol and model transmembrane proteins on drug
[72]
[73]
[74]
[75]
[76]
[77]
[78]
[79]
[80]
partitioning into lipid bilayers as analysed by immobilized-liposome
chromatography, J Pharm Pharmacol 53 (2001) 1477-87.
X.Y. Liu, Q. Yang, N. Kamo and J. Miyake, Effect of liposome type
and membrane fluidity on drug-membrane partitioning analyzed by
immobilized liposome chromatography, J Chromatogr A 913 (2001)
123-31.
E. Corvera, O.G. Mouritsen, M.A. Singer and M.J. Zuckermann,
The permeability and the effect of acyl-chain length for phospholipid bilayers containing cholesterol: theory and experiment, Biochim
Biophys Acta 1107 (1992) 261-70.
E.C. Yan and K.B. Eisenthal, Effect of cholesterol on molecular
transport of organic cations across liposome bilayers probed by second harmonic generation, Biophys J 79 (2000) 898-903.
C. Giaginis, S. Theocharis and A. Tsantili-Kakoulidou, Contribution
to the standardization of the chromatographic conditions for the
lipophilicity assessment of neutral and basic drugs, Anal Chim Acta
573 (2006) 311-318.
R.C. MacDonald, F.D. Jones and R. Qiu, Fragmentation into small
vesicles of dioleoylphosphatidylcholine bilayers during freezing and
thawing, Biochim Biophys Acta 1191 (1994) 362-70.
S. Sriwongsitanont and M. Ueno, Effect of freeze-thawing and polyethylene glycol (PEG) lipid on fusion and fission of phospholipid
vesicles, Chem Pharm Bull 52 (2004) 641-642.
M. Traikia, D.E. Warschawski, M. Recouvreur, J. Cartaud and P.F.
Devaux, Formation of unilamellar vesicles by repetitive freeze-thaw
cycles: characterization by electron microscopy and P-31-nuclear
magnetic resonance, Eur Biophys J Biophys Lett 29 (2000) 184-195.
M. Ueno and S. Sriwongsitanont, Effect of PEG lipid on fusion and
fission of phospholipid vesicles in the process of freeze-thawing,
Polymer 46 (2005) 1257-1267.
V.P. Torchilin and V. Weissig, Liposomes, Oxford University Press,
New York, 2003.
61
Acta Universitatis Upsaliensis
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