Università Politecnica Delle Marche
FACOLTÀ DI SCIENZE
Scuola di Dottorato di Ricerca
IX ciclo n.s.
Curriculum “Scienze Biomolecolari”
Coordinatore: Prof. Mario Orena
STUDY OF CUBIC MONOOLEIN-CYTOCHROME C-WATER
PHASE: PROTEIN CONCENTRATION ,TEMPERATURE AND
PRESSURE EFFECTS
Dottorando
Serena Mazzoni
Relatore
Prof. Paolo Mariani
…..Il processo di una scoperta
scientifica è, in effetti, un continuo
conflitto di meraviglie……
Albert Einstein
Table of Contents
Introduction
I-VI
CHAPTER 1
Lipids
1.1 Significance of liquid crystal………………………………………………………2
1.2
Lipids ………………………………………………………………………….4
1.3
Polimorphism…………………………………………………………………...5
1.3.1 Lamellar phase…………………………………………………………..7
1.3.2 Hexagonal phase………………………………………………………..8
1.3.3 Cubic phase……………………………………………………………..10
1.4.Interfacial Curvature……………………………………………………………15
1.4.1 Curvature and Frustration…………………………………………………...18
1.5 Lyotropic phase diagram……………………………………………………….22
1.6 Lipid-Protein interaction………………………………………………………..24
CHAPTER 2
Application of lipid system
2.1. New application of lipid systems……………………...……………………….28
2.2 Drug delivery…………………………………………………………………...31
2.3. Cubosomes……………………………………………………………………..37
2.4 Solid Lipid Nanopaticules………………………………………………………41
CHAPTER 3
Description of monoolein-cytochrome c-water system
3.1. History of monoolein-cytochrome c-water system………………………………48
CHAPTER 4
Material and methods
4.1 Diffraction X-Ray Scattering…………………………………………………..56
4.2 The Law and Bragg equation…………………………………………………...57
4.2.1 X Ray diffraction methods for structure determination……………………...63
4.2.2 Phase identification…………………………………………………………..64
4.3 Production of X-Ray
4.3.1 The X-Ray Tube
4.3.1.1 Diffractomer in the SAIFET laboratory…………………………………67
4.3.1.2 Diffractomer in the USP laboratoty……………………………………..69
4.3.2 Synchrotron………………………………………………………………..70
4.3.2.1 Pressure Cell…………………………………………………………….73
4.3.2.2 Temperature System…………………………………………………….75
4.4 Spectrophotometry……………………………………………………………...75
4.4.1 The Beer-Lambert law………………………………………………………..77
4.5 Sample preparation……………………………………………………………...80
CHAPTER 5
Data analysis: Cytochrome c concentration effects………………………………….82
5.1 Monoolein and Cytochrome c 1 mg/ml……………………………………………..84
5.2. Monoomein and Cytochrome c 5 mg/ml…………………………………………..87
5.3. Monoolein and Cytochrome c 10 mg/ml…………………………………………..90
5.4. Monoolein and Cytochrome c 25 mg/ml…………………………………………..92
5.5. Monoolein and Cytochrome c 50 mg/ml…………………………………………..95
5.6. Monoolein and Cytochrome c 100 mg/ml…………………………………………96
CHAPTER 6
Data analysis:Temperature Effects…………………………………………………..99
6.1 Monoolein and Cytochrome c 1mg/ml…………………………………………….100
6.2 Monoolein and Cytochrome c 5mg/ml……………………………………………103
6.3 Monoolein and Cytochrome c 10 mg/ml…………………………………………..107
6.4 Monoolein and Cytochrome c 25 mg/ml…………………………………………..111
6.5 Monoolein and Cytochrome c 50 mg/ml…………………………………………..115
CHAPTER 7
Data analysis: mechanical pressure Effects………………………………………...121
7.1 Monoolein and Cytochrome c 1mg/ml…………………………………………….122
7.1.1 Monoolein and Cytochrome c 1mg/ml (bis)…………………………………126
7.2 Monoolein and Cytochrome c 5mg/ml……………………………………………127
7.3 Monoolein and Cytochrome c 10 mg/ml…………………………………………..128
7.4 Monoolein and Cytochrome c 25 mg/ml…………………………………………..131
7.5 Monoolein and Cytochrome c 50 mg/ml…………………………………………..132
7.6 Monoolein ad Cytochrome c 100 mg/ml…………………………………………..136
CHAPTER 8
Discussion and conclusion……………………………………………………………138
8.1 Discussion Data of Concentration Effects………………………………………139
8.2 Discussion Data of Temperature Effects………………………………………..142
8.2 Discussion Data of Mechanical Pressure Data………………………………….152
CHAPTER 9
Applications of cubic phases…………………………………………………………153
9.1 Nanoparticulate lipidic dispersions for bromocriptine delivery:
a comparative study………………………………………………………………156
9.1.1 Introduction………………………………………………………………….157
9.1.2 Materials and methods……………………………………………………….160
9.1.2.1 Materials…………………………………………………………………...160
9.1.2.2. MAD preparation………………………………………………………….161
9.1.2.3 SLN preparation……………………………………………………………162
9.1.2.4 Characterization of lipidic dispersions………………………………………...163
9.1.2.4.1 Photon Correlation Spectroscopy (PCS)………………………………...163
9.1.2.4.2 Cryo-Transmission Electron Microscopy (Cryo-TEM)………………….163
9.1.2.4.3 X-ray diffraction measurements…………………………………………..164
9.1.2.5 Drug Content of Dispersions………………………………………………......165
9.1.2.5.1 Sedimentation Field Flow Fractionation Analysis…………………………..165
9.1.2.6 HPLC Procedure……………………………………………………………….166
9.1.3. Results…………………………………………………………………………..166
9.1.3.1 Production and characterization of lipidic dispersions………………………...166
9.1.3.2 Efficiency of BC encapsulation………………………………………………..179
9.1.4. Discussion
181
9.2 X-ray Diffraction Analysis of Nucleotides Effects on
Monoolein-Based Liquid Crystals ……………………………………………………183
9.2.1. Introduction……………………………………………………………………..183
9.2.2.Materials and methods…………………………………………………………...187
9.2.2.1 Materials……………………………………………………………………….187
9.2.2.2 SAXRD Experiments………………………………………………………….188
9.2.3 Results…………………………………………………………………………...190
9.3.3.Conclusions……………………………………………………………………...203
Bibliography
Acknowledgements
Introduction
INTRODUCTION
I
Introduction
Biological systems often show so complex molecular architectures that
questions about their structure, stability and function are very demanding
problems for molecular biophysics. Biological membranes are an
example of complex molecular systems: they comprend various types of
lipids and protein as major constituents.
Moreover, lipids exhibit a rich lyotropic and thermotropic phase
behaviour, due to their amphipatic character : this is a very important
point, as any variation of the physico-chemical properties of the lipid
component will largely modify the state and function of the membrane,
for example influencing protein activities.
In this context, it can be observed that even the phase behaviour and
structural properties of monoacylglycerides (e.g., monoolein) in water
have been investigate for a long time, because on the extended
polymorphism. For example, monoolein in water shows several
mesophases, characterized by a highly disordered conformation of the
hydrocarbon chain. Varying the water concentration a lamellar L-alfa, an
inverted (type II) hexagonal phase HII and two bicontinuos inverted
cubic phases with space group Pn3m (Q224) and Ia3d (Q230) have been
identified [1].
The structure of bicontinuos cubic phase has been described in terms of
Infinite Periodic Minimal Surface (IPMS), the crystallographic space
II
Introduction
group of the cubic phase determining the type of the IPMS: in particular,
the cubic phases observed in the monoolein – water system (space group
Ia3d and Pn3m) are based on G (gyroid) and D (diamond ) surface.
The cubic phases are also unique in their ability to accommodate
proteins as compared with other lipid-water phases. A wide range of
globular protein with molecular weight 5000-15000 is known to
intimately mix in lipid cubic phases, even if in some cases the protein
causes phase transition.
Only few ternary lipid-protein-water phase diagrams have been
completely determined, but the role of protein on the phase transition
remains unclear. This is an interesting point, because cubic bicontinuous
lipid mesophases have been recently proved to be useful media growing
crystal of membrane proteins (in-cubo crystallization) [2] or have been
indicated as possible drug delivery systems [3].
Considering in-cubo crystallization, the mechanism promoting protein
crystallization is not known: apparently, the presence of the protein
affects the cubic structure and induces the coexistence of cubic and
lamellar phases. On the other side, aqueous dispersions of cubic lipid
phases led to the development of new nanoparticulate delivery systems,
the so-called ‘cubosomes’, characterized by high biocompatibility and
III
Introduction
bioadhesivity. Because their properties, these versatile delivery systems
can be administered percutaneously.
In both cases, structure destabilization may render the lipidic phases
unsuitable to act as crystal growing systems or as stable delivery system:
therefore, the evaluation of the effects that proteins have on the phase
behaviour of lipid systems has a significant impact on the application of
lipidic systems.
We were in particular attracted by the monoolein-cytochrome C-water
ternary system, that was studied some time ago. In this system, a cubicto-cubic phase transition occurs when the monoolein is left to
equilibrium for several days in excess of a cytochrome C solution: this
protein has the unique effect to induce the transformation from the Pn3m
to Im3m cubic phase.
This cubic phase is also bicontinuos, inverse (type II), and is based on
the P-surface belonging to IPMS. Noticeable is that the structural
characteristics of the Im3m phase are not completely defined, and also
its bicontinuity has been the subject of debate. The monoolein forms the
Pn3m cubic phase in excess water, while it forms the Im3m cubic phase
in excess of cytochrome C solutions.
Cytochrome c, or cyt c is a small, water-soluble heme protein associated
with the inner membrane of the mitochondrion. It is an essential link in
IV
Introduction
the electron transport chain through which cells perform the controlled
"burning" of glucose and capture much of that released energy by storing
it in ATP, the cell's primary energy distribution molecule. Each
cytochrome c carries one electron between two different electron
transport complexes embedded in the inner membrane. In doing this,
cytochrome c repetitively undergoes either oxidation or reduction, but it
does not bind oxygen. Cytochrome c has been particularly throughly
studied because its small size (about 100 amino acids) and its water
solubility permit researchers to isolate it from other mitochondrial
proteins, which tend to be not only larger than cytochrome c but also fat
soluble and embedded in the membrane. These factors combined have
led researchers to determine the amino acid sequences for the
cytochrome c occurring in many organisms from yeast to humans.
In this thesis the result of a structural investigation of the monooleincytochrome C- water system under variation of concentration of protein,
temperature and pressure is discussed. In particular, We take advantage
of the structural properties of monoolein and cytochrome-c to
extensively analyze the temperature, pressure and protein concentration
effects on the cubic transition from Pn3m to Im3m by means of smallangle X-ray diffraction techniques (SAXD) and absorption spectroscopy
(EAS).
V
Introduction
Experiments were performed in our laboratory, by using a standard Xray generator, at the Physics Institute of the University of São Paulo
(Brazil), by using a NanoStar X-ray generator, and at different
synchrotron beam-lines as ID02 and ID21 BL at ESRF (Grenoble), A2 at
DESY (Hamburg) and SAXS at LNLS (Campinas).
To do so, we made samples composed of monoolein (50 mg/ml) in the
presence of 1,5, 10,25,50 and 100 mg/ml of cytochrome-c.
VI
CHAPTER 1
Lipids
CHAPTER 1
LIPIDS
-1-
CHAPTER 1
Lipids
1.1 Significance of liquid crystals
Although liquid crystals were discovered as an interesting phenomenon
at the end of the 19th century, it took nearly 30 years of research to
establish their true identity [4].
Georges Friedel in the 1920's recognized that liquid crystals are indeed a
new state of matter that is intermediate in structure and molecular
organisation between the amorphous liquid state and the solid crystalline
state [4].
The discovery of liquid crystals coincided with a period of rapid
expansion in terms of the physical techniques available to study
materials. This, with great advances in the theory of condensed matter,
made liquid crystals an exciting area for scientific research.
Liquid crystals are partially ordered systems without a rigid, long-range
structure [4]. They are intermediate in symmetry and structure between
the solid crystalline state and the amorphous liquid state. These
substances do not pass directly from liquid to solid, but under certain
conditions are able to organize into intermediate phases (mesophases)
which have characteristics of both the liquid crystalline and solid.
Actually, such duality justifies the name of these compounds: liquid
crystals.
-2-
CHAPTER 1
Lipids
These mesophases are actually different conformations that the
molecules can assume: from crystalline solid state to the random
arrangement of the liquid state.
Among the different classes of liquid crystals, the liotropic one is the
most studied, due to its biological interest. The molecules aggregate into
mesophases when dissolved in an appropriate solvent. One can observe
different liotropic mesophases at different concentrations, and there is a
critical value below which the solution is isotropic.
The degree of organization of these molecules increases with the
concentration, micellar aggregation, for instance, being classified
according to the aggregate geometry, as:
* Spherical
* Columnar
* Cubic
* Lamellar
1.2. Lipids
Lipids are a class of molecules that display a wide diversity in structure
and biological function. A primary role of lipids in cellular function is its
ability to form the membrane of cells and organelles [5], acting as a
barrier too [5].
-3-
CHAPTER 1
Lipids
Fig.1.1: Example of lipid
They are amphiphilic molecules and possess two distinct parts (polar as
well as nonpolar) with rather different properties in the same molecule.
The hydrophilic (polar) head attracts water, while the lipophilic tail
(nonpolar) avoids water (Fig.1.1).
The properties of lipids to form liquid-crystalline phases are the basis of
the structure of cell membranes.
Lipid polymorphism appears to offer a more acceptable framework to
characterize the physical properties of lipids and their functional roles in
biological membranes.
In this chapter we describe the type of liquid-crystalline phases
(mesophases) adopted by lipid in water (Fig.1.2) [6].
-4-
CHAPTER 1
Lipids
Fig.1.2: Lipid shape and supramolecular
1.3 Polimorphism
An essential property of lipid molecules in aqueous solution is their
ability to arrange into compartments (structure elements) and to selforganize in stable structures which combine long range order among the
structure elements with disorder at molecular distances (lyotropic
phases). A variety of different phases can exist as a function of
concentration and temperature for a particular lipid (this property is
called polimorphism), and a small change in sample condition is
sufficient to cause a transformation from one form or structure to another
one [4].
-5-
CHAPTER 1
Lipids
Fig.1.3: Principal Lyotropic Mesophases
These different mesophases result from an optimization of the
hydrophobic effect with a variety of intra- and intermolecular
interactions, in combination with a number of geometry packing
constrains. In some structures, a topological distinction between the
inner and the outer parts of the structure elements could be made.
Therefore, two chemically distinct topologies for the molecular
distribution should be considered: in the type I (direct phase. Oil-inwater), the structural elements are filled by the paraffinic chains and are
embedded in the water matrix; in the type II (inverse phase, water-in-
-6-
CHAPTER 1
Lipids
oil), the structural elements are filled by water and are embedded in a
paraffin medium.
In both cases, the lipid polar headgroups lie on the polar/apolar interface.
It should be observed that other molecules of biological interest, like
DNA and derivates, show a lyotropic polymorphism characterized by the
presence of the type I phase.
In the lipidic system at least three different lyotropic mesophases occur:
all these phases display one-parameter structured and belong to onedimensional (1-D) lamellar (L). two-dimensional (2-D) hexagonal (H)
and
three-dimensional
(3-D)cubic
(Q)
crystallographic
systems.
Moreover, all the observed phases are characterized by a disordered
short-range organization of the hydrocarbon chains (the lipids show the
so-called alpha conformation). The different phases are here described.
11
1.3.1 Lamellar phase
The fluid lamellar L alpha phase is the simplest and largely studied
lyotropic mesophase, being the structural model for any biological
membrane. In this structure, the lipid molecules are associated in
lamellae, each lamella is filled by paraffin chains and covered on both
sides by the hydrophilic lipid group.[4,7] The lipid layers are separated
by water (Fig.1.4).
-7-
CHAPTER 1
Lipids
Fig.1.4: Lamellar phase
The most commonly observed structure is the fluid lamellar phase which
consists of a periodic stacking of lipid bilayers separated by water
channels. It is this phase that forms the basic building block of all
biological membranes.
More complex structures such as the hexagonal, micellar cubic and
bicontinous cubic phases with order in more than one dimension can also
be formed under suitable conditions.
1.3.2 Hexagonal phase
In the hexagonal phase, the structure elements are rigid rods, infinitely
long, all identical, crystallographic equivalent and packed in a 2-D
hexagonal lattice. It is obvious that two different topologies are possible,
and then H I and H II phases have been described (Fig.1.5) [4,7].
-8-
CHAPTER 1
Lipids
Fig.1.5: Hexagonal phase
In the HI phase, the cylinders are filled with the hydrocarbon chains and
the lipids expose the polar group to the water. In the HII phase, which is
the inverse of HI, the cylinders are filled with the water and are
dispersed in a continuous medium constituted by the hydrocarbon
chains; the polar group is located at the water/hydrocarbon chain
interface. It is interesting to note that it is possible to distinguish the two
topologies directly from the diffraction patterns. A variety of methods
may be used to obtain this information. The simplest one is based on the
analysis of the area-per-lipid at the lipid/water interface during a
swelling experiments: the area-per-molecule at the polar/apolar interface
is expected to increase (or at least not to decrease) as temperature and
water content increase [7,8]. Only when the good topology is assumed
during
calculations,
the
expected
behaviour
will
be
detected
experimentally. Moreover, as a general rule, upon water dilution a type I
-9-
CHAPTER 1
Lipids
phase will eventually transform to a micellar solution, whereas type II
phase are frequently stable in the presence of a large excess water phase.
1.3.3 Cubic phase
Nowadays, at least six different lyotropic phases with 3-D long-range
ordered structure have been identified and characterized: in all cases they
belong to the cubic symmetry and are called cubic phases. Their
identification is based on the analysis of the spacing ration of the
reflections observed in the X-ray low-angle diffraction profile: the
extinction symbol defines the aspect of the phases and then the
crystalline lattice and the symmetry of the structure (Fig.1.6) [7,8].
Fig.1.6: Cubic phases
- 10 -
CHAPTER 1
Lipids
According to the presence of the continuous polar and hydrocarbon
media, cubic phases have been separated in two different families:
bicontinuous, in which both polar hydrocarbon regions are continuous,
and micellar, in which only one of the two media is continuous. It is
evident that also in such phases two different topologies are possible:
noticeable is the fact that bicontinuous cubic phases of type II can be
considered as a topological generalization of the biological membrane
(two bicontinuous water media separated by a continuous hydrocarbon
septum).
The structure can be described as composed of two crystallographic
equivalent 3-D labyrinths. Adopting the skeletal graph representation,
the labyrinths lie on a pair of 3-D network of rods, having equal length
intertwined and unconnected the surface of the rods sit at the
polar/apolar interface. The three bicontinuous cubic phases belong to the
crystallographic space group Pn3m (Q224), Im3m (Q229), Ia3d (Q230)
[8,9].
The inverse bicontinuous cubic phases are particularly interesting,
consisting of bilayers draped over gyroid (G), double diamond (D) and
primitive (P) periodic minimal surfaces [8].
The phase Ia3d (Q230) is the first lipidic cubic phase whose structure
was determined: the two 3-D networks are formed by rods joined in the
- 11 -
CHAPTER 1
Lipids
same plane: 3 by 3. In some lipid systems (soap, detergent), the topology
of the chain and the interstices between the rods are filled by water,
while in other lipid systems (e.g. monoglycerides) the relative
distribution of the hydrocarbons and water is the reverse and the
topology is of type II.
The phase Pn3m (Q224) shows a structure very similar to the Ia3d, being
described in terms of two 3-D networks of rods, each one has a diamond
symmetry; in that structure, the rods are joined tetrahedrally 4 by 4. In all
cases reported, the structure Pn3m phase is a type II (water-in-oil)
(Fig.1.7) [7].
The last bicontinuous cubic phase is the Im3m (Q229): in this case the
two 3-D networks are formed by rods orthogonally connected 6 by 6
(Fig.1.8). Also this phase has been observed to exist in both type I and II
topologies. Concerning the micellar cubic phase, they are formed by
closed structure elements embedded in a matrix of inverted polarity.
The Pm3n (Q224) is one of these, and consist of disjoined micelles (type
I) embedded in a continuous water matrix. The micelles belong to two
different classes: those of one class are quasi spherical in shape while
those of the other class are disc-shaped.
- 12 -
CHAPTER 1
Lipids
Fig.1.7:Structure of cubic phase Pn3m (Q224)
Fig.1.8: Structure of cubic phase Im3m (Q229).
It should be observed that the fluid lyotropic phases that will be deal in
this thesis have a relevance to the structure and function of
biomembrane. The present work has been performed on pure, welldefined synthetic lipids, although natural lipids extracted from
membranes often exhibit the same phase structures [7].
Q212 is the phase discovered in the system composed of Monoolein
(MO), Cythocrome (Cyt) and water, one of the topics of this thesis. This
symmetry, however, was not observed previously in lipid-containing
systems. The extinction symbol unambiguously specifies space group
212. This space group is non-centrosymmetric; Q3 is in fact the first
- 13 -
CHAPTER 1
Lipids
unequivocal example of lipid-containing phase, with chains in the
disordered (alpha) conformation, whose structure is chiral.
By analogy with the phases Q224 and Q230 of the system MO-water, it
may be presumed that the rods are filled by the polar moiety, coated by
the polar headgroup of the lipid molecules and embedded in the
hydrocarbon matrix, and that each of the quasi-spherical globules of
Q212 contains one hydrated protein molecule, surrounded by lipid
molecules whose polar headgroup are oriented towards the protein.
The ability of encapsulation of hydrophilic, hydrophobic and
amphiphilic additives, together with the capability to protect and slowly
release the entrapped molecule make lipid mesophases, and in particular
cubic phases, potential candidates for drug delivery systems [7].
1.4. Interfacial Curvature
In order to describe and characterize the various lyotropic phases, it is
important to focus our attention on the interface between the polar and
non-polar regions of the phases, corresponding to the plane at which the
interfacial tension acts within a monolayer.
Near to this interfacial plane should lie the so-called neutral or pivotal
surface. From a geometrical point of view, each curved interface is
characterized by its mean and Gaussian curvature, H and K. These are
- 14 -
CHAPTER 1
Lipids
related to the principal curvatures c1 and c2 at a given point on the
surface by:
H = c1+c2/2
K = c1*c2
Different phases have different values of mean and/or Gaussian
interfacial curvatures and these may or may not be uniform at different
points on the interface within a single phase. For a lipid monolayer, the
convention is that H > 0 when the layer curves towards the hydrocarbon
chains, and H < 0 when the layer curves towards the water region.
The mean curvature H of a monolayer can be changed simply by
bending, without stretching the interface. However, changing the
Gaussian curvature K necessarily involves stretching or contracting the
interface. Both of these types of determination involve an associated
curvature elastic energy costs. It should be observed that the Gaussian
curvature K is a more fundamental property of the interface than H since
it determines the qualitative nature of the surface. Surface for which K is
positive are known as elliptic, and bend round to form closed shells. A
micelle, or an inversed micelle, are examples of this elliptic surface
(Fig.1.9).
- 15 -
CHAPTER 1
Lipids
Fig.1.9: Increasing negative curvature
When the principal curvature is zero, the Gaussian curvature is zero and
the surface is known as parabolic. A lamellar or a hexagonal phases are
examples of parabolic surfaces. The third possibility arises when the
principal curvature c1 and c2 are of opposite sign, leading to a negative
Gaussian curvature: these surfaces are known as hyperbolic and an
example is the saddle surface.
The Gaussian curvature is more negative at the saddle point and increase
smoothly to zero at four apices. When the principal curvatures are
everywhere equal in magnitude but opposite sign, then the surface has
zero mean curvature at all points and is known as a minimal surface.
Such surface can be extended to fill space, forming an infinite periodic
array of minimal surfaces, the Infinite Periodic Minimal Surface, IPMS,
which form a single septum, dividing space into two congruent subvolumes. Noticeable, bicontinuous cubic phases are based in such
underlying minimal surface [9,13-18]. Several IPMS were discovered by
- 16 -
CHAPTER 1
Lipids
Scwarz in the 19 century: these include the primitive cubic P surface and
the tetragonal D surface.
For the first time, the extraordinary complexity of these structures was
revealed [19].
More recently, Schoen discovered 13 more IPMS by using a
computerized numerical search. This thesis will be concerned with
Schwarz’s P and D surface (which are related to the Im3m and Pn3m
cubic structure, respectively) and with Schoen’s Gyroid (G), which is
related to the Ia3d cubic phase.
Larsson and co-workers [20] proposed that in bicontinuous phases the
lipid monolayers are draped on either side of the minimal surface, with
the terminal part of the lipid hydrocarbon chains lying on the surface.
1.6 Curvature and Frustration
With the exception of the lamellar phase, all the showed structures are
characterized by curved interfaces. In order to understand how such
phases form and how they are stabilized, we will need to explain the
causes of this interfacial curvature.
The factor responsible for controlling phase stability is the lateral stress
which occurs around the headgroup region, the polar/apolar interface ,
and the hydrocarbon region (Fig.1.10-11-12).
- 17 -
CHAPTER 1
Lipids
Fig.1.10: (a) The fluid lamellar La phase. (b) Curvature frustration in a fluid bilayer upon
heating.
Fig.1.11: (a) The inverse hexagonal HII phase. (b) Packing frustration in the inverse
hexagonal phase where a is the lattice parameter; rw, the water core radius; and
Lmax and Lmin, the maximum and minimum chain extension, respectively.
- 18 -
CHAPTER 1
Lipids
Fig.1.12 :The underlying minimal surfaces and skeletal graphs (centres of water channels)
for the inverse bicontinuous cubic phases. (a) Pn3m (D), (b) Ia3d (G) and (c) Im3m
(P).
The repulsive lateral pressure (Fc) in the chain region is due to thermally
activated cis-trans rotations in the C-C bonds [21]. The hydrophobic
effect results in an interfacial tension (F gamma) which tends to
minimize the interfacial area, arise from unfavourable hydrocarbonwater contact. The lateral stress around the headgroup region (Fh) arises
from steric, hydration and electrostatic interactions, all of them are
usually repulsive. The various interactions occur at different depths
within the monolayer and then may lead to a tendency for bending.
When the lateral stresses in the headgroup region outweigh that in the
chain region, the monolayer will bend towards the chain region. For the
opposite case, when the repulsive lateral chain pressure is larger than
lateral pressure in the headgroup region, the curvature will be towards
the aqueous region.
- 19 -
CHAPTER 1
Lipids
For a bilayer, which consists of two oppositely oriented monolayers
placed back-to-back, any tendency for the spontaneous curvature is
frustrated. In a planar bilayer, the cross-sectional area per molecule is
constant at all depths across the bilayer, both at the lipids/water interface
and at the surface where the terminal methyl group are located.
However, as parameters such as temperature or water concentration
changes, the optimal area of the two regions will tend to become
different. The individual monolayer would like to become curved to
optimise both the headgroup and the chain packing. To remain planar,
the cross-sectional area-per-molecule remains equal at all depths: thus, at
one side the area is reduced while at the other is increased away from the
optimal values. This state of compromise is called frustration, and means
that the system is internally stressed [9,20,22-23], the planar
configuration arising from a balance between two opposing stresses. The
build up the frustration is energetically unfavourable and the system
must find some way to reduce..
Sadoc and Charvolin have identified four different topological ways of
relieving this stress:
1. When the desire for a curved interface is not too great, the bilayer will
remain flat. This solution leaved the topology unchanged, but it costs
stretching elastic energy; the stretching energy per unit area, gA, is
- 20 -
CHAPTER 1
Lipids
gA = 1/2Ka (A/A0 -1)2
where: A is the actual molecular area, A0 is optimum area per molecule,
Ka is the isothermal lateral compression modulus. The lipid bilayers are
quite stiff to stretch and beyond this limit the material has no choice
other than to undergo a phase transition where the stored curvature
energy can be relaxed.
2. A different choice is to form a single continuous bilayer of negative
Gaussian curvature. This corresponds to the porous structure of the
inverse bicontinuous cubics.
3. A further choice is to form an infinite number of infinite disconnected
aggregates. This corresponds to the cylindrical aggregates of the HII
phase.
4. Finally, when the desire for interfacial curvature is at its strongest, the
system forms an infinite number of finite disconnected aggregates. This
corresponds to inverse micellar solutions, eventually packed on a
specific cubic lattice.
The explanation of the forces which drive the lamellar phase towards
phases with structures with curved interfaces is only qualitative. It is
necessary to derive an expression which relates the described lateral
interaction with the local geometry.
- 21 -
CHAPTER 1
Lipids
In other words, it is necessary an expression for the curvature free energy
which can be related to the local molecular interactions. The curvature
free energy has been expressed in a number ways by different authors.
1.5 Lyotropic phase diagram
Water content and temperature are the primary system variables for
binary lipid/water systems. A theoretical binary lipid/water diagram, in
which transitions are driven mainly by the former, is shown in Fig.1.13.
Fig.1.13: Lyotropic phase diagram
There is a natural sequence in which the various possible fluid phases
occur, determined by average mean curvature of polar/non polar
interface. In the central position there is the lamellar L alpha, which an a
flat interface (H =0, K= 0). On either side of it, the interfacial mean
curvature of successive phases is increasing in magnitude.
- 22 -
CHAPTER 1
Lipids
To the left, the interfacial curvature is towards the water, and as above
described the mean curvature is negative. The following phases are
known as type II, or inverse phases. Such phases are commonly formed
by double-chain amphiphiles such as phospholipids. Conversely, to the
right of the lamellar phase, the curvature is towards the hydrophobic
region, the mean curvature is positive and the occurring phases are
known as type I, or normal phases. Such phases are typically formed by
single-chain amphiphiles. The regions marked a, b, c and d contain
intermediate phases, the majority of which are cubic: those occurring in
regions c and d are of type I, whereas those forming in regions a and b
are inverse, type II. This thesis will be concerned an inverse phases
(monoolein –water system).
1.4 Lipids-protein interaction
The cubic phases are also unique in their ability to accommodate
proteins as compared with other lipid-water phases. [23] A wide range of
globular proteins with molecular weights 5000-15000 Dalton is known
to intimately mix in lipidic cubic phases. In some cases, the protein can
even cause a phase transition in the lipid. Only few ternary lipid-proteinwater phase diagrams have been completely determined, but the role of
the protein on the phase transition is still unclear. This is an interesting
- 23 -
CHAPTER 1
Lipids
point, because cubic bicontinuous lipid mesophases have been proved to
be an useful media for growing crystals membrane proteins [7,9].
The mechanism by which cubic phases promote crystallization is not
known: apparently, the presence of proteins affect the cubic structure and
induces the coexistence of cubic and lamellar phases, which seem crucial
for protein crystallization. However, the destabilization may render the
lipid phases unsuitable to act as crystal growing systems: therefore, the
evaluation of the effects that proteins have on the phase behaviour of the
cubic phases has a significant impact on the application of the in cubo
protein crystallization method.
The interaction of proteins with lipids, with special regard to the
formation of aqueous liquid crystalline phases, has been studied in
various systems.
As reported below proteins with a wide variation in size (about 14-150
kDa) are able to form a cubic phase with lipids, one of the best known
examples is the interaction between monoolein and various proteins.[10]
Little is known from earlier studies about the protein structure in such
lipid-protein liquid-crystalline phases. In a Raman spectroscopy study
[20] of insulin-phospholipid-water phases, it was reported that the
protein kept its native structure provided the lipid chains were below a
certain length.
- 24 -
CHAPTER 1
Lipids
When it was realized that the cubic lipid-water phase consists of open
water-channel systems of reasonable dimensions separated by the
infinite lipid bilayer, it was natural to examine whether this phase could
accommodate protein.
Ericsson [20] et al have reported that lysozyme and other globular
proteins with a considerable variation in size are able to form cubic
phases with monoolein.
Monoolein-based cubic phases containing casein and gliadin have also
been described. Luzzati and co-workers have reported a detailed X-ray
diffraction study of the cubic monoolein-cytochrome c (cyt c)-water [7].
A remarkable feature in the monoolein-protein-water systems was the
observation that large amounts of protein in its native conformation can
be incorporated to form the cubic phases without an ionic interaction
with the lipid bilayer.
Razumas and co-workers have shown that the enzymes with molecular
weights of up to 59 KDa can be entrapped and stabilized in the
monoolein cubic phases [12].
They have chosen, as a model protein, cyt c. Considering the role of cyt
c in the mitochondrial oxidative phosphorylation and the self-assembly
of the lipid in a curved bilayer within the cubic phase, this system can
also serve as model of the inner mitochondrial membrane.
- 25 -
CHAPTER 2
Application of Lipid Systems
CHAPTER 2
APPLICATION OF LIPID SYSTEMS
- 27 -
CHAPTER 2
Application of Lipid Systems
Within the range of self-assembled phases surfactant-like lipid systems,
the monoglyceride-based lyotropic liquid crystalline phases are relatively
unique owing to their rich polymorphism in water [12] and the potential
application as drug nanocarriers. Various studies have focused on
understanding their self-assembling behaviour [12] studying the effects
of loading hydrophilic or hydrophobic guest molecules and exploring the
impact of varying temperature [25] or pressure [26].
Among these monoglycerides, monoolein (MO) and monolinolein
(MLO) are well studied. They self-assemble in water to form various
well-ordered inverted type nanostructures: a fluid isotropic micellar
solution (L2), H2 (Hexagonal phase), and V2 ( cubic phases) [27]. In
addition, much effort has been devoted in the past two decades to various
nanostructured aqueous dispersions with these model lipids such as
cubosomes, hexosomes, and micellar cubosomes by confining with
suitable stabilizers the corresponding fully hydrated crystalline phases in
kinetically particles.
2.1. New application of lipid systems
In recent developments surfactant and colloidal science has become the
basis for bioscience and nanotechnology .The knowledge of surfactant
self-assemble and the awareness of the interplay of hydrophobic–
hydrophilic intermolecular interaction play a crucial role in the
- 28 -
CHAPTER 2
Application of Lipid Systems
projection of new systems for highly specific applications. In particular,
making hierarchically ordered materials represents an important
challenge to engineer intelligent biomaterials for bio-nanotechnological
applications such as biosensors and drug delivery [28]. Focusing our
attention on drug delivery systems, the advances in drug discovery have
been huge and would have been unpredictable 20 years ago.
However, the high specificity, in terms of efficacy, obtained in drug
production has not been accompanied by a specific targeting of the
delivery systems: substantially efficacious drugs are available,
pharmacologists know suitable drugs to attack the disease but the drug
cannot be often delivered to the most suitable receptors.
The acceleration in the discovery of new therapies based on chemical,
biological, genetic and radiological moieties has brought an increasing
demand for delivery systems able for protecting, transporting and
selectively releasing the therapeutic agents to the desired receptor site
[26].
Surface properties and interfacial interactions with the biological
environments are crucial to determine the bio-adhesion and then the
release performance of the drug [29,30]. Various steps and parameters
involved in the drug delivery should be controlled: first the
bioavailability (that is, the amount of therapeutic agent really available
- 29 -
CHAPTER 2
Application of Lipid Systems
for the therapeutic action), the time dependent bio-distribution at the
specific receptor sites, then the pharmacokinetic and pharmacodynamic
parameters, which affect the therapeutic effectiveness, and finally the
circulation lifetime and the immune response from phagocytic cells
[30,31]. These steps and parameters are controlled by intermolecular
interactions due to surface charges, steric stabilization, phase behaviour,
particle size, and hydrophilic–hydrophobic surface coating (Fig.2.1).
Fig.2.1: Nanoparticles used for the drug delivery
Most of the innovative drug delivery formulations are obtained
exploiting nanoscience and nanotechnology advances. The biologically
active nanoparticles may be of different types depending on the target.
They
include
microsphere
hydrogels
(0.5–20
μm)
based
on
polysaccharides, emulsions and microemulsions, liposomes, micelles,
lipid nanoparticles such as cubosomes and hexosomes [29-30].
- 30 -
CHAPTER 2
Application of Lipid Systems
2.2. Drug delivery
In order to be used as a drug delivery system, cubic phase has to be able
to dissolve or disperse drugs of various polarities, from low to
moderately high concentrations to accommodate higher doses. In this
respect, cubic phase reveals a great flexibility, since drugs of very
different polarity and size may be incorporated. Typically hydrophilic
drugs can be dissolved in water and this aqueous drug solution can be
used to form the cubic phase [31].
Lipids have been used extensively for drug delivery in various forms
such as liposomes (Fig.2.2), and solid-matrices. The focus of this review
is evaluation of liquid crystalline cubic phases, spontaneously formed
when amphiphilic lipids are placed in aqueous environment, for drug
delivery. Cubic phases have an interesting thermodynamically stable
structure consisting of curved bicontinuous lipid bilayers in three
dimensions, separating two congruent networks of water channels [32].
The unique structure of cubic phase has been extensively studied using
various spectroscopic techniques and their resemblance to biomembranes
has prompted many scientists to study behaviour of proteins in cubic
phases. The ability of cubic phase to incorporate and control release of
drugs of varying size and polar characteristics, and biodegradability of
lipids make it an interesting drug delivery system for various routes of
- 31 -
CHAPTER 2
Application of Lipid Systems
administration. Cubic phases have been shown to deliver small drug
molecules and large proteins by oral and parenteral routes in addition to
local delivery in vaginal and periodontal cavity [29-31]. A number of
different proteins in cubic phase appear to retain their native
conformation and bioactivity, and are protected from chemical and
physical inactivation perhaps due to the reduced activity of water and
biomembrane-like structure of cubic phase.
Bicontinuous cubic phase liquid crystals are newly discovered exotic
materials originally found in the most unassuming places. The original
observations of cubic liquid crystalline phase came during the study of
polar lipids, such as monoolein that are used as food emulsifiers.
Bicontinuous cubic liquid crystalline materials are an active research
topic because their unique structure lends itself well to controlled release
applications. Amphiphilic molecules form bicontinuous water and oil
channels, where “bicontinuous” refers to two distinct (continuous, but
non-intersecting) hydrophilic regions separated by the bilayer.
Incorporation of drug in cubic phase can cause phase transformation to
lamellar or reversed hexagonal phase depending on the polarity and
concentration of the drug, which may affect the release profile.
- 32 -
CHAPTER 2
Application of Lipid Systems
Relatively hydrophobic lipids in the solid state have been used primarily
as matrix material as carriers of hydrophilic drugs to provide sustained
release orally and as a drug delivery carrier in solid implants.
Liposome is one kind of lipid organization in a closed circular lipid
bilayer enclosing an aqueous phase, and they have been extensively
studied for drug, protein and gene delivery. However, the spontaneous
reorganization of amphiphilic lipids in aqueous environment can result in
other three-dimensional structures such as the lamellar phase, the cubic
phase, and transferosomes, which can be used for, drug delivery. The
structure of cubic phase has generated a lot of interest and is yet another
exciting lipid-based system beginning to be explored for drug, protein
and vaccine delivery [29-32].
The therapeutic potential of peptide and protein drugs, as well as their
clinical application, are often hampered by a number of obstacles to their
successful delivery [24,33-35].
Protein stability is the balance resulted between destabilizing and
stabilizing forces. The formation and stability of the secondary, tertiary
and quaternary structures of proteins are based on weak non-covalent
interactions (e.g. electrostatic interactions, hydrogen bonding, van der
Waals forces and hydrophobic interactions). Disruption of any of these
interactions will shift this delicate balance and destabilize the proteins
- 33 -
CHAPTER 2
Application of Lipid Systems
[24, 34-35]. Therefore, the chemical and physical stability of proteins
can be compromised by environmental factors such as pH, ionic
strength, temperature, high pressure, non-aqueous solvents, metal ions,
detergents, adsorption, and agitation and shearing. Most of these factors
are present in common manufacturing processes, including sterilisation
and lyophilisation, which may damage the proteins, reducing their
biological activity, inducing aggregation and render the proteins
immunogenic, leading ultimately to precipitation [36,37].
Regardless the administration route many therapeutic proteins do not
possess the required physicochemical properties to be absorbed, and
reach or enter target cells, needing delivery and targeting systems that
aim to overcome these limitations, and improve drug performance. In
order to fulfil this requirement, particulate carriers such as liposomes,
microspheres, micelles and nanoparticles, etc., are currently under
development.
- 34 -
CHAPTER 2
Application of Lipid Systems
Fig.2.2: Liposomes
One of the more important drug properties to consider is potency.
Additional properties such as stability, solubility, size (molecular
weight), and charge are also important. As a general rule, the fewer
molecules that can carry (i.e., the lower the drug: carrier ratio), then the
more potent the drug must be. For some types of drug delivery that can
carry only a few molecules of a drug (such as immunotoxins and
immunoconjugates) or a few tens of molecules (such as polymer
conjugates), drugs with higher potencies are needed in order to deliver
therapeutically relevant amounts of drug.
- 35 -
CHAPTER 2
Application of Lipid Systems
The use of unreasonably high quantities of the carrier can lead to
problems
of
carrier
toxicity,
metabolism
and
elimination,
or
biodegradability.
For example, if the free drug is already in clinical use, the advantages of
the drug delivery system compared to the free drug can be directly
evaluated in well established indications, potentially resulting in more
rapid clinical development.
In addition, although the DDS (Drug Delivery System) can result in new
toxicities compared to the free drug, the toxicity profiles of DDS are
usually similar to those of the free drug, the differences being in degree
rather than in kind. As a result, procedures used to treat the side effects
of the free drug can often be applied to the DDS. The mechanism of
action of a drug may also dictate its suitability for delivery in a particular
DDS.
The unique structure and physiochemical properties of liquid crystalline
cubic phase make it suitable as a drug delivery matrix. The ability to
incorporate and slowly release a variety of drugs with different
physicochemical properties by a variety of routes of administration has
been demonstrated [31]. The similarity of cubic phase to physiological
lipid membranes and its ability to incorporate and maintain protein in
their native bioactive conformation is a unique attribute, extremely
- 36 -
CHAPTER 2
Application of Lipid Systems
desirable for macromolecule drug delivery. However, it is not without
certain limitations and disadvantages. One of the major obstacles in the
direct administration of drug-incorporated cubic phase is its extremely
high viscosity (this is the particular case for the cubic phase). Another
complicating factor is the solubilization and/or incorporation of the drug
molecules in the hydrated bilayer of the amphiphilic monoglyceride, and
thus hydrophilic and lipophilic drugs can cause different phase
transformations. This could possible affect the release characteristics of
the drug and have a side effect on the physical stability of the matrix.
Although cubic phase offers tremendous potential in the field of the drug
delivery, it may be limited to specific applications such as periodontal,
mucosal, vaginal and short acting oral and parental drug delivery due to
some of the above-mentioned disadvantages.
2.3 Cubosomes
Cubosomes are bicontinuous cubic phase liquid crystals and have many
properties that make them appealing as a universal vehicle for drug
delivery (Fig.2.3). Luzzati et al. [38] first documented its geometric
model supplied later by Scriven [39]. The surfactant assembles into
bilayers that are twisted into a three dimension, periodic, minimal
surface forming tightly packed structure, like “honeycombed” with
bicontinuous domains of water and lipid [7,40].
- 37 -
CHAPTER 2
Application of Lipid Systems
Fig.2.3 : Cubosomes
Cubosome particles are first prepared by mechanical fragmentation of
the cubic lipid-water phase in a three-phase region containing a
liposomal dispersion and to differentiate from liposomes, these particles
have been termed as cubosomes.
Its structure is different from liposomes because its structure can
simultaneously
accommodate
water-soluble,
amphiphilic molecules.
- 38 -
lipid-soluble,
and
CHAPTER 2
Application of Lipid Systems
Fig.2.4: Types of cubosome
Three structure of cubosomes have been proposed by Luzzati et al [7].;
(i) Pn3m (D-surface) (Diamond surface), (ii) Ia3d (G-surface) (Gyroid
surface), and (iii) Im3m (P-surface) (Primitive surface), in terms of nodal
surfaces (Fig.2.4). The structure generally maintains the efficacy;
stability of actives such as vitamins and proteins. Cubosomes are
thermodynamically stable; lasting indefinitely. Colloidal dispersions of
cubosomes can be stabilized by the addition of polymers. They also
possess the potential for controlled delivery of actives, where diffusion is
governed by the tortuous diffusion of the active through the “regular”
channel structure of the cubic phase. Cubosomes possess a sufficient
average degree of molecular orientation order to characterize by
structural symmetry, and often form in aqueous surfactant system at
relatively high amphiphile concentrations [7,37,39].
- 39 -
CHAPTER 2
Application of Lipid Systems
Luzzati and Husson and Luzzati et al. first recognized the existence of
cubic phases in lipid-water system using X-ray scattering measurement
[7].
Three macroscopic forms of cubic phase are typically encountered;
precursor, bulk gel and particulate dispersion. The precursor form exists
as a solid or liquid material that forms cubic phase in response to a
stimulus, such as contact with liquid [7,37].
Bulk cubic phase gel is an optically isotropic, stiff, and solid like
material in equilibrium with water can be dispersed into particles called
cubosomes.
Bicontinuous cubic phases are found in natural lipids, cationic and nonionic surfactants, and polymer systems, although the lipid most widely
used to construct bicontinuous cubic phases is the monoglyceride
monoolein, monoglycerides spontaneously form bicontinuous cubic
phases upon the addition of water, are relatively insoluble (allowing the
formation of colloidal dispersions of cubosomes), and are resistant to
changes in temperature [40].
Cubic phase of cubosomes is attractive for controlled release because of
its small pore size (5-10 nm); its ability to solubilize hydrophobic,
hydrophilic, and amphiphilic molecules; and its biodegradability by
- 40 -
CHAPTER 2
Application of Lipid Systems
simple enzyme action. Cubic phase is strongly bioadhesive and is
thought to be a skin penetration enhancer with excellent
compatibility with topical and mucosal deposition and delivery of active
ingredients [41].
Cubosomes prepared in dispersions possess a nanometer scale structure
identical to bulk cubic phase, but the dispersion itself has much lower,
water like viscosity. Compared to liposomes or vesicles, cubosomes
possess much higher bilayer area-to-particle volume ratios as well as
higher viscous resistance to rupture. Although bulk cubic phase has
sufficient length scale to allow controlled release of solutes, cubosomes
are too small and have too high a surface area for such performance,
exhibiting instead burst release [42].
2.4 Solid Lipid Nanoparticles (SLN)
The first generation of solid lipid carrier systems in nanometer range,
Solid Lipid Nanoparticles (SLN), was introduced as an alternative to
liposomes. SLN are aqueous colloidal dispersions, the matrix of which
comprises of solid biodegradable lipids. SLN are manufactured by
techniques like high pressure homogenization, solvent diffusion method
etc. They exhibit major advantages such as modulated release, improved
bioavailability, protection of chemically labile molecules like retinol,
- 41 -
CHAPTER 2
Application of Lipid Systems
peptides from degradation, cost effective excipients, improved drug
incorporation and wide application spectrum.
Fig.2.5: Evolution of Nanoparticules
However there are certain limitations associated with SLN, like limited
drug loading capacity and drug expulsion during storage, which can be
minimized by the next generation of solid lipids, Nanostructured lipid
carriers (NLC) (Fig.2.5-2.6) [43].
Fig. 2.6: Solid Lipid Nanoparticules
- 42 -
CHAPTER 2
Application of Lipid Systems
NLC are lipid particles with a controlled nanostructure that improves
drug loading and firmly incorporates the drug during storage. Owing to
their properties and advantages, SLN and NLC may find extensive
application in topical drug delivery, oral and parenteral administration of
cosmetic and pharmaceutical actives. Cosmeceuticals is emerging as the
biggest application target of these carriers. Carrier systems like SLN and
NLC were developed with a perspective to meet industrial needs like
scale up, qualification and validation, simple technology, low cost etc. In
this chapter is present status of SLN and NLC as carrier systems with
special emphasis on their application in Cosmeceuticals; it also gives an
overview about various manufacturing techniques of SLN and NLC
(Fig.2.7).
Fig.2.7: Differnce beetwen SLN and NLC
They are increasing in significance as alternative drug carriers to
polymeric nanoparticles. Controlled drug delivery, enhancement of
bioavailability of entrapped drugs via modification of dissolution rate
[44] and/or improvement of tissue distribution and targeting of drugs
[45] by using SLN have been reported in various application routes:
- 43 -
CHAPTER 2
Application of Lipid Systems
– Parenteral (intravenously, intramuscularly or subcutaneously) [46,47]
– Oral [48]
– Rectal [49]
– Opthalmic [50]
– Topical (in cosmetics and dermatological preparations) [51]
Indeed, nanoparticles were initially thought to be designed as carriers for
Vaccines and anticancer drugs when they were first developed in about
1970. In the strategy of drug targeting in order to enhance tumor uptake,
researchers focused on the development methods to reduce the uptake of
the nanoparticles by the cells of the reticuloendothelial system (RES) as
the first important step.
Several innovative reviews on solid lipid nanotechnology for drug
delivery are available in the literature which describes extensive
preparation techniques, characterization and types of SLN, investigation
of their structural properties, factors affecting their formation and storage
stability, drug loading principles and drug release characteristics.
SLN are produced by using several methods extensively described in the
literature:
– High pressure homogenization (cold and hot homogenization)–
Breaking of o/w microemulsion [52-57].
- 44 -
CHAPTER 2
Application of Lipid Systems
– Solvent emulsifi cation-evaporation [58,59] or solvent emulsifi cation–
diffusion
– Solvent injection [60]
– Preparation via water-in-oil-in-water double emulsion (w/o/w)
[59-61]
– High shear homogenization [62] and/or ultrasound dispersion [63]
– Preparation by using membrane contactor as a new reported
technique for SLN production [64].
Today, the high pressure homogenization technique has been
demonstrated to be the most effective technique due to some advantages
such as narrow particle size distribution of the product with a low
content of microparticles (> 5 μm is requested for injections), higher
particle content in the dispersions, avoidance of organic solvents,
acceptability of the homogenization equipment by the regulatory
authorities (even for parenteral products), scale-up feasibility and the
availability of homogenization lines in industrial. Depending on the size
of production-scale homogenizers, a wide production range can be
possible.
Factors affecting loading capacity of a drug in lipid are:
– solubility of drug in lipid melt,
– miscibility of drug melt and lipid melt,
- 45 -
CHAPTER 2
Application of Lipid Systems
– chemical and physical structure of solid matrix lipid,
– polymorphic state of lipid material.
In particular, there is an inverse relationship between solubility of the
drug and loading capacity. Enhancement in aqueous solubility of drug
leads lower to entrapment efficiency [65].
SLN are interesting for drug delivery for several reasons, they have a
high loading capacity for hydrophobic drugs, hydrolytic degradation is
limited, the drug release rate can be controlled by the particle size and
composition, and burst release is largely absent. The latter is particularly
interesting in applications involving toxic drugs and resulting sideeffects, e.g., cancer therapies, where high peak concentrations must be
avoided. Due to this, and the hydrophobic nature of several anticancer
drugs, SLN constitute an interesting formulation option for cancer
therapy. Furthermore, while the cytotoxicity of SLN in the absence of
incorporated drugs is very low, the cytotoxicity of SLN containing
incorporated anti-cancer agents has been found to be substantial. In fact,
for several drugs, SLN formulations have been found to be significantly
more efficient even than the free drug in solution, which suggests that
particle-mediated uptake plays a role [66].
- 46 -
CHAPTER 3
Description of the Monoolein-Cytochrome c-Water System
CHAPTER 3
DESCRIPTION OF THE MONOOLEINCYTOCHROME C- WATER SYSTEM
- 47 -
CHAPTER 3
Description of the Monoolein-Cytochrome c-Water System
3.1. History of monoolein-cytochrome c-water system
Monoglycerides are amphipatic neutral lipid molecules in which a
hydrophobic fatty acid is attached at the rac-1 position of a hydrophilic
glycerol backbone via an ester bond. Despite their relatively simple
chemical structure, monoglycerides (Fig.3.1) can form various phases
found in membrane phospholipid/water systems. The ability of
monoglycerides to form bilayer as well as nonbilayer structures offers
many interesting opportunities for studies of membrane lipid
organization.
Fig.3.1: Structure of Monoolein
One of the most commonly used of all monoacylglycerols is monoolein.
Monoolein (MO) is a lipid which forms a wide variety of self-assembly
structures when mixed with water [67] (Fig.3.2).
Upon increasing the water content the MO/W binary system shows a
small region of reverse micellar (L2) phase followed by a lamellar (Lα)
phase, and by a CG (Ia3d space group) and a CD (Pn3m space group)
bicontinuous cubic phase. The CG phase evolves towards a reverse
- 48 -
CHAPTER 3
Description of the Monoolein-Cytochrome c-Water System
hexagonal (H2) phase at high temperature, whereas the CD phase can
coexist with water excess.
Fig.3.2: Diagram Phase of monoolein-water
Since the extensive pioneering work of K. Larsson [68] in which the
monoolein (MO) phase behaviour in water (W) was clarified, and its
similarity to the physiological lipid membrane organization was found,
monoolein has received great interest for applications in the
pharmaceutical area.
The ability of encapsulation of hydrophilic, hydrophobic and
amphiphilic additives, together with the capability to protect and slowly
release the entrapped molecule make monoolein mesophases, and in
particular cubic phases, potential candidates for drug delivery systems.
- 49 -
CHAPTER 3
Description of the Monoolein-Cytochrome c-Water System
The binary system monoolein-water was also investigated for the effects
of changes of temperature [25].
The phase diagram represented in this work showed equilibrium
behaviour and that the assorted phase boundaries have been determined
accurately. The interpreted phase diagram is based on close to 400
discrete measurements in temperature-composition space recorded as a
function of temperature in 5 °C increments (3 °C in the HIT phase) and
of composition in 2% (w/w) water increments on average. The various
mesophases have been characterized structurally as a function of
temperature and hydration, and the corresponding thermal and
composition expansion coefficients are reported. These and related data
show that the average radius of water channels in the fully hydrated
bicontinuous cubic Pn3m phase is remarkably sensitive to temperature
and to monoacylglycerol chain identity.
More recently the phase stability
transitions are related to pressure
changes [69].
Hydrostatic pressure can be used to influence the structural properties
and then to obtain an extended description of the phase behaviour,
stability and energetics of cubic phases. Small-angle X-ray diffraction is
a powerful tool for elucidation of the symmetry as well as the topology
of these structures.
- 50 -
CHAPTER 3
Description of the Monoolein-Cytochrome c-Water System
The results show that the pressure induces a transition from the Ia3d
cubic to the lamellar Lα phase and then to the lamellar crystalline phase
for a 30% hydrated sample [69]. In the more hydrated samples, a cubicto-cubic phase transition (Pn3m to Ia3d) was observed.
The crystalline phase is the only one that survives above 3 kbar.
A number of studies has been conducted in which protein has been
incorporated into the cubic phases formed by mixture of lipids and water
[31].
In one of the first studies of protein in cubic phase amphiphlic protein Agliadin was incorporated into cubic phases formed by mixture of Mo and
water [70]. The authors were able to solubilise a substantial amount of
protein, about 10% (wt/wt), in the Mo cubic phases.
In another study a variety of soluble proteins ranging in size from 14-150
kDA were incorporated in MO- water cubic phases [20].
The protein: lysozyme (14 kDa), alpha-lactalbumin (14 kDa), myoglobin
(17 kDa), bovine serum albumine (67 kDa) and glucose oxidase (150
kDa) were studied in cubic phase composed in 40 % Mo, 18 % protein
and 42 % water (wt/wt) [20].
It was assumed that the proteins would be located in the aqueous channel
of the cubic phases and not in the lipid bilayers.
- 51 -
CHAPTER 3
Description of the Monoolein-Cytochrome c-Water System
It was found that MO cubic phases containing proteins reached
equilibrium within 24 hours and formed a viscous transparent optically
isotropic phase with the same characteristics as Mo cubic phases
prepared in absence of protein.
The ternary phase diagram determined Mo:protein:water is different to
the classic diagram of Mo-water system.
In the present work, we dedicate our efforts in better understand the
system composed of monoolein in excess of water in the absence and
presence of cytochrome-c (Fig.3.3).
Fig.3.3: Cytochrome c
The first work in which they are treated together is from Mariani and
Luzzati. They published a partial phase diagram of the Mo\cytochrome
c\water system. In this work the authors proposed the appearance of a
phase (Q229) that is not normally present in the diagram of monoolein in
water [9].
Subsequently Larson et al focused his work on a better understanding of
the interplay between the cubic lipid-aqueous phase and the protein
- 52 -
CHAPTER 3
Description of the Monoolein-Cytochrome c-Water System
entrapped within it [67,68]. As a model protein, we have chosen cyt c.
Considering the role of cyt c in the mitochondrial oxidative
phosphorylation and the self-assembly of the lipid in a curved bilayer
within the cubic phase, this system can also serve as a model of the inner
mitochondrial membrane.
In 2000 Caboi and co-workers observed a cubic-to-hexagonal phase
transition and the MO hydrolysis plays a crucial role in this transition
with the release of free oleic acid that favours the reverse interfacial
curvature [71].
Few years later (2003) Lendermann has approached this issue by
validating what has been said in previous years or that the presence of
cytochrome monoolein within the stages of producing a change in threedimensional structure of lipid valuation also, the impact of temperature
and pressure [72].
In 2005 Kraina et al elucidated the structure and equilibrium of the
temperature-pressure phase diagram of the system monoolein/cyt c at
limited hydration conditions as well as to study the kinetics and
mechanisms of the various lamellar and non-lamellar phase transitions of
the system. We mixed the small protein cyt c (rmax ) 17 Å with a
MO/water system exhibiting the cubic Ia3d (Q230) phase that forms
spontaneously at 20 wt % water [73].
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Description of the Monoolein-Cytochrome c-Water System
In this context my PhD work is undertaken. The first step was done to
evaluate the effect of different concentrations of cytochrome also using
different concentrations of monoolein to assess which conditions are best
to see a change in the structure of three-dimensional phase.
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MATERIALS AND METHODS
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Materials and Methods
4.1 Diffraction X-Ray Scattering
In this chapter we give an overview of all lipid mesophases which play a
role in nature and illustrate how X-ray scattering techniques contribute to
the determination of their structural as well as mechanical properties.
Research on liquid-paraffin water-containing phases with small angle Xray scattering goes as far back as to 1939, when Stauff published first
structural data on different soap types. However, it was not before the
end of the 1950s when Luzzati [7-9] and co-workers started to
investigate systematically the underlying structures, and later especially
classified the vast amount of different lipid-based lyotropic phases.
Lipid/water
systems
are
known
for
their
extraordinary
rich
polymorphism, which form liquid crystalline structures with 1-D, 2-D or
3-D periodicities. The biological impact of this structural diversity has
been widely discussed.
An integral part of the process of crystal structure determination is an
experiment and the techniques used to collect the experimental data. The
experiment consists of scattering radiation from lipids, the radiation that
are usually employed being X-ray, electron or neutron.
It is important to describe the geometric condition under which the
constructive interferences of radiation scattered from a triply periodic
arrangement of material unity takes place. These conditions, known as
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Materials and Methods
diffraction conditions, are the basis of any experiment in which
intensities of diffracted radiation are measured.
These measurements have many purposes but two are outstandingly
important:
1. to determine of periodicity, symmetry and orientation of a lipids
2. to obtain accurate estimates of the intensities of diffracted
radiation, in order to elucidate from them the atomic arrangement
within the asymmetric units or to determine its structure.
4.2 The Law and Bragg equation
Let us assume the above “crystal” of point charges is irradiated with
monochromatic X-ray. Since X-Ray electromagnetic radiation can be
described, at a large distance from the source, in term of plane waves,
with appropriate wavevector. The electric field of the incident X-Ray
wave varies with time and therefore accelerated the point charges it
encounters. Electromagnetic theory tells us that an accelerated charge
emits energy in the form of electromagnetic radiation, with the same
frequency as that of in the incident wave.
This may require some small correction for quantum effects, such as the
Compton effect, but this will be neglected in the present treatment.
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Materials and Methods
We can therefore say that the incident X-Ray are reemitted, with an
unchanged wavelength, the efficiency of reemission been determined by
scattering cross section of the electron for electromagnetic radiation.
We consider an X-Ray wave , with wavevector S0 ,falling on a crystal,
and a reemitted (or scattered ) X-Ray wave with vector S, In view of
assumption unchanged wavelength, the magnitude of wavevector S0 and
S will be indicated, and we shall take them as :
S = S0 = 1/λ
We assume further that the lattice of our crystal can be described in
terms of the basis (a b c). The question to be answered is : for what
geometrical relationship between the wavevector of the radiation and
the basis vectors of the lattice will maximum constructive interference of
the scattered X-Ray wave occur. Because of the assume strict periodicity
of the arrangement, it is sufficient to consider two points charges related
by translation through a lattice vector ru,v,w
It follow that the required condition is:
ru,v,w * h = integer (eq.4.1)
where h= S- S0 is called the diffraction vector. This can be also rewritten
as (ua + vb+ wc)*h = integer.
Since the coefficients u, v, w can be any integers and the eq 4.1 is
equivalent to the three equation:
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Materials and Methods
a*h = h
b*h = k
c*h = l
which have to be simultaneously satisfied for maximum interference to
occur; here h, k, l are any integers. This equation are knows as Laue
equations.
As seen above each lattice thus arises from the infinite space and regular
repetition of these identical structural units (called cells elementary unit
cells) transferred according to the three dimensions of space in
accordance with a number of symmetry operations. Because of this
smooth motion, a crystal may show the presence of regular lattice, called
atomic plans. The different set of plans respect the symmetry of the
crystal and are named according to the so-called Miller indices (h, k, l)
representing the coordinates in reciprocal space. Consider a crystal
formed by spatial repetition of a basic three-dimensional cells. The unit
cell is given when known its three dimensions a, b, c, along the three
axes x, y, z, while the crystal structure is known at the symmetry
operations are known that determine the spatial repetition of the regular
cell. The atomic planes of the crystal can trap axis passing along a, b, c,
or be parallel to them. The intercepts are at h, b / k, c / l, since h, k, l
Miller indices indicating how many floors of the series considered
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Materials and Methods
cutting boards in various sections of length a, b, c. A family of lattice is
then defined by triple h, k, l, where the first index refers to the size, the
second to the third a b and c. If a plane is parallel to an axis, the index is
obviously invalid. A simple interpretation of the angles of deviation of
the diffracted beams was provided by demonstrating that the result of
Bragg diffraction determined by a crystal lattice can be reduced to the
study of reflections given by the various atomic planes of the crystal.
Considering a series of lattice of a crystal separated by the same distance
"d" and supposing that the incident radiation is diffuse elastically by the
crystal, so that the wavelength of the photon or neutron reflection does
not vary. There will be a diffracted beam only when the reflections of
parallel planes interfere constructively. The beam reflected by the lower
lattice plane along a path greater than the reflection from the upper deck
and the path difference for rays reflected from nearby surfaces is 2dsinθ
(Fig.4.1).
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Materials and Methods
Fig.4.1 : Diffraction Pattern
Constructive interference occurs whenever the path difference between
the two beams is an integer number n of wavelengths and this occurs
when:
n λ =2 d sinθ
where λ is the wavelength of incident radiation (in our case X-rays), d is
the distance between parallel lattice planes (defined by the nature of the
crystal examined), θ is the angle between the incident beam and crystal
planes (Note that only some values of θ reflections are added with equal
phase to give rise to a diffracted beam) and n is an integer positive
natural. This report is called Bragg's law.
The direction of the diffracted radiation therefore depends on shape
(symmetry) and the size of the unit cell in the lattice and its intensity
depends on the distribution of atoms in the cell. Furthermore, X-rays
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Materials and Methods
diffracted by a crystal can be treated as reflections by all the atomic
planes in which the condition occurred Bragg.
Only when that law is satisfied there is reflection. One can therefore say
that an X-ray diffraction profile contains two types of information. The
first is obtained from analysis of the position of observed peaks: the
Bragg law shows how these positions are directly related to the type of
lattice of the sample, or rather with the symmetry of the crystal lattice,
three-dimensional features. Considering the type of symmetry, it is
possible to calculate the size of the unit cell. For example, for the cubic
phases the position of peaks (Fig.4.2) is:
Fig.4.2 : Position peaks of cubic phases
From the second information derived from analysis of the diffracted
intensity, we obtain the profiles of electron density of the sample and
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Materials and Methods
because it allows to determine the type of atoms present, addresses the
structure.
The diffraction spectrum (Fig.4.3) obtained by liquid crystalline systems
can be analyzed by dividing it into two areas that provide different
information: a low angle (q <0.25) and a high angle (q >0.25 °).
Intensity (a.u.)
cubic phase Im3m
1000
100
0.05
0.10
0.15
0.20
0.25
Å
q ( -1)
Fig.4.3:Diffraction spectrum obtained by liquid crystalline systems.
4.2.1 X Ray diffraction methods for structure determination
Diffraction methods, in particular X-Ray scattering, are the most reliable
way of carrying out lyotropic phase identification. Spectroscopic
techniques such as NMR and freeze-fracture electron microscopy, when
used in conjunction with X-ray diffraction, can yield useful
complementary data. Because the large number of phases that can be
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Materials and Methods
observed as a function of composition and temperature [11] in this
section we will in particular discuss the case of lipid-water systems. In
fact, a common property of lipids is the segregation of polar and
paraffinic moieties into distinct regions. A direct consequence is the
ability of lipids to take up a wide variety of structure when mixed with
water. In the lipid diffraction, there are two regions of the diffraction
pattern that are used to identify the phase. The diffraction pattern
observed in the low-angle region (from several hundred Å to 10 Å)
specifies the crystalline lattice, identifies the symmetry of the structures
and gives information about the long range organization of the phase,
whereas from the diffraction observed in the wide-angle region (centred
around 4 Å) information on the molecular packing and the short-range
organization of the lipids can be observed. It must be noticed that the
phases considered by one parameter and belong to one- (lamellar phase),
two- (hexagonal phase) and three- dimensional (cubic phase) systems.
Moreover, in the case discussed, the aliphatic chains are melted, and
only a diffuse band will be observed in the high-angle region of X-ray
spectra.
4.2.2. Phase identification
The first step in the structural analysis of a multicomponent system is to
construct the phase diagram, to characterized the different phases and to
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Materials and Methods
determine their temperature and composition domain of existence [11].
Very intricate phase diagrams can be obtained, in particular when lipid
mixtures are complex, as in the case of lipids extracted from biological
systems where the number of components is large. It appears very
important to perform X-ray scattering experiments as a function of water
composition and temperature. In each experiment, a set of reflections are
observed, and it is essential to unambiguously identify the reflections
corresponding to each phase. The problem is to index the reflections and
to determine the symmetry of reflections can be systematically absent for
reasons other than that of symmetry [10].
From an experimental point of view, data consist of a set of reflections
characteristic of one phase. If the X-ray diffraction experiment is
performed on unorientated samples, only the spacing and the intensities
of the different reflections are known. The problem is to index the
reflections and to determine the symmetry of the lattice and, finally, the
phase structure. The equation that define the spacings of reflections for
the three different symmetry systems are given in [10].
S h,k,l
d
a
reciprocal spacing (Å-1) of the reflection of the indices h,k and l
repeat distance of the lamellar phase
dimension of the unit cell in the hexagonal and cubic phases
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Materials and Methods
S h= h/d
one-dimension lamellar phase
S h,k= 2/√3/√h2+ k2-hk/a
two-dimensional hexagonal phase
S h,k,l=√ h2+ k2+l2/a
three-dimensional cubic phase
Table 1: Equation relevant to some different symmetry systems
In table 1 we report only equations relevant to one-dimensional lamellar,
two-dimensional hexagonal and three-dimensional cubic symmetry
systems. The symmetry of the lattice is easily determined by finding the
equation which agrees with all observed peaks.
It can be observed that the spacing ratio of the reflections characteristic
of a hexagonal lattice is 1:√3: √4: √7: √9, while the patterns becomes 1:
2: 3: 4…..when a lamellar structure is considered. Concerning the cubic
structure, until now six different phase with cubic symmetry have been
observed [10,76]. The identification of the permitted reflections defines
unambiguously the cubic aspect of the phase, while it has been shown
that the most satisfactory space group among those compatible with the
extinction symbol is the one of highest symmetry [10]. Some spacing
rations of the characteristic reflections observed in different cubic phase
are reported in Fig.4.2.
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Materials and Methods
When the symmetry is found, the dimension of the unit cell and other
structural parameters can be calculated [11]. In fact, if all chemical data
(phase composition, molecular weight and lipid density) are available,
only a few hypothesis are sufficient to obtain the area-per-chain at the
polar/apolar interface and the dimensions of structure elements [11,76].
The hypothesis usually considered can be summarized as follows [10].
First, the water is excluded from the paraffinic regions; second, the
polar/apolar interface is covered by the hydrophilic groups of lipid
molecules; third, the structure element shape can have higher symmetry
than that permitted from the space group (in the hexagonal phase, the rod
section is considered to be circular, for example). However, if the
structure elements are not fully defined from the lattice symmetry, as in
the case of cubic phases, their shape can be obtained only from an
analysis of the intensities of the reflections [11, 77].
4.3 Production of X-Ray
4.3.1 The X-ray tube
4.3.1.1. Diffractomer in the SAIFET laboratory
The traditional method of producing X-Ray in laboratory is by means of
an X-ray tube. This device and improved technically during the
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Materials and Methods
following century, is still being used and as an interesting physical
background that marks major scientific development.
In general, an X-ray diffractometer consists of an X-ray generator
system and collimation of the incident beam by a sample holder and a
detection system of the diffracted beam.
• The generator consists of a tube inside which a heated tungsten
filament releases electrons that are accelerated by heating an electric
field collide with high potential and a metal anode. Following this
interaction are so X-rays emitted at different wavelengths characteristic
of each substance. The system consists of monocromator of quartz
crystal, which have the function to select the desired radiation (usually
with Kα λ = 1.54Å). The system allows collimation to define the shape
and size of the radius, is used for this purpose a series of cracks that
control the beam divergence angle. The X-ray beam hits the sample so
generated, contained in a specimen holder should diffract X-rays in
different directions.
The sample is inserted into the hole of a Teflon disk (A) 1 mm thick and
sealed by two thin sheets of Mylar that are the windows through which
radiation passes and two disks of aluminum (B) too 'they pierced. Filling
the cell with the solution in which the sample is dissolved with a small
spatula that allows you to precisely control the amount introduced.
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Materials and Methods
This must be done very carefully to avoid formation of air bubbles that
may interfere with the spectra of X-ray diffraction.
To protect the detector, corresponding to the transmitted beam is placed
a small slab of lead (BeamStop) in order to shade the minimum angular
region can plan for registration, which would otherwise be blinded by
the high intensity of the transmitted beam.
4.3.1.2 Laboratory Diffractometer of USP
SAXS experiments were performed at the NanoStar machine from
Bruker (Karlshure- Germany) (Fig.4.4), with radiation wavelength
lambda = 1.542 Å and sample-to-detector distance of 670 mm. Samples
were set in between two mica windows and a 1 mm spacer, handled in a
liquid sample-holder. This was placed perpendicular to the primary Xray beam. The obtained curves (data collection of 120 min) were
corrected for detector homogeneity (two-dimension position sensitive
detector). The parasitic background (buffer solution) was subtracted
considering the sample’s attenuation.
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Materials and Methods
Fig.4.4: NanoStar machine from Bruker (Karlshure- Germany)
4.3.2 The synchrotron
All the diffraction techniques to be outlined below are of widespread
availability ; they can be found in crystallographic laboratories, and
serve ad the basic tools for the collection of diffracted intensity data. A
popular instrument is the four-circle diffractometer, because
of its
accuracy and sophisticated automation. Its main limitation , when a
selected X-Ray tubes is used , at the relatively low intensity of incident
radiation that can be obtained and the necessity for collecting the
diffracted intensities from one reflection at a time. The first of these
results in time-consuming experiments , and the second add the danger
of crystal determination due to radiation damage. Ideally would like to
be able to collect a large number of diffracted intensities in a short time.
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Materials and Methods
The best answer to the latter requirement is offered by synchrotron
radiation which is produced in special installation.
Some of the diffraction experiments, particularly those regarding the
influence of temperature and pressure were conducted using beam-line
source of synchrotron radiation synchrotron in Grenoble (ESRF France) (Fig.4.7), the DORIS III Desy Hamburg in Germany (Fig.4.6)
and LNLS (Fig.4.5) at Campinas (Sao-Paulo, Brazil).
Fig.4.5: LNLS at Campinas (Sao-Paulo, Brazil)
Fig.4.6: DORIS III Desy Hamburg in Germany
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Materials and Methods
Fig.4.7: Synchrotron in Grenoble (ESRF - France)
In a synchrotron X-rays are emitted by a beam of electrons forced to
move through a system of magnets at a speed close to that of light on a
nearly circular path consists of a steel tube-shaped ring held vacuum . A
component called injector LINAC (linear accelerator), takes the
electrons and accelerates them up to a speed approaching that of light,
then a transfer line picks up these electrons and transfers them fast in the
ring center. Electrons, injected high-energy accumulation in the ring, if
left to themselves tend to move along a straight line and leave the ring
for the tangential direction. This is prevented by a system of magnets
which bend the path of the electrons causing them to remain on a nearly
circular path. The electrons are then accelerated as they are forced to
continually change the direction of their velocity. The electrons inside
the ring, moving grouped into packages emitting electromagnetic
radiation (in a wide range of wavelengths) in the direction tangent to the
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Materials and Methods
ring. These waves are known as "bremsstrahlung". Special devices
issuers (Wiggler and undulator), inserted outside of the ring into sections
including two magnets , increase the flow and the brightness of the
radiation and reduce the wide band of wavelengths, concentrating in the
region of the rays X Each undulator branches then a line of light which is
collected in the thin, intense beam of X-ray Note that during the
movement of electrons, the line of light continues to receive intense
flashes of synchrotron light and the result is prolonged illumination
rather than a series of short pulses. The thin beam of X-rays produced is
FWHM optical systems to filter the wavelengths and select the one
needed for each application. This beam is sent into a chamber of use
(experimental hutch) at the end of the line of light and equipped with all
necessary equipment. Many beamlines branch From the ring center and
provide light instruments dedicated to specific uses. What is a
synchrotron light source is therefore the production of a wide stream,
concentrated in a collimated beam. Technically speaking, a concentrated
source and a collimated source is "high brightness", ie small size and
high collimation angle. High brightness means that the light emitted can
be entirely concentrated on the use of with great intensity and without
waste. This is the basic requirement for most uses of X-ray sources
In order to obtain more detailed information about the properties of
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Materials and Methods
cytochrome c and monoolein , we decided to take advantage of X-ray
sources of electricity of Desy, ESRF and LNLS.
4.3.2.1 Pressure Cell of ESRF
Diffraction experiments were performed at the ID02 beamline at the
European Synchrotron Radiation Facility, ESRF, Grenoble (France),
using a SAXS-WAXS setup.6 The wavelength of the incident beam was
λ= 1 Å, and the investigated Q-range was between 0.03 and 0.6 Å-1 (Q )
4π sin θ λ /, where 2 θ is the scattering angle) on the SAXS detector and
between 2.8 and 13.2 Å-1 on the WAXS detector. For high-pressure
measurements, a NovaSwiss pressure-control system was used. The
pressure cell has two diamond windows (3.0 mm diameter and 1 mm
thickness) and allows measurement of diffraction patterns at hydrostatic
pressures up to 3 kbar.
X-ray diffraction measurements were performed at 25 °C for different
pressures, from 1 bar to about 3 kbar, with steps of about 100 bar. To
avoid radiation damage, the exposure time was kept as low as 0.2-1
s/frame, and a fast beam shutter was used to protect the sample from
irradiation when data were not being acquired. Particular attention was
paid to checking forequilibrium conditions and monitoring radiation
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Materials and Methods
damage. Measurements were repeated several times (up to 10) at the
same constant pressure to account for stability in position and intensity
of the Bragg peaks [76].
The sample holder inside the cell are small cylinders made of teflon.
The high brightness (B) SAXS of the light line is defined by the
following formula
B = (ΔN / Dt) / (ΔS * * ΔΩ ΔE / E)
ΔN / Dt = photon flux
ΔS = area source
ΔΩ = solid angle
ΔE / E = bandwidth energy
The flow of photons is equal to 5x1012 photons / s (2 GeV, 200 mA, 8
keV) and the size of the beam is <5.4 x 1.8 mm2 (HxV).
4.3.2.2 Temperature System of DESY
The SAXS experiments were performed at the DESY synchrotron
facility in Hamburg, Germany, on the A2 beamline. The investigated
q-range (q ) 4π sin θ/λ, where 2θ is the scattering angle and λ ) 1.50 Å
the X-ray wavelength) was 0.02-0.35 Å-.
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Materials and Methods
Scattering data were recorded on a bidimensional CCD camera of 1024
× 1024 pixels, radially averaged and corrected for the dark, detector
efficiency and sample transmission [77].
Diffraction data were collected from 25 °C up to 90 °C.
4.4 Spectrophotometry
Spectrophotometry is one of the most useful tools available to the
biochemist. It offers a high degree of precision, sensitivity, and accuracy.
In addition, it is inexpensive and applicable to the measurement of a
variety of substances.
Organic compounds that absorb light are pigments.
The absorption properties of an organic substance are constant,
wherever.1 A spectrophotometer is an instrument that contains (a) a light
source(s), (b) a means of isolating a particular wavelength band of the
light source, (c) a sample holder, and (d) a device to measure light
intensity.
To measure organic compound content of the extracts above, we would
select a proper wavelength on the spectrophotometer. Selection of
wavelength is important because an extract or any solution may contain
many compounds that absorb light.
The spectral properties of a substance may change, depending on the
chemical environment and modifications to the molecule. In some cases,
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Materials and Methods
“simply” changing the pH will convert a colored substance to a colorless
one. In other cases, the oxidation state of the molecule will cause
dramatic changes in the spectral properties.
Given constant conditions, the absorption by a particular species at a
particular wavelength is constant. Although the instrument itself is
essentially measuring transmittance (the percentage of incident radiation
that passes through the sample), it is not convenient to use transmittance
because the concentration of the absorbing species is not linear with
transmittance. However, a simple mathematical conversion, to Optical
Density (O.D.) or Absorption, creates a linear relationship:
O.D. = - log (T)
The so-called mM (say “millimolar extinction coefficient”) at 340 nm
with a 1-cm light path.
4.4.1.The Beer-Lambert law
In optics, the Beer-Lambert law is an empirical relationship that
correlates the light absorption properties of absorbent material. Recall
that the light intensity affecting in a material (half) is called, precisely,
and is usually denoted by accident while entering in the middle is said
transmitted intensity, and finally, what is reflected from the interface is
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Materials and Methods
'intensity reflected. The Beer-Lambert law states that the intensity of
transmitted light fades exponentially passing through a medium. The
most common form in which that law is written is as follows where is
the extinction coefficient, which depends on the characteristics of the
medium than the wavelength of incident light and the relationship
between the intensity is called transmittance. This is a form, as
mentioned, is a very general common law into consideration, but, in
spectroscopy, particularly applied to biology, the expression above has
been made more convenient for practical use in laboratories, by
exponential passing under natural that in decimal base and setting the
standard and constant quantities useful for the calculations. It will,
therefore, a new expression, suitable to treat liquid assets (with further
solid and gas): where alpha is the molar absorption coefficient of the
solute, l is the thickness of the medium and c is the concentration of
solute. We can define now absorbed as the logarithm base 10 of the
report above: Note that the expression of the absorbance, the only
unknown is the concentration of the formula because a, ie the molar
absorption coefficient for each solute are tabulated and the length is
unknown or at least measurable. So, as far as we have seen can be traced
back to the concentration of a solution or suspension using a beam of
light but there is one final aspect to which attention. The terms set out
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Materials and Methods
applies, naturally light of any wavelength (and thus also for white light),
but the absorption coefficients, as we have seen, vary depending on the
wavelength and for each parcel of experimental interest, are tabulated
only one or a few absorption values at wavelengths well defined. Usually
choose the wavelengths in which the coincidence has a high absorption
and are different from those for maximum absorption of water or other
buffers or solvents used in its own laboratory to distinguish the solutes
from solvent.Perform analysis with monochromatic light (not necessarily
be a laser) is practically indispensable and also allows to distinguish and
measure the concentrations of most solutes in solution, in case they have
quite different absorption spectra of (or at least that everyone has a very
low absorption at wavelengths characteristic of the other). A new
laboratory facility for the calculations comes from having established the
optical path l The vials used in spectrophotometers, that specimen
containers to be inserted into the instrument are thick compared to 1-cm
optical beam. Once you choose the wavelength with which to analyze
and using an instrument with a standard tubes (the normal situation in
the laboratory) can be traced very quickly at this concentration the
optical density, defined as:
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Materials and Methods
The optical density is nothing but the absorbance wavelength and optical
path set. Theoretically it is said that the road should be 1 cm (ie with
units cm-1) but, as we said, this is the standard length. Indeed, when the
ODL is taken in cm-1 is indicated by odulating. In fact even when it is
only ODL, if not explicitly expressed, the unit is deemed cm-1.
4.5 Sample Preparation
Important in the experimental part of this thesis was to deal how to
prepare the sample. During sample preparation is needed to pay attention
to the priority of placing reagents in solution of water and cytochrome c.
It was determined that the last component to be included must be the
monoolein.
Then you need to prepare the solution of cytochrome c and water at
different concentrations (1-5-10-25-50 and 100 mg/ml) of protein and
then finally enter the fixed amount of monoolein (50 mg/ml).
The sample has been stabilized for several hours at room temperature or
in a thermal bath at 32 ° C. Both situations have been tested in order to
understand if the temperature could play an important role in facilitating
or not phase transition.
Once stabilized, samples were used for diffraction experiments that were
performed every day to monitor the kinetics of the transition and
incorporation.
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Materials and Methods
Daily was then taken a little portion of the lipid phase (that have shown
consistency in the semi-solid solution) and was used for diffraction
experiments.
Spectrophotometric analysis for the same, every day was taken a number
of cytochrome c-water solution and, then calculate the difference, how
much protein was entered into the channels of the cubic phase.
Accordingly, the first stage which is based solely on the evaluation of
the effects of concentration, until you get to take as a condition better
than seeing the monoolein at 50 mg / ml and cytochrome concentrations
ranging from 1 up to 50 and in some cases 100 mg / ml.
Then we moved to the second step where the focus has fallen on the
variable temperature. The aim was to assess what was the effect on the
system monoolein cytochrome a wide temperature range (25 to 90 ° C).
The tests were multiple and attempted to investigate what might happen
within the stage during the raising of the temperature, and subsequent
return to the stage.
The final phase focused on assessing the effect of pressure on the
system.
The pressure has been exerted through the use of cell pressure supplied
at the synchrotron in Grenoble (ESRF) with a maximum of 3000 bar.
All tests were carried out on kinetics of time (sometimes up to the first
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Materials and Methods
21 days) or samples were measured at different days as the cytochrome
takes time to enter the channels of monoolein and cause the change.
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CHAPTER 5
Data Analysis: Cytochrome c Concentration Effects
CHAPTER 5
DATA ANALYSIS: CYTOCHROME C
CONCENTRATION EFFECTS
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CHAPTER 5
Data Analysis: Cytochrome c Concentration Effects
The first stage of our work had as its subject the study the cytochrome
c-monoolein system varying the concentration before lipid and then
protein.
To determine which was the best concentration of lipid in solution, we
have made several tests using different lipid concentration.
We started with 25 mg / ml but the concentration is not allowed during
the various measurements in an optimal amount of time monoolein can
also be used in the last days of measurement.
Finally we decided to use a fixed lipid concentration of 50 mg / ml (Cv
mo = 0.614) and varying only the concentration of cytochrome c.
The concentrations of cytochrome c were 1-5-10-25-50-100 mg / ml
solution.
The measurements were performed at times from the first to the twentyfirst day after sample preparation, so we could evaluate and measure
every day the kinetics of the transition.
Will be shown below the diffraction profiles and various studies on the
different systems studied, analyzed as the general course of the
experiments is not the same for everyone.
Diffraction measurements were performed at the SAXS equipment of the
Laboratory of the University of Sao Paulo, Brazil.
- 83 -
CHAPTER 5
Data Analysis: Cytochrome c Concentration Effects
5.1. Monoolein and Cytochrome c 1 mg/ml
The image below shows a plot of the first sample containing 50 mg / ml
of monoolein and 1 mg / ml cytochrome.
Fig.5.1: X-Ray diffraction patterns from monoolein-Cytochrome c at concentration
50 mg/ml of lipid and 1 mg/ml of protein at different days.
Measurements were performed on the first day of preparation until the
tenth day (Fig.5.1).
As you can see from the figure 5.1, with the passing of the days we see a
transition phase that becomes visible on the seventh day (purple profile).
So the monoolein passes from a Q224 (Pn3m) visible from first to sixth
day to measure a phase Q229 (Im3m) presented on the seventh day.
- 84 -
CHAPTER 5
Data Analysis: Cytochrome c Concentration Effects
The seventh day the appearance of the new peak value at q 0.06 A-1 also
indicates an increase in cell size as seen from the bottom plot (Fig.5.2).
The phase change is also indicated by the increase in cell size that goes
from typical values of Q224 (about 104 Å) to reach values of 140 Å (as
showed in the figure below).
Q224
Q229
160
150
unit cell︵ )
Å
140
130
120
110
100
90
1
2
3
4
5
6
7
8
9 10
day
Fig.5.2: Day dependence of the unit cell dimension of the cubic Pn3m and Im3m
The data obtained confirm that cytochrome c needs some days to enter
into the cubic phase and then be able to change the structure leading to
increased cell size (Fig.5.2).
This phase change occurs abruptly and then remain constant in the
following days.
During the transition phase we see the emergence of arising Q229
accompanied by a second peak Im3m, at q value of about 0.07 Å-1.
- 85 -
CHAPTER 5
Data Analysis: Cytochrome c Concentration Effects
This new phase we thought might be a phase that arises simultaneously
Intensity (a.u.)
with Q212and Q229 (Fig.5.3).
0.10
0.15
1
-
0.05
Å
q(
0.20
0.25
)
Fig.5.3: Detail of the monoolein-cytochrome (1mg/ml) at 7 day.
The presence of this second phase simultaneously with the Q229 may
indicate that there is a coexistence of Im3m and other areas where the
provision is to stage a P4332 (Q212).
In parallel with diffraction analysis, we performed spetrophotometry to
evaluate how much cytochrome c remains in solution and how much has
entered inside the tubes of monoolein.
Daily was taken an amount of cytochrome c-water solution to evaluate
the amount of protein inside the liquid crystal phase (Fig. 5.4).
- 86 -
CHAPTER 5
Data Analysis: Cytochrome c Concentration Effects
cytochrome (mg/ml)
0.8
0.6
0.4
0.2
0.0
0
5
10
day
Fig.5.4: Concentration of cytochrome in the phase of monoolein
From the plot (Fig.5.4) we can see that the quantity mg / ml cytochrome
within the channels increases with each passing day.
Specifically, the increase is beginning to see the fifth / sixth day with a
small increase, which becomes significant in the days following.
5.2. Monoolein and Cytochrome c 5 mg/ml
The second sample examined is the one containing 50 mg / ml of
monoolein and 5 mg / ml of cytochrome in solution (Fig.5.5).
- 87 -
CHAPTER 5
Data Analysis: Cytochrome c Concentration Effects
1° day
2° day
Intensity (a.u.)
5° day
7 day
9° day
11° day
1
-
0.00 0.05 0.10 0.15 0.20 0.25 0.30
Å
q(
)
Fig.5.5: X-Ray diffraction patterns from monoolein-Cytochrome c at concentration
50 mg/ml of lipid and 5 mg/ml of protein at different days.
The sample was measured over time, from first to sixth day after
preparation.
In this case you notice changes in phase, or the Q224 remains constant
from the fluid up to eleventh day. The only change you may notice is the
slight movement of the first reflection (110) to longer q values.
The increase in cell dimension is observed from the graph below in the
first day with a Q224 of 104 Å up to the sixth day at 114 Å (Fig.5.6).
- 88 -
CHAPTER 5
Data Analysis: Cytochrome c Concentration Effects
Unit Cell ( )
Å
116
114
112
110
108
106
104
102
100
98
96
0
2
4
6
8
10
12
day
Fig.5.6: Day dependence of the unit cell dimension of the cubic Pn3m
One can notice the spectrophotomer analysis are in accordance with the
diffraction data. (Fig.5.7).
conc.cito
Cytochrome (mg/ml)
2.5
2.0
1.5
1.0
0.5
0.0
0
2
4
6
8
10
12
day
Fig.5.7: Concentration of cytochrome in the phase of monoolein
- 89 -
CHAPTER 5
Data Analysis: Cytochrome c Concentration Effects
In the graph we can see that a number of cytochrome is reduced to the
solution and then entered into the monoolein but failed to change its
structure at least until the eleventh day.
5.3. Monoolein and Cytochrome c 10 mg/ml
The next sample contains 50 mg / ml of monoolein and 10 mg / ml
cytochrome c (Fig.5.8).
The measurements were performed from first to tenth day after sample
preparation.
Intensity(a.u.)
1 day
2 day
3 day
4 day
6 day
7 day
9 day
10 day
0.05
0.10
Å
q(
0.15
1
-
0.00
0.20
0.25
)
Fig.5.8: X-Ray diffraction patterns from monoolein-Cytochrome c at concentration
50 mg/ml of lipid and 10 mg/ml of protein at different days.
- 90 -
CHAPTER 5
Data Analysis: Cytochrome c Concentration Effects
Here, as in the previous sample, it is not possible to evidence any change
in the monoolein mesophase, which means that cytochrome is not able to
change the three-dimensional arrangement of monoolein.
115
unit cell ( )
Å
110
105
100
0
5
10
day
Fig.5.9: Day dependence of the unit cell dimension of the cubic Pn3m .
The increase in cell size is comparable to the previous sample (5 mg / ml
cytochome c) 107 Å, in fact, switching from the first day of
measurement, up to the tenth day to 114Å.
By analysis of spectophotometry, it turns out that even here there is a
small entrance of cytochrome into the cubic phase but perhaps not
sufficient to bring about change. The decrease of cytochrome in solution
is visible from the sixth day (Fig.5.10).
- 91 -
CHAPTER 5
Data Analysis: Cytochrome c Concentration Effects
cytochrome (mg/ml)
6
5
4
3
2
0
5
10
day
Fig.5.10: Concentration of cytochrome in the phase of monoolein
5.4. Monoolein and Cytochrome c 25 mg/ml
The fourth sample studied was that containing 25 mg / ml cytochrome c
always in the presence of the same amount of monoolein (50 mg / ml)
(Fig.5.11).
- 92 -
CHAPTER 5
Data Analysis: Cytochrome c Concentration Effects
2 day
3 day
4 day
6 day
7 day
9 day
10 day
100000
Intensity (a.u.)
10000
1000
100
10
1
0.1
0.10
0.15
Å
0.05
0.20
0.25
0.30
q( )
Fig.5.11: X-Ray diffraction patterns from monoolein-Cytochrome c at concentration
50 mg/ml of lipid and 25 mg/ml of protein at different days.
The evolution of this sample is anomalous, as can be seen from the
diffraction profile shown above.
Q224 phase remains constant, with some variation in cell size, until the
sixth day, then changed into a Q229 with a large cell size.
Until the sixth day the unit cell measured was 115 Å to suffer a drastic
increase in the seventh day, at the onset of Q229, up to 145 Å, before
returning to cell values around 110 Å. In the following days, however, it
is possible to see a new occurrence of reflection (110) belonging to
- 93 -
CHAPTER 5
Data Analysis: Cytochrome c Concentration Effects
Pn3m stage, as if the reorganization was only a temporary Im3m
(Fig.5.12).
Q224
Q229
150
140
Unit cell ( )
Å
130
120
110
100
2
4
6
8
10
day
Fig.5.12: Day dependence of the unit cell dimension of the cubic Pn3m and Im3m
The
cytochrome
comes
slowly,
as
you
can
see
from
the
spectophotometric analysis then the seventh day to reach higher levels
and decrease again in the days following (Fig.5.13).
- 94 -
CHAPTER 5
Data Analysis: Cytochrome c Concentration Effects
cytochrome (mg/ml)
14
12
10
8
6
4
2
0
0
5
10
day
Fig.5.13: Concentration of cytochrome in the phase of monoolein
5.5. Monoolein and Cytochrome c 50 mg/ml
The fifth sample has the same concentration of monoolein and
cytochrome or 50 mg / ml for both (Fig.5.14).
Intensity (a.u.)
1 day
2 day
3 day
4 day
6 day
7 day
10 day
21 day
0.00 0.05 0.10 0.15 0.20 0.25 0.30
1
-
Å
q( )
Fig.5.14: X-Ray diffraction patterns from monoolein-Cytochrome c at concentration
50 mg/ml of lipid and 50 mg/ml of protein at different days.
- 95 -
CHAPTER 5
Data Analysis: Cytochrome c Concentration Effects
Diffraction profiles of the sample are illustrated in several days. The
phase Q224 is up to the twenty-first day, but simultaneously appear
reflections belonging to Q229.
Such coexistence phase begins to be evident from the sixth day of
measurement (Fig.5.15).
Q224
Q229
160
150
unit cell ( )
Å
140
130
120
110
0
5
10
15
20
25
Day
Fig.5.15: Day dependence of the unit cell dimension of the cubic Pn3m and Im3m
We do not have spectophotometric analysis because of problems during
the experiment.
5.6. Monoolein and Cytochrome c 100 mg/ml
The latter sample contains 100 mg / ml cytochrome and was measured at
the synchrotron of Campinas, Brazil.
- 96 -
CHAPTER 5
Data Analysis: Cytochrome c Concentration Effects
The sample was measured seven days after preparation. The presence of
the Q229 (123 Å)is clear in comparation with the plot of monoolein pure
(Q224 and 104 Å).
Graph is shown in red in the profile of the same sample measured on the
first day of preparation, while in the black sample after seven days
(Fig.5.16).
Intensity (a.u.)
monoolein
mo+ cyt 100 mg/ml
0.00 0.05 0.10 0.15 0.20 0.25 0.30
Å
-1
q( )
Fig.5.16: X-Ray diffraction patterns from monoolein-Cytochrome c at concentration
50 mg/ml of lipid and 100 mg/ml and monolein –water system.
- 97 -
CHAPTER 6
Data Analysis: Temperature Effects
CHAPTER 6
DATA ANALYSIS: TEMPERATURE EFFECTS
- 98 -
CHAPTER 6
Data Analysis: Temperature Effects
The Influence of Temperature on the Lipid Phase Transition
The second step was to evaluate the influence of temperature on the
system monoolein-water-cytochrome, and to observe the difference with
the traditional phase diagram of monoolein (Fig.3.3).
The thermotropic structural behavior of the MO/cytochrome was
investigated by diffraction techniques with the temperature between 25
and 90°C. Diffraction measurements were performed at the SAXS
beamline of the DESY Light Source, Hamburg (Germany)at different
days of sample preparation, from the first up to the thirteen day.
The temperature analysis is interesting because it provides outline the
lipid and cytochrome behaviour at room temperature up to temperature
that goes over its denaturation status.
The denaturation temperature of cytochrome is around 60 ° C, but in this
system, perhaps the protein is protected from the lipid structure at high
temperatures.
The first sample sequences are :
• mono 50 mg/ml + 1 mg/ml cytochrome c
• mono 50 mg/ml + 10 mg/ml cytochrome c
• mono 50 mg/ml + 25 mg/ml cytochrome c
• mono 50 mg/ml + 50 mg/ml cytochrome c
These samples were prepared 5 days from the preparation.
- 99 -
CHAPTER 6
Data Analysis: Temperature Effects
• mono 50 mg/ml + 1 mg/ml cytochrome c
• mono 50 mg/ml + 10 mg/ml cytochrome c
• mono 50 mg/ml + 25 mg/ml cytochrome c
• mono 50 mg/ml + 50 mg/ml cytochrome c
• mono 50 mg/ml + 5 mg/ml cytochrome c
These samples were prepared 8 and 13 days from the preparation.
6.1.Monoolein and Cytochrome c 1mg/ml
The first sample containing 1 mg / ml was measured at 5 days of
preparation at temperatures from 25° to 65°C. (Fig.6.1).
mono
mono
mono
mono
mono
Intensity (a.u.)
10000
25°C
35°C
45°C
55°C
65°C
1000
100
10
1
0.1
0.00
0.05
0.10
0.15
0.20
0.25
q (Å -1)
Fig.6.1: X-Ray diffraction patterns from monoolein-Cytochrome c at concentration
50 mg/ml of lipid and 1 mg/ml of protein at different temperature.
- 100 -
CHAPTER 6
Data Analysis: Temperature Effects
Selected low-angle X-Ray diffraction patterns, showing a number of
reflections, are reported in Figure 6.1
At 5 day, the X-Ray diffraction profiles confirm the presence of the
Pn3m cubic phase and do not exhibit a cubic-to-cubic phase transition,
from Pn3m to the Im3m cubic phase.
The temperature effect is visible only in the small shift of the position
peak versus q value around 0.11 Å-1 .
Å
unit cell ( )
100
95
90
85
80
20
30 40 50 60
temperature (°C)
70
Fig.6.2:Temperature dependence of the unit cell dimension of the monooleincytochrome
The position of the cubic phase Bragg peak corresponds to a repeat
spacing d 110 = 101 Å at 25°C , which decreases to d 110 = 83 Å upon
heating to 65°C (Fig.6.2).
In the figure above is possible to see the decrease of unit cell dimension,
congruent with the previous studies present in literature [22].
After 8 days the situation is not changed, so the only cubic phase present
in the system is the Q224 up to a high temperature of 90°C (Fig.6.3).
- 101 -
CHAPTER 6
Data Analysis: Temperature Effects
105
100
95
Å
Unit cell ( )
Intensity (a.u.)
mono 25°C
mono 30°C
mono 35°C
mono 40°C
mono 45°C
mono 50°C
mono 55°C
mono 60°C
mono 65°C
mono 70°C
mono 75°C
mono 80°C
mono 85°C
mono 90°C
90
85
80
75
70
20 30 40 50 60 70 80 90 100
0.00 0.05 0.10 0.15 0.20 0.25
Temperature (°C)
q (Å -1)
Fig.6.3:(a)X-Ray diffraction patterns from monoolein-Cytochrome c at concentration
50 mg/ml of lipid and 1 mg/ml of protein at different temperature.
(b) Temperature dependence of the unit cell dimension of the monoolein-cytochrome
.
In this plot is interesting to note the presence of Q224 up to 90°C,
unusual behaviour of monoolein that in the its phase diagram shows the
presence of hexagonal phase from 60°C (Fig,3,3) [25]. In this case the
cytocrome c appears to prevent the new structural disposition (hexagonal
phase) and it allows monoolein to remain in the cubic phase Pn3m,
varying only the unit cell.
This parameter, like the samples measured as function of temperature,
decreases for loss of hydration in the channel of lipid.
- 102 -
CHAPTER 6
Data Analysis: Temperature Effects
mono 25°C
mono 35°C
mono 45°C
mono 55°C
mono 65°C
Q224
Q229
130
Å
unit cell ( )
Intensity (a.u.)
120
110
100
90
80
20
0.00
0.05
0.10
0.20
0.25
30
40
50
60
70
temperature (°C)
-1
Å
q(
0.15
)
Fig.6.4: (a)X-Ray diffraction patterns from monoolein-Cytochrome c at
concentration 50 mg/ml of lipid and 1 mg/ml of protein at different temperature.
(b) Temperature dependence of the unit cell dimension of the monoolein-cytochrome
At the 13th day one can see the presence of both cubic phase Q224 and
Q229 from the 25°C up to 55°C (Fig.6.4).
While the temperature increases it can be seen that the peaks of the Q224
will begin to lose intensity up to 65 °C in which it appears the peak of
the hexagonal phase.
The unit cell dimension decreases both for Pn3m and Im3m.
6.2 Monoolein and Cytochrome c 5mg/ml
The second sample composed of 5 mg / ml cytochrome were measured
at 8 and 13 days (Fig.6.5).
- 103 -
Data Analysis: Temperature Effects
Intensity (a.u.)
CHAPTER 6
mono 25°C
mono 30°C
mono 35°C
mono 40°C
mono 45°C
mono 50°C
mono 55°C
mono 60°C
mono 65°C
mono 70°C
mono 75°C
mono 80°C
mono 85°C
mono 90°C
1E9
1E8
1E7
1000000
100000
10000
1000
100
10
1
0.1
0.00 0.05 0.10 0.15 0.20 0.25
q (Å-1)
Fig.6.5: X-Ray diffraction patterns from monoolein-Cytochrome c at concentration
50 mg/ml of lipid and 5 mg/ml of protein at different temperature.
The diffraction profile shows a constant trend with the Q229 presence
form 25° up 90°C, at once proceeding to being a new peak at q value
around 0.13 Å, that is the first hexagonal reflection Bragg with the unit
cell dimension of 55 Å.
- 104 -
CHAPTER 6
Data Analysis: Temperature Effects
Q229
H
120
110
Unit cell︵ )
Å
100
90
80
70
60
50
20 30 40 50 60 70 80 90 100
Temperature (°C)
Fig.6.6:Temperature dependence of the unit cell dimension of the monooleincytochrome
The presence of the hexagonal phase at the high temperature in this
system, it is comparable to the diagram phase of monoolein-water
system that shows this phase at temperature over 80°C (Fig.6.6).
The dimension unit cell decreases both Q229 and hexagonal.
- 105 -
CHAPTER 6
Data Analysis: Temperature Effects
Intensity (a.u.)
mono 25°C
mono 35°C
mono 45°C
mono 55°C
mono 65°C
0.00 0.05 0.10 0.15 0.20 0.25
q(Å-1)
Fig.6.7: X-Ray diffraction patterns from monoolein-Cytochrome c at concentration
50 mg/ml of lipid and 5 mg/ml of protein at different temperature
The plot above represents measurements at 13 day according to trend of
the analysis previously showed (Fig.6.7).
Unit cell ( )
Å
140
130
120
110
100
90
80
70
60
50
40
20
Q229
H
30
40
50
60
70
Temperature (°C)
Fig.6.8:Temperature dependence of the unit cell dimension of the monooleincytochrome
- 106 -
CHAPTER 6
Data Analysis: Temperature Effects
The cubic phase Q229 is persistent from 25-45°C, while the following
temperature presents the Q229 coexisting with the hexagonal phase at
55°C (Fig.6.8).
6.3 Monoolein and Cytochrome c 10 mg/ml
Figure 6.9 shows the plot of the sample composed of 50 mg/ml of
monoolein and 10 mg/ml of cytochrome at 5- 8 -13 days (Fig.6.9).
Intensity(a.u.)
mono 25°C
mono 35°C
mono 45°C
mono 55°C
mono 65°C
0.00 0.05 0.10 0.15 0.20 0.25
q (Å-1)
Fig.6.9: X-Ray diffraction patterns from monoolein-Cytochrome c at concentration
50 mg/ml of lipid and 10 mg/ml of protein at different temperatures.
Here it possible to note the presence of Q229 phase up to 45°C, this
phase coexist with a new phase that could be hexagonal.
- 107 -
CHAPTER 6
Data Analysis: Temperature Effects
Q229
hex
120
110
unit cell ( )
Å
100
90
80
70
60
50
20
30
40
50
60
70
Temperature (°C)
Fig.6.10:Temperature dependence on the unit cell dimension of the monooleincytochrome
At 55°C the second order of Im3m shows splitting in two peaks, one of
cubic phase and one hexagonal phase (Fig.6.10).
Then 8 days:
Intensity(a.u.)
mono 25°C
mono 30°C
mono 35°C
mono 40°C
mono 45°C
mono 55°C
mono 60°C
mono 65°C
mono 70°C
mono 75°C
mono 80°C
mono 85°C
mono 90°C
mono 95°C
0.00 0.05 0.10 0.15 0.20 0.25
q (Å-1)
Fig.6.11: X-Ray diffraction patterns from monoolein-Cytochrome c at concentration
50 mg/ml of lipid and 10 mg/ml of protein at different temperature.
- 108 -
CHAPTER 6
Data Analysis: Temperature Effects
In this sample the transition phase happens whith the presence just of
Q229 at 25°C (Fig.6.11-12).
Q229
H
110
100
Unit cell ( )
Å
90
80
70
60
50
20
30
40
50
60
70
80
90
Temperature (°C)
Fig.6.12:Temperature dependence of the unit cell dimension of the monooleincytochrome
This structure is long lasting up to 55°C, when it begins to appear the
splitting of second peak of Im3m for the hexagonal up to 90°C.
- 109 -
CHAPTER 6
Data Analysis: Temperature Effects
Then 13th days (Fig.6.13):
Intensity (a.u.)
mono 25°C
mono 35°C
mono 45°C
mono 55°C
mono 65°C
0.00 0.05 0.10 0.15 0.20 0.25
q (Å-1)
Fig.6.13: X-Ray diffraction patterns from monoolein-Cytochrome c at concentration
50 mg/ml of lipid and 10 mg/ml of protein at different temperature.
After 13 days the sample shows the same trend observed in the previous
day: at 25°C Q229 is alone and then coexists (up to 55°C) whith
hexagonal phase (Fig.6.14).
Unit cell ( )
Å
120
115
110
105
100
95
90
85
80
75
70
65
20
H
Q 229
30
40
50
60
70
T e m p era tu re (°C )
Fig.6.14:Temperature dependence of the unit cell dimension of the monooleincytochrome
- 110 -
CHAPTER 6
Data Analysis: Temperature Effects
6.4 Monoolein and Cytochrome c 25 mg/ml
This sample contains 25 mg / ml cytochrome and was measured at 5
days (Fig.6.15).
Intensity(a.u.)
mono 25°C
mono 35°C
mono 45°C
mono 55°C
mono 65°C
0.00 0.05 0.10 0.15 0.20 0.25
q (Å-1)
Fig.6.15 : X-Ray diffraction patterns from monoolein-Cytochrome c at concentration
50 mg/ml of lipid and 25 mg/ml of protein at different temperature.
This sample shows the cubic phase Q229 up to 65°C, whith increase of
unit cell; at 55°C is important to note the Q212 that coexist with
hexagonal phase. This phase is present starting at low temperature but
becomes visible at 45°C, its unit cell is 55 Å (Fig.6.16).
- 111 -
CHAPTER 6
Data Analysis: Temperature Effects
Å
Unit cell ( )
Q229
H
170
Q212
160
150
140
130
120
110
100
90
80
70
60
50
40
20 25 30 35 40 45 50 55 60 65 70
Temperature (°C)
Fig.6.16:Temperature dependence of the unit cell dimension of the monooleincytochrome
Intensity (a.u.)
After 8th days (Fig.6.17):
mono_c_25
mono_c_30
mono_c_35
mono_c_40
mono_c_45
mono_c_50
mono_c_55
mono_c_60
mono_c_65
mono_c_70
mono_c_75
mono_c_80
mono_c_85
mono_c_90
1E7
1000000
100000
10000
1000
100
10
1
0.1
0.00 0.05 0.10 0.15 0.20 0.25
q (Å-1)
Fig.6.17: X-Ray diffraction patterns from monoolein-Cytochrome c at concentration
50 mg/ml of lipid and 25 mg/ml of protein at different temperature.
- 112 -
CHAPTER 6
Data Analysis: Temperature Effects
This sample shows the cubic phase Q212 at low temperature (barely
visible), Q229 and Hexagonal phase.
The unit cell parameters are respectively 164 Å, 115.1 Å and 72.5 Å.
This parameter change increasing the temperature: at 60°C is present the
cubic phase Q229 (115 Å) and Q212 (161 Å), while at higher
temperature remains only lamellar phase with the unit cell of 43.4 Å
(Fig.6.18).
a (Q229)
Q212
L
180
160
140
Unit cell ( )
Å
120
100
80
60
40
20
30
40
50
60
70
80
90
Temperature (°C)
Fig.6.18:Temperature dependence of the unit cell dimension of the monooleincytochrome
- 113 -
CHAPTER 6
Data Analysis: Temperature Effects
After 13th days (Fig.6.19):
mono_c_25
mono_c_35
mono_c_45
mono_c_55
mono_c_65
Intensity (a.u.)
1000
100
10
1
0.1
0.00 0.05 0.10 0.15 0.20 0.25
q (Å-1)
Fig.6.19: X-Ray diffraction patterns from monoolein-Cytochrome c at concentration
50 mg/ml of lipid and 25 mg/ml of protein at different temperature.
In this case is possible to note the present of Q212 and hexagonal phase,
that appear from 25°C, with unit cell of 165 Å and 73 Å (Fig.6.20).
Q 212
H
180
160
Unit cell ( )
Å
140
120
100
80
60
20
30
40
50
60
70
Temperature (°C)
Fig.6.20:Temperature dependence of the unit cell dimension of the monooleincytochrome
- 114 -
CHAPTER 6
Data Analysis: Temperature Effects
The hexagonal phase disappears at 55°C and remains only the cubic
phase that does not change in the unit cell up to 65°C.
6.5 Monoolein and Cytochrome c 50 mg/ml
In the 5th day (Fig.6.21):
Intensity (a.u.)
mono 25°C
mono 35°C
mono 45°C
mono 55°C
mono 65°C
0.00 0.05 0.10 0.15 0.20 0.25
q (Å-1)
Fig.6.21: X-Ray diffraction patterns from monoolein-Cytochrome c at concentration
50 mg/ml of lipid and 50 mg/ml of protein at different temperature.
At 25° and 35°C is the coexistence of Q224 (unit cell 87.9 Å) and Q229
(unit cell 113.9 Å ). When the temperature increases the hexagonal phase
take cubic phase’s place, in fact at 45°C we have Q229 (109.4 Å), Q224
(87.8 Å) and hexagonal phase (56.7 Å).
At 55°C the cubic phases Q224 and Q229 vanish and it appears the
Q212 (160.1 Å) and H (52.2 Å) that remain up to 65°C (Fig.6.22).
- 115 -
CHAPTER 6
Data Analysis: Temperature Effects
Q229
Q212
H
Q224
180
160
140
Unit Cell ( )
Å
120
100
80
60
40
25 30 35 40 45 50 55 60 65
Temperature (°C)
Fig.6.22:Temperature dependence of the unit cell dimension of the monooleincytochrome
After 8th days (Fig.6.23):
Intensity (a.u.)
mono 25°C
mono 30°C
mono 35°C
mono 40°C
mono 45°C
mono 50°C
mono 55°C
mono 60°C
mono 65°C
mono 70°C
mono 75°C
mono 80°C
mono 85°C
mono 90°C
0.00 0.05 0.10 0.15 0.20 0.25
q (Å-1)
Fig.6.23: X-Ray diffraction patterns from monoolein-Cytochrome c at concentration
50 mg/ml of lipid and 50 mg/ml of protein at different temperature.
- 116 -
CHAPTER 6
Data Analysis: Temperature Effects
In this case at low temperature is present the cubic phase Q212 (164 Å)
and hexagonal phase (73.3 Å) that with heat disappears.
Indeed at 70°C we have only the cubic phase Q212 with the unit cell of
157.7 Å. At higher temperature is present only the lamellar phase
(42.7Å) that at 90°C changes in FI (fluid isotropic) (Fig.6.24).
Q212
H
L
160
140
Unit cell ( )
Å
120
100
80
60
20
30
40
50
60
70
80
Temperature (°C)
Fig.6.24:Temperature dependence of the unit cell dimension of the monooleincytochrome
- 117 -
CHAPTER 6
Data Analysis: Temperature Effects
After 13th days (Fig.6.25):
Intensity (a.u.)
mono 25°C
mono 35°C
mono 45°C
mono 55°C
mono 65°C
0.00 0.05 0.10 0.15 0.20 0.25
q (Å-1)
Fig.6.25: X-Ray diffraction patterns from monoolein-Cytochrome c at concentration
50 mg/ml of lipid and 50 mg/ml of protein at different temperature.
In this sample we have note the presence of Q212
(160 Å) and
hexagonal phase (72.9 Å ) at low temperature. At 55°C remains the
cubic phase Q212 and the hexagonal phase but it changed the unit cell,
respectively of 156.7 Å and 71.8 Å. Increasing the temperature we have
the transformation in the fluid isotropic (Fig.6.26).
- 118 -
CHAPTER 6
Data Analysis: Temperature Effects
Q212
H
180
160
Unit cell ( )
Å
140
120
100
80
60
20 25 30 35 40 45 50 55 60
Temperature (°C)
Fig.6.26:Temperature dependence of the unit cell dimension of the monooleincytochrome
- 119 -
CHAPTER 7
Data Analysis:Mechanical Pressure Effects
CHAPTER 7
DATA ANALYSIS:MECHANICAL PRESSURE
EFFECTS
- 120 -
CHAPTER 7
Data Analysis:Mechanical Pressure Effects
The Influence of Pressure on the Lipid Phase Transitions
The last step of the work was to evaluate the influence of pressure on the
monoolein-water-cytochrome c system, and to observe the difference
with the results reported in the literature.
Lyotropic liquid crystalline phases originating from the self-assembly of
biologically relevant lipids in water are hierarchical complex assemblies.
Their nanostructures strongly depend on hydration level, lipid molecular
structure, and composition and depend of course on the experimental
conditions [4-9]. Among the experimental parameters, several reports on
the modulation of the nanostructure by pressure [6,12-19] temperature
[4,5,7,8,20-22] and pH value [24] were published. It has also been
demonstrated that salt concentration [25,27] and presence of peptides
[29-30] and proteins [32-34] play a vital role in controlling the lipidbased nanostructures.
In a review, Luzzati et al. issued [8] different pressure-dependent
processes and gave particular interest in the high-pressure effect on
different biologically relevant systems. Typical examples were the
pressure induced lipid bilayer-protein interactions and the unfolding
kinetics of protein [11].
Indeed, pressure significantly influences the fully hydrated lyotropic
liquid crystalline phases, as well as lipid membrane and lipid phase
- 121 -
CHAPTER 7
Data Analysis:Mechanical Pressure Effects
transitions [7,9,12,13]: as a general result, it can be observed that
pressure shows an opposite effect on structural transition in lipid systems
as compared to the influence of temperature [7,19].
The experiments related to the pressure effects on the monooleincytochrome c-water system have been performed at ESRF synchrotron in
Grenoble, using the pressure cell described in Chapter 4. Different
samples were prepared (see the list here reported) and measured after 2
and 10 days from the preparation. The following samples were prepared:
• mono 50 mg/ml + 1 mg/ml cytochrome c
• mono 50 mg/ml + 10 mg/ml cytochrome c
• mono 50 mg/ml + 25 mg/ml cytochrome c
• mono 50 mg/ml + 50 mg/ml cytochrome c
• mono 50 mg/ml + 100 mg/ml cytochrome c
and measured in a pressure range from 1 to 3000 bar. The results will be
here discussed considering separately the different concentrations.
7.1 Monoolein and Cytochrome c 1 mg/ml
Fig. 7.1 shows the X-ray diffraction profiles obtained from the sample
prepared with 1 mg/ml cytochrome c solution and measured after 2th
days from the preparation.
- 122 -
CHAPTER 7
Data Analysis:Mechanical Pressure Effects
At this composition, two different measures were made, the first using a
sample stored at room temperature (Mono_1A) and the second with a
sample stored at – 4 °C (Mono_1bis).
Mono1A sample shows at low pressure (128-512 bar) a cubic structure
belonging the 224 space group, with a unit cell of 95 Å. Increasing the
pressure, an unusual behaviour is detected, namely the presence of two
cubic phases with the same symmetry (Q224) but with different unit cell;
that can be seen as indication of inhomogeneity in the lipid system (e.g.,
the presence of low cytochrome c and high cytochrome c concentration
regions). Indeed, the Q224 cubic phases have unit cell of 103.3 Å and of
94.5 Å, perhaps due to the cytochrome c location into the rods. By
further increasing the pressure, only the low unit cell Q224 phase (95 Å)
remains.
- 123 -
CHAPTER 7
Data Analysis:Mechanical Pressure Effects
Intensity (a.u.)
Mono128
Mono256
Mono512
Mono1104
Mono1240
Mono2176
Mono2688
Mono3200
Å
0.1
-1
0.2
0.3
q( )
Fig.7.1: X-Ray diffraction patterns from monoolein-Cytochrome c at concentration
50 mg/ml of lipid and 1 mg/ml of protein at different pressure.
Q224 (*)
Q224
104
Intensity (a.u.)
102
100
98
96
94
0
700
1400 2100 2800 3500
Pressure (bar)
Fig.7.2: Pressure dependence of the unit cell dimension of the monooleincytochrome
- 124 -
CHAPTER 7
Data Analysis:Mechanical Pressure Effects
After 10th days from preparation (Fig.7.3), the monoolein sample shows
at low pressure (0-128 bar) the presence of a Q224 cubic phase (unit cell
of 100.9 Å) that coexists up to 512 bar whit a non-biconitnuous Q212
cubic phase (100.4 Å). When increasing the pressure up to 1400 bar, the
Q212 remains alone, whit a unit cell of 83.8 Å. At higher pressure (2016
bar), the hexagonal phase appears (the unit cell being 59.4 Å), but it
remains in coexistence whit the Q212 (83.8 Å) up to the higher
investigated pressures.
Intensity (a.u.)
Mono 128
Mono 512
Mono 768
Mono 1024
Mono 1408
Mono 1728
Mono 2016
0.1
0.2
0.3
0.4
Å
q ( -1)
Fig.7.3: X-Ray diffraction patterns from monoolein-Cytochrome c at concentration
50 mg/ml of lipid and 1 mg/ml of protein at different pressure.
- 125 -
CHAPTER 7
Data Analysis:Mechanical Pressure Effects
Unit cell ( )
Å
110
105
100
95
90
85
80
75
70
65
60
55
50
Q212
Q224
H
0
400
800
1200
1600
Pressure (bar)
Fig.7.4: Pressure dependence of the unit cell dimension of the monooleincytochrome
7.1.1 Monoolein and Cytochrome c 1mg/ml (bis)
This sample (Fig.7.5), preserved at -4°C, shows several transitions
including a Q230 cubic phase and a lamellar phase. At low pressure (128
bar) it possible to see only the Q224 (103 Å), which coexists with a
lamellar phase (40 Å) when the pressure increases up to 250 bar (the cell
parameter of the cubic phase becomes 106 Å). This transition is followed
at 1300 bar by the formation of a Q230 cubic phase (unit cell of 154 Å)
which coexists with a Q224 cubic phase (100 Å) and a lamellar phase
(40 Å). At pressures of 1500-1600 bar, both Q224 and lamellar phases
disappear and only the Q230 (with a unit cell of 145 Å) remains.
- 126 -
CHAPTER 7
Data Analysis:Mechanical Pressure Effects
Intensity (a.u.)
Mono 128
Mono 512
Mono 1280
Mono 1536
Mono 1664
Mono 2048
Mono 2368
0.2
q ( -1)
Å
0.1
0.3
Fig.7.5: X-Ray diffraction patterns from monoolein-Cytochrome c at concentration
50 mg/ml of lipid and 1 mg/ml of protein at different pressure.
Intensity (a.u.)
The plot below shows the trend of unit cell.
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0
Q224
Q230
L
0
400 800 1200 1600 2000 2400
Pressure (bar)
Fig.7.6: Pressure dependence of the unit cell dimension of the monooleincytochrome
- 127 -
CHAPTER 7
Data Analysis:Mechanical Pressure Effects
7.2 Monoolein and Cytochrome c 5 mg/ml
After 2th days, the sample prepared with the Cytochrome c solution at 5
mg/ml (Fig.7.7) has a unique structural behaviour, showing at each
pressure a Q224 cubic phase (101 Å). Probably, the cytochrome c was
unable to enter the phase. The sample after 10 days from preparation was
not analysed.
Intensity (a.u.)
Mono 128
Mono 512
Mono 1104
Mono 1536
Mono 2048
Mono 3200
0.1
0.2
1
-
0.0
Å
q(
0.3
)
Fig.7.7: X-Ray diffraction patterns from monoolein-Cytochrome c at concentration
50 mg/ml of lipid and 1 mg/ml of protein at different pressure.
7.3 Monoolein and Cytochrome c 10 mg/ml
The sample measured after 2th days from preparation shows several
transitions, which include the formation of a Q230 cubic phase and of a
high-pressure Q224 cubic phase characterized by a small unit cell
(Fig.7.8).
- 128 -
CHAPTER 7
Data Analysis:Mechanical Pressure Effects
Intensity (a.u.)
Mono 16
Mono 128
Mono 768
Mono 1408
Mono 2048
0.2
q ( -1)
0.3
Å
0.1
Fig.7.8: X-Ray diffraction patterns from monoolein-Cytochrome c at concentration
50 mg/ml of lipid and 1 mg/ml of protein at different pressure.
At low pressures, it is possible to note the presence of a Q224 (unit cell
around 103 Å). By increasing the pressure, the Q224 unit cell suddenly
decreases (up to 99 Å), but a Q230 cubic phase (unit cell 154 Å) appears.
At high pressures, only the Q224 cubic phase (97 Å) is present.
The plot below shows the trend of unit cell.
Q224
Q230
160
Intensity (a.u.)
150
140
130
120
110
100
90
0
500
1000
1500
2000
Pressure (bar)
Fig.7.9: Pressure dependence of the unit cell dimension of the monooleincytochrome
- 129 -
CHAPTER 7
Data Analysis:Mechanical Pressure Effects
In the X-ray diffraction profiles of the sample measured after 10th days
from preparation (Fig.7.10) it is possible to notice the presence of a
Q229 cubic phase (unit cell of 130.6 Å) existing from low pressures up
to 500 bar. By increasing the pressure, we observe the coexistence of the
Q229 (130.6 Å) and Q212 (120.3 Å) cubic phases. This situation
changes when the pressure is around 1500 bar, as only the Q212 cubic
phase (143.3 Å) is detected.
Intensity (a.u.)
Mono 1
Mono 128
Mono 512
Mono 1500
Mono 2899
0.1
0.2
0.3
0.4
Å
q ( -1)
Fig.7.10: X-Ray diffraction patterns from monoolein-Cytochrome c at concentration
50 mg/ml of lipid and 1 mg/ml of protein at different pressure.
- 130 -
CHAPTER 7
Data Analysis:Mechanical Pressure Effects
Q212
Q229
145
140
Unit cell ( )
Å
135
130
125
120
0
400
800
1200
1600
Pressure (bar)
Fig.7.11: Pressure dependence of the unit cell dimension of the monooleincytochrome
7.3 Monoolein ad Cytochrome c 25 mg/ml
The X-ray diffraction plots of the sample measured after 2th day from the
preparation (Fig.7.12) shows a different transition path leading to the
formation of the Q212 cubic phase. At low pressure, a Q224 cubic phase
(with unit cell of 107 Å) is present. On increasing pressure, at 800 bar,
the Q212 cubic phase (unit cell around 118 Å) appears, and persists in
equilibrium with the Q224 cubic phase up 2300 bar. A further increase
of pressure induces the formation of a hexagonal phase (54 Å) that is still
detected up to 3000 bar.
- 131 -
CHAPTER 7
Data Analysis:Mechanical Pressure Effects
Intensity (a.u.)
Mono 1
Mono 128
Mono 768
Mono 1536
Mono 1998
Mono 2210
Mono 2976
0.2
q ( -1)
0.3
Å
0.1
Fig.7.12: X-Ray diffraction patterns from monoolein-Cytochrome c at concentration
50 mg/ml of lipid and 1 mg/ml of protein at different pressure.
The plot below shows the trend of unit cell.
Q212
Q224
H
140
Intensity (a.u.)
120
100
80
60
40
0
700
1400
2100
2800
pressure (bar)
Fig.7.13: Pressure dependence of the unit cell dimension of the monooleincytochrome
- 132 -
CHAPTER 7
Data Analysis:Mechanical Pressure Effects
The sample after 10th days from preparation was not analysed.
7.4 Monoolein and Cytochrome c 50 mg/ml
The X-ray diffraction profiles of the sample measured after 2th days from
preparation are shown in Fig.7.14. Different transitions that lead to the
formation of lamellar and Q230 cubic phases can be observed.
Intensity (a.u.)
Mono 128
Mono 512
Mono 1104
Mono 1536
Mono 2048
Mono 3200
0.0
0.1
0.2
0.3
Å
q ( -1)
Fig.7.14: X-Ray diffraction patterns from monoolein-Cytochrome c at concentration
50 mg/ml of lipid and 1 mg/ml of protein at different pressure.
We start with the Q224 cubic phase (112 Å), observed pure at low
pressures, but increasing the pressure a coexistence with hexagonal (58.9
Å) and Q230 cubic (167 Å) phases is detected. At 1000 bar, the Q224
(113.9 Å) tends to disappear and only the hexagonal phase (58 Å) and
the Q230 cubic phase (162 Å) remain. At higher pressures, it is possible
- 133 -
CHAPTER 7
Data Analysis:Mechanical Pressure Effects
to notice three phases: the hexagonal, the lamellar and the Q230 cubic
phases.
The plot below shows the trend of unit cell.
Unit cell︵ )
Å
200
180
160
140
120
100
80
60
40
Q230
L
H
Q224
0
400
800
1200
1600
2000
Pressure (bar)
Fig.7.15: Pressure dependence of the unit cell dimension of the monooleincytochrome
Data for the sample measured after 10th days, reported in Fig.7.16, show
that at low pressures (0-512 bar) we have the presence of the Q224 cubic
phase (99.5 Å) and of the hexagonal phase (55.5 Å). These are
supplemented by the formation of a lamellar phase (49.3 Å) when the
pressure reaches 1024 bar. The Q224 transforms in a Q212 1500 bar and
then everything remains unchanged until a over high pressure.
- 134 -
CHAPTER 7
Data Analysis:Mechanical Pressure Effects
Intensity (a.u.)
Mono 128
Mono 512
Mono 1024
Mono 1400
Mono 2000
Mono 2988
0.1
0.2
0.3
0.4
Å
q ( -1)
Fig.7.16: X-Ray diffraction patterns from monoolein-Cytochrome c at concentration
50 mg/ml of lipid and 1 mg/ml of protein at different pressure.
Q224
H
L
110
Intensity(a.u.)
100
90
80
70
60
50
40
0
700
1400
2100
2800
Pressure (bar)
Fig.7.17: Pressure dependence of the unit cell dimension of the monooleincytochrome
7.4 Monoolein and Cytochrome c 100 mg/ml
This sample has been analysed only 10th days after preparation. Fig.7.18
shows that a hexagonal phase (72.5 Å) exists at low pressures (1-512
bar) and then a Q212 cubic phase (158 Å) forms when the pressure
- 135 -
CHAPTER 7
Data Analysis:Mechanical Pressure Effects
increases up to 500 bar. At pressures higher than 1000 bar the hexagonal
phase is only barely visible.
Intensity (a.u.)
Mono 1
Mono 128
Mono 512
Mono 1000
Mono 2000
Mono 2988
0.1
0.2
0.3
0.4
Å
q( -1)
Fig.7.18: X-Ray diffraction patterns from monoolein-Cytochrome c at concentration
50 mg/ml of lipid and 1 mg/ml of protein at different pressure.
Q212
H
160
Unit cell ( )
Å
140
120
100
80
60
0
700
1400
2100
2800
Pressure (bar)
Fig.7.19: Pressure dependence of the unit cell dimension of the monooleincytochrome
- 136 -
CHAPTER 7
Data Analysis:Mechanical Pressure Effects
In the plot above it shows the progression of unit cell of sample with the
highest concentration of cytochrome c.
- 137 -
CHAPTER 8
Discussion and Conclusion
CHAPTER 8
DISCUSSION AND CONCLUSION
- 138 -
CHAPTER 8
Discussion and Conclusion
8.1 DISCUSSION ON CYTOCHROME C CONCENTRATION
EFFECTS
The addition of cytochrome c in a solution containing monoolein has
different effects, which depend on protein concentration. According to
the structural properties, the protein needs many days (up to 7) to
promote a phase transition from a Q224 (Pn3m) to a Q229 (Im3m) in the
monoolein/water system, even though his solubilisation in the water
regions inside the cubic phase (e.g., the aqueous rods) already takes
place since the first time after sample preparation.
In samples containing various amounts of cytochrome c, the time
kinetics and then the occurrence of phase transitions have its own timing,
and in a few cases not necessarily we notice the presence of Q229 (ex.510 mg/ml of cytochrome c). This demonstrates the metastability of the
different phases and the non-equilibrium state of the whole system.
It is possible to start the discussion from the monoolein/water sample
prepared in the presence of 1 mg/ml of cytochrome c (Fig. 5.1). By
successive sampling, the structure of the sample has been studied as a
function of time. At the beginning, the sample showed a Q224 cubic
structure, which maintains the characteristic unit cell of 104-105 Å, as
observed in monoolein fully hydrated in pure water. The Q224 cubic
phase is observed up 6 days from the preparation. After 7th days, in the
diffraction profile appears a new cubic phase, with Im3m symmetry and
- 139 -
CHAPTER 8
Discussion and Conclusion
with a unit cell dimension of 140 Å. The scattering profile and the unit
cell parameter remain constant until 10 days of measurement.
This behaviour indicates that the cytochrome c has entered into the
phase so that the structure has been modified: the phase is still
bicontinuous, but the rod intersection has been changed from 4x4
(observed in the Q224) to 6x6 (observed in the Q229), with the
consequent increase of the unit cell [9]. The spectroscopic analysis
(Fig.5.4) reveals that the cytochrome c concentration inside the cubic
phase increases as a function of time, until a constant value of 0.8 mg/ml
is observed after 7 days.
The same transition does not occur in other samples, as previously
mentioned. In particular, monoolein samples prepared in excess
cytochrome c solutions at 5 and 10 mg/ml (Fig.5.5 -5.8) show the Q224
cubic structure, even if there is an increase of the unit cell parameter
which demonstrates the entrance of the protein in the aqueous rods (also
confirmed by spectrophotometry). Very probably, in these cases the
cytochrome c would have needed of larger time to cause the phase
transition.
Data from the 25 and 50 mg/ml samples are interesting: in both cases,
there is the prevalence of the Q224 phase, but in few profiles the Q229
cubic phase appears. In any case, however, the Q224 unit cell increases,
- 140 -
CHAPTER 8
Discussion and Conclusion
suggesting the presence of the cytochrome c inside the phase (Fig.5.135.15).
It should be observed that the appearing or disappearing of Q224 or
Q229 cubic phases should not be considered so strange: in fact, the many
diffraction measurements done indicate that inside the same monoolein
sample there are a few parts with a Pn3m structure and separate parts
with an Im3m structure. Therefore, according to the sample preparation,
we could meet a phase or the other one. In other words, samples are nonhomogeneous, as the complete transition probably occurs only after a
longer time.
In samples at the higher cytochrome c concentrations, the lack of a stable
transition is due to an excess of protein, which probably induces a series
of local and dramatic structural events (transition to H phase or to other
non-bicontinuous cubic phase), which prevents the entrance of the
protein in the rods and then the occurrence of a homogeneous and
continuous phase transition.
These results are in fact in compliance with the measurements make on
the Campinas Synchrotron (LNLS) using a monoolein sample prepared
in a 100 mg/ml cytochrome c solution. The structure of the sample was
investigated after 7 days from the preparation. In this case, a definite and
stable transition is present (Fig.5.16). From the plot, it is possible to
- 141 -
CHAPTER 8
Discussion and Conclusion
notice the difference of the structure between monoolein in water (Q224)
and the monoolein-water-cytochrome c system (Q229).
8.2. DISCUSSION ON TEMPERATURE EFFECTS
The structural analyses performed as a function of temperature have
pointed out that also the thermal effects depend on the passing of time
from sample preparation. Time kinetics was indeed important as we can
notice that there is a structure evolution, which takes the formation of the
Q229 cubic phase and of the other phases according to the sample taken
into consideration.
As a result, it has been possible to draft the time-temperature phase
diagram for the monoolein-water-cytochrome c system to various
concentrations of protein, and every sample has its own behaviour.
From the phase diagram observed in the monoolein sample prepared in
the presence of 1 mg/ml cytochrome c solution (Fig.8.1), it is possible to
notice the initial presence, until 8 days, of the diamond phase, which
remains stable also at high temperatures (65°C). This is a rather
anomalous event, as monoolein in water (Fig.3.3) showed the formation
of an hexagonal phase in equilibrium with water already at 50°C. In this
case, the cytochrome c seems to stabilize or physically block the rods,
- 142 -
CHAPTER 8
Discussion and Conclusion
preventing the arrangement in long cylinders, which are characteristic of
the hexagonal phase.
Afetr 13th days from preparation, a coexistence of Q224 and Q229 cubic
phases is detected: the transition is then acting, but does seem to be
influenced by temperature, as at 65°C the Im3m phase disappears and
monoolein reorganizes in a Pn3m.
1 mg/ml
Q224
Q224 + Q229
Temperature (°C)
60
50
40
30
20
4
6
8
10
12
14
Days
Fig.8.1: Phase Diagram of sample 1 mg/ml cytochrome c (50 mg/ml Monoolein)
The phase diagram for the sample prepared with a 5 mg/ml cytochrome c
solution (Fig.8.2) shows a more complex behavior. After 8 days from the
preparation, a Q229 cubic phase, which remains stable up to 55 °C when
it comes in coexistence with a Hexagonal phase (as reported the phase
- 143 -
CHAPTER 8
Discussion and Conclusion
diagram of monoolein Fig.3.3), is detected. At 65 °C, only the hexagonal
phase remains, losing the co-existence with the primitive phase. The
behavior is different after 13 days from preparation: the sample shows a
coexisting Q224 and Q229 cubic phases at room temperature, while by
increasing the temperature, the Q229 becomes predominant. At 55 °C, a
hexagonal phase appears. In this case, the temperature advances the
hexagonal phase compared with the measurement made in previous
days.
5 mg/ml
Q229
Hex
Hex + Q229
Q224 + Q229
Temperature (°C)
60
50
40
30
20
4
6
8
Days
10
12
14
Fig.8.2: Phase Diagram of sample 5 mg/ml cytochrome c (50 mg/ml Monoolein)
The phase diagram for the sample prepared in the presence of 10 mg/ml
cytochrome c solution is shown in Fig.8.3. It can be observed that after 5
days from preparation the structure is Q229 at room temperature, and
- 144 -
CHAPTER 8
Discussion and Conclusion
this structure is maintained up to 45 °C. At this temperature, an
hexagonal phase forms, but it coexists with the cubic phase on heating,
as happened in the previous sample (Fig.8.2). After 8 days from
preparation, the trend is the same, but reaching higher temperatures (up
to 85 °C) we can notice only the presence of the hexagonal phase. As
previously observed, after 13 days from the preparation, the Q229 cubic
phase coexists with the Q224 cubic phase. Also at this concentration, the
increase of temperature induces the disappearing of the Q224 cubic
phase, and, at higher temperature, the formation of a hexagonal phase.
70
10 mg/ml
Q229
Hex
Hex + Q229
Q224 + 229
Temperature (°C)
60
50
40
30
20
4
6
8
Days
10
12
14
Fig 8.3: Phase Diagram of sample 10 mg/ml cytochrome c (50 mg/ml Monoolein)
- 145 -
CHAPTER 8
Discussion and Conclusion
The phase diagrams of monoolein samples prepared at the higher
cytochrome c concentrations are very complex. Fig.8.4 shows that after 5
days, the sample containing the 25 mg/ml cytochrome c solution at low
temperature has a Q229 structure. As previously, heating induces the
formation of a hexagonal phase, followed however by the appearance of
a Q212 cubic structure. After 8 days from the preparation, the Q229 and
the hexagonal phases are already coexistent at room temperature, and the
phase Q212 immediately form by increasing temperature. At higher
temperatures, only the Q212 cubic phase remains. After 13 days at
preparation, the sample shows a clear predominance of the phase Q212,
which coexists with a hexagonal phase at low temperatures and occurs
pure at temperatures higher than 40 °C.
70
25 mg/ml
Q229
Q212
Q212 + Q229
Hex + Q229
Hex + Q212
Hex + Q212 +Q229
Temperature (°C)
60
50
40
30
20
4
6
8
10
12
14
Days
Fig 8.4: Phase Diagram of sample 25 mg/ml cytochrome c (50 mg/ml Monoolein)
- 146 -
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Discussion and Conclusion
Finally, the sample with the highest concentration of protein (Fig.8.5)
shows a Q224 and Q212 cubic phases at room temperature, and,
increasing the temperature, a hexagonal and a Q212 phases. Over 55 °C,
only the Q212 cubic phase remains, probably mixed with an isotropic
fluid. As a function of time, the phase behavior is similar, with the
exception that the co-existence between the hexagonal and the Q212
cubic phases, appears at high temperatures for the sample measured after
8 days from preparation, but anticipated in the last sample (measured
after 13 days from preparation).
50 mg/ml
Q212
Hex + Q212
Hex + Q229
Q224 + Q212
Temperature (°C)
60
50
40
30
20
4
6
8
Days
10
12
14
Fig 8.5: Phase Diagram of sample 50 mg/ml cytochrome c (50 mg/ml Monoolein)
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Discussion and Conclusion
To summarize what has been dicussed until now, the different phase
diagrams obtained at different concentrations as a function of time from
sample preparation and temperature were constructed.
At 5th days from preparation, the phase diagrams is shown in Fig.8.6,
while those referring to 8th days and 13th days after preparation are
reported in Fig. 8.7 and 8.8.
70
Q224
Q229
Q212
Q229+Q212
Hex+Q212
Hex+Q212+Q229
Temperature (°C)
60
50
40
30
20
0
10
20
30
40
Concentration (mg/ml)
Fig 8.6: Phase Diagram at 5 days.
- 148 -
50
60
CHAPTER 8
Discussion and Conclusion
70
Q224
Q229
Q212
Hex
Q229+Q212
Hex+Q229
Hex+Q212
Hex+Q212+Q229
Temperature (°C)
60
50
40
30
20
0
10
20
30
40
50
60
Concentration (mg/ml)
Fig 8.7: Phase Diagram at 8 days.
70
Temperature (°C)
60
Q224
Q212
Q229
Hex+229
Hex+Q212
Q224+Q229
50
40
30
20
0
10
20
30
40
50
60
Concentration (mg/ml)
Fig 8.8: Phase Diagram at 13 days.
As a general result, and with a large prudence due to metastability
effects, it can be observed that cytochrome c definitively induces a
rearrangement of the Q224 cubic phase observed in the pure monoolein
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Discussion and Conclusion
in excess of water. The structural effects occur even when the protein
concentration is very low, providing a sufficient time for penetration of
the protein into the water region inside the cubic phase (e.g., inside the
water rods of the cubic phase).
The first phase transition is related to the formation of the Q229 cubic
phase, which is quite stable even at high temperature. It can be observed
that the time required to induce the formation of the Q229 cubic phase is
in inverse proportion to the protein concentration of the bathing solution,
suggesting that the phase transition is controlled by the slow diffusion of
the protein in the lipid medium. However, the presence of cytochrome c
inside the lipid phase also stabilizes the hexagonal phase, which in fact
occurs at lower temperature than that detected in the monoolein-water
system.
Further extending the equilibrium time, a second phase transition occurs:
indeed, the cytochrome c induces the formation of the Q212 cubic phase.
It can be recalled that the structure of phase Q212 can be described
conveniently by reference to phase Q230. As depicted in the first
chapter, one of the two 3-D rod networks of Q230 is preserved in Q212,
whereas the other network is replaced by a set of identical quasispherical globules, each centered on every second three-rod junction:
four globules are contained in the unit cell. By analogy with the phases
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Discussion and Conclusion
Q224 and Q230 of the system MO-water, it may be presumed that the
rods are filled by the polar moiety, coated by the polar headgroups of the
lipid molecules and embedded in the hydrocarbon matrix, and that each
of the quasi-spherical globules of Q212 contains one (or, on the average,
a fraction of) hydrated protein molecule, surrounded by lipid molecules
whose polar head-groups are oriented towards the protein. This phase is
stable at the higher investigated temperatures, while, rather surprisingly,
it coexists with a hexagonal phase at low temperature.
At present it is difficult to deduce the mechanism for the phase
transitions, as well as to derive the role of the different molecular
parameters (as monoolein surface area at the lipid-water interface, cross
section at the pivotal plane and monolayer thickness, Gaussian or Mean
curvatures, which have been all calculated in all the different analyzed
experimental conditions, but non reported in the present thesis for the
sake of brevity) or of the hydration in the stability of the different
phases. Further model analysis will be necessary: at moment, we can
only suggest that cytochrome c modifies the curvature energy by direct
interaction, stabilizing phases with larger curvatures, and possibly
without saddle surfaces.
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Discussion and Conclusion
8.3. DISCUSSION OF MECHANICAL PRESSURE EFFECTS
The structural analysis performed on the monoolein-cytochrome c-water
system as a function of pressure showed different structural behaviours,
both in samples prepared at different concentrations of protein and
measured after different times from the sample preparation.
As a result, it has been possible to sketch the pressure-dependent phase
diagram for monoolein samples prepared with different cytochrome c
solutions and analyzed by X-ray diffraction after 2 and 10 days from
preparation.
The phase diagram derived from data obtained after 2 days from sample
preparation is reported in Fig.8.9. The large persistence of the Q224
cubic phase can be noticed. Moreover, it should be recalled that at the
lower cytochrome c compositions a Q224-Q224 cubic phase transition is
detected, as also indicated by an abrupt decrease of the unit cell
parameter. It can be suggested that pressure determines a kind of
squeezing out of the protein from the lipid media, which determines a
drop of the cubic unit cell due to a reduction of the steric hindrance
inside the aqueous compartment and/or to an increase of the osmotic
pressure exerted by the cytochrome c bulk solution. The diamond phase
is also present in the samples prepared using the higher concentrated
cytochrome c solutions, but pressure induces the appearing of a series of
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Discussion and Conclusion
different phases, as the Q230 cubic phase (expected, if the temperatureconcentration phase diagram of the monoolein in water is considered)
and hexagonal and lamellar phases.
The phase diagram describing the structural behavior observed under
mechanical pressure after 10 days from the sample preparation is
reported in fig. 8.9. At low pressures, the phase diagram is dominated by
the two Q224 and Q229 bicontinuous cubic phases, which are relatively
unstable under compression. Indeed, at any cytochrome c concentration,
pressure induces the formation of the non-bicontinuous Q212 cubic
phase (alone or in the presence of hexagonal or lamellar phases), which
curiously results stabilized both by high temperature and by high
pressure. Accordingly, at very high cytochrome c content, the phase
sequence observed by increasing temperature or by increasing pressure is
very similar.
As previously discussed, the different molecular parameters calculated as
a function of pressure in all the different detected structures (data not
shown for the sake of brevity) are not useful to establish the mechanism
for the observed phase transition or to derive the factors affecting or
controlling the stability the different phases. In particular, the
exceptional stability of the Q212 cubic phase is really astonishing, and
can be only explained considering that cytochrome c can modify the
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Discussion and Conclusion
curvature energy both by direct interactions and by the osmotic pressure
exerted by the bulk solution. Saddle surfaces appear very unstable; since
the lipid component is chemically homogeneous in this phase, it can be
concluded that the heterogeneity of the structure elements induced by
pressure (e.g., the transition form bicontinuous cubic phases to the Q212
cubic phase) can indeed be explained by a difference in composition
between the two type of elements (rods and micelles) probably related to
a different affinity of cytochrome c to surfaces with negative Gaussian
curvature with respect to convex/elliptical surfaces which have positive
Gaussian curvature. The squeezing out observed in the Q224 at low
equilibrium time seems to confirm the following picture: the
compression of samples equilibrated for a long time induces the
removing of cytochrome c mainly from one of the two aqueous
continuous media, so that one of the two 3-D networks is preserved
(even if dehydration determines the transition to a Q230-type network),
while the local increased protein concentration determine the formation
of inverse micelle sets which replace the second network. Once formed,
this phase appears very stable. Further model analyses will be necessary
to confirm these suggestions.
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Discussion and Conclusion
2500
Q224
Q224*
Hex+Q230
Hex+Q224+Q230
Q224+Q230
Hex+Q230+Lam
Pressure (bar)
2000
1500
1000
500
0
0
20
40
60
Concentration (mg/ml)
Fig 8.9: Phase Diagram at 2 days.
2500
Q224+Q212
Q212
Q224
Q229
Q229+Q212
Hex+Q229
Hex+Q224
Hex+Q224+L
Hex+Q212+L
Hex
Hex+Q212
Q224+Q224*
Q224+Q224*+Hex
Q224*+Hex
Pressure (bar)
2000
1500
1000
500
0
-20
MO 0
20
40
60
80
Concentration (mg/ml)
Fig 8.10: Phase Diagram at 10 days
- 155 -
100
120
CHAPTER 9
Application of Cubic Phase
CHAPTER 9
APPLICATIONS OF CUBIC PHASES
- 156 -
CHAPTER 9
Application of Cubic Phase
Finally, we have studied several applications of cubic phases: there are
two examples, the first work was done in collaboration with the
Department of Pharmaceutical Sciences University of Ferrara (9.1), the
next study was performed in collaboration with the Department of
Science Chemistry, University of Cagliari (9.2).
The part of the work that our session was occupied is the x-ray
diffraction and data analysis.
9.1 Nanoparticulate lipidic dispersions for bromocriptine
delivery: a comparative study
9.1.1 INTRODUCTION
Lipidic dispersions have attracted significant attention in the literature
due to their potential application as matrixes able to solubilize active
molecules, to deliver them in a controlled fashion, reducing side effects
and improving bioavailability [78,79].
Solid lipid nanoparticles (SLN) dispersions are a new generation of
delivery systems whose nanodisperse phase has a solid matrix of
crystalline solid lipids, able to protect encapsulated molecules from
degradation and to modulate their release [80-81]. A particular type of
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Application of Cubic Phase
SLN is represented by nanostructured lipid carriers (NLC), composed of
a solid lipid matrix with a certain content of a liquid lipid phase [78]. For
instance the use of tricaprin, liquid at room temperature, in mixture with
a solid lipid such as tristearin leads to the formation of solid carriers with
homogenous lipid nanocompartments (NLC) [82-83].
The emulsification in water of surfactant-like lipid gives rise to aqueous
nanostructured dispersions of complex lyotropic liquid crystalline phases
(lamellar, hexagonal, and cubic structure) [84]. In particular monoolein
aqueous dispersions (MAD) stabilized by the addition of a block
copolymer like pluronic F127 are mainly constituted of dispersed
nanoparticles such as cubosomes and hexosomes often in coexistence
with vesicles [85]. The predominance of one nanostructure in respect to
another is related to the temperature of the system that induces phase
transition [86].
Cubosomes are nanostructured particles of cubic liquid crystalline
phases dispersed in water.
The inner structure of cubosomes has a cubic crystallographic
symmetry, due to the self-assembly of amphiphilic or surfactant-like
molecules [87]. Cubosomes can be defined as thermodynamically stable
bicontinuous structures with two distinct regions of water separated by a
contorted bilayers. Cubosomes often coexists with vesicles [88].
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Application of Cubic Phase
Hexasomes are particles of hexagonal shape with an inner structure with
hexagonal symmetry and/or curved concentric striations [89].
MAD represent a newer attractive delivery system. The methods of
MAD production [90-91] and the inner structure of dispersed
nanoparticles [92-93] have been widely investigated, nevertheless few
work has been done until now to study drug release from these systems
[94-96].
Moreover to our knowledge there is a lack of data about comparison
between various type of nanoparticulate lipidic dispersions as drug
delivery systems [95].
Recently the development of lipid nanosystems have been proposed in
the field of brain disease therapy [97-98]. The pharmacological treatment
of central nervous system diseases, such as brain tumors, neurological
and psychiatric disorders, is often confined by the inability of potent
drugs to pass the blood brain barrier (BBB) [99-100] . BBB significantly
restricts water-soluble, charged and high molecular weight therapeutics
to the vascular space while allowing brain parenchyma penetration of
small and/or lipophilic molecules. Multiple strategies have been
employed to circumvent the BBB. An emerging approach is the use of
colloidal carriers [101-102], which allow brain penetration to nontransportable drugs by masking their physico-chemical characteristics. In
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Application of Cubic Phase
fact when drug is loaded, colloidal carriers offer clinical advantages such
as decreased drug dose, reduced drug side effects, increased drug
viability, non invasive routes of administration and improved patient
quality of life [103].
In a recent study [104] the group of University in Ferrara have
demonstrated the potential of using SLN as bromocriptine (BC) delivery
system to treat Parkinson’s disease (PD).
The aim of this study is to compare the potential of different lipidic
dispersions namely MAD and SLN, as delivery systems for BC, keeping
particular attention to their performances for PD therapy. The feasibility
of using MAD or SLN as BC controlled delivery formulation is
demonstrated through intensive characterization of morphology, size, BC
encapsulation, BC release and in vivo activity in parkinsonian rats.
9.1.2 Materials and methods
9.1.2.1 Materials
The glyceryl monooleate RYLO MG 19 (MO) was a gift from Danisco
Cultor (Grindsted, Denmark). Pluronic F127 (Poloxamer 407) (PEO98POP67-PEO98) was obtained from BASF (Ludwigshafen, Germany).
Carbopol 934P (CTFA: Carbomer) was from BFGoodrich (Cleveland,
OH, USA).
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Application of Cubic Phase
Lutrol F 68, oxirane, methyl- , polymer with oxirane (75;30) (poloxamer
188) was a gift of BASF ChemTrade GmbH (Burgbernheim, Germany).
Tristearin, stearic triglyceride (tristearin) was provided by Fluka (Buchs,
Switzerland). Miglyol 812, caprylic/capric triglycerides (tricaprin) was
purchased from Eigenmann & Veronelli (Rho, Milano, Italy). Mivaplex
600, stearic monoglyceride (monostearin) was kind gift of Eastman Ch.
Company (USA). Compritol 888 ATO is a mixture of approximately
15% mono-, 50% di- and 35% triglycerides of behenic acid (C22)
(tribehenin); it was provided by Gattefossé (Saint Priest-France).
Bromocriptine mesylate (2-Bromo-∝-ergocriptine methansulfonate salt)
(BC) was obtained from Sigma (Steinheim, Germany). Amphetamine
and 6-OHDA were purchased from Sigma Chemical Company (St Louis,
MO, USA).
9.1.2.2 MAD preparation
Production of dispersions was based on the emulsification of MO (4.5%
w/w) and Poloxamer 407 (0.5% w/w) in water (90%, w/w), as described
by Esposito et al. [105]. In the present study, after emulsification, the
dispersion was subjected to homogenization at 15,000 rev min-1 (Ultra
Turrax, Janke & Kunkel, Ika-Werk, Sardo, Italy) at 60°C for 1 min;
afterwards, it was cooled and maintained at room temperature in glass
vials.
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Application of Cubic Phase
In the case of BC-containing dispersions, 12.5 mg of the drug (0.27%
w/w with respect to the monooleine, 0.025% w/w with respect to the
dispersion) was added to the molten MO/ poloxamer solution and
solubilized before adding to the aqueous solution.
The dispersion was then filtered through mixed esters cellulose
membrane (0.6-mm pore size) to separate big MO/poloxamer
aggregates. Dispersion characterization as well as in vitro and in vivo
experiments were performed on the MO dispersions after filtration,
without taking into account the fraction of larger particles whose
dimensions have been measured by laser diffraction (Horiba, LA-920,
Horiba Ltd., Tokyo, Japan). For in vitro and in vivo experiments, a blank
MO formulation and free drug were used to prepare controls. In
particular, a weighted amount of BC has been added to 50 ml of a
filtered MO dispersion and subjected to magnetic stirring (250 rev min-1)
for 1 h.
9.1.2.3 SLN preparation
SLN were prepared by stirring, followed by ultrasonication [104].
Briefly, 1g of lipidic mixture was melted at 75°C. The lipidic mixture
was constituted of tristearin/tricaprin 2:1 w/w. The fused lipid phase was
dispersed in 19 ml of an aqueous poloxamer 188 solution (2.5 % w/w).
The obtained emulsion was subjected to ultrasonication (Microson
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TM
,
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Application of Cubic Phase
Ultrasonic cell Disruptor) at 6.75 kHz for 15 min and then cooled down
to room temperature by placing it in a water bath at 22 °C. SLN
dispersions were stored at room temperature.
In the case of BC-containing dispersions, 5 mg of the drug (0.025% w/w
with respect to the total dispersions, 0.5% w/w with respect to the lipid
phase) were added to the molten lipidic mixture and dissolved before
adding to the aqueous solution.
9.1.2.4 Characterization of lipidic dispersions
9.1.2.4.1 Photon Correlation Spectroscopy (PCS)
Submicron particle size analysis was performed using a Zetasizer 3000
PCS (Malvern Instr., Malvern, England) equipped with a 5 mW helium
neon laser with a wavelength output of 633 nm. Glassware was cleaned
of dust by washing with detergent and rinsing twice with water for
injections. Measurements were made at 25 °C at an angle of 90°. Data
were interpreted using the “method of cumulants” [106].
9.1.2.4.2 Cryo-Transmission Electron Microscopy (Cryo-TEM)
Samples were vitrified as described in a previous study by Esposito et al.
[104]. The vitrified specimen was transferred to a Zeiss EM922
transmission electron microscope for imaging using a cryoholder
(CT3500, Gatan). The temperature of the sample was kept below -175
°C throughout the examination. Specimens were examined with doses of
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Application of Cubic Phase
about 1000-2000 e/nm2 at 200 kV. Images were recorded digitally by a
CCD camera (Ultrascan 1000, Gatan) using a image processing system
(GMS 1.4 software, Gatan). A drop of dispersion prepared for TEM
measurements was placed on a bare copper grid and plunge frozen in
liquid ethane at approximately 100 K. The sample was transferred into a
cryo electron microscope (CEM902a, Zeiss, D-Oberkochen, Philips
CM120, NLEindhoven) operated at 80 kV respectively 120 kV. Samples
were viewed under lowdose conditions at a constant temperature around
77–100 K. Images were acquired by a Dage SIT low intensity TV
camera system and processed by a Kontron IBAS image processing
system in the case of the Zeiss CEM902A and a Tietz Fastscan CCD
camera system for the Philips CM120.
9.1.2.4.3 X-ray diffraction measurements
Low angle X-ray scattering experiments were performed at the DESY
synchrotron facility in Hamburg, Germany, on the A2 beamline. The
investigated Q-range (Q = 4ππ sin θ / λ, where 2θ is the scattering angle
and λ = 1.50 Å the X-ray wavelength) was 0.02-0.35 Å-1. Experiments
were run on the different samples as a function of the temperature, in the
physiological range from 20 to 40 °C. Scattering data were recorded on a
bidimensional CCD camera of 1024 x 1024 pixels, radially averaged and
corrected for the dark, detector efficiency and sample transmission .
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Application of Cubic Phase
A few wide-angle X-ray diffraction experiments were performed using a
laboratory 3.5 kW Philips PW 1830 X-ray generator equipped with a
Guinier-type focusing camera operating with a bent quartz crystal
monochromator (λ = 1.54 Å). Diffraction patterns were recorded on
GNR Analytical Instruments Imaging Plate system. Samples were held
in a vacuum tight cylindrical cell provided with thin mylar windows.
Diffraction data were collected at 20°C.
In each experiment, a number of Bragg peaks were observed in the low
angle X-ray diffraction region, and their spacings were measured. The
peak indexing was performed considering the different symmetries
commonly observed in lipid phases. From the averaged spacing of the
observed peaks, the unit cell dimension, a, was finally calculated by the
Bragg law. The nature of the short-range lipid conformation was derived
analyzing the high-angle X-ray diffraction profiles.
9.1.2.5 Drug Content of Dispersions
With the aim to quantify drug content of dispersions after production, a
sample of filtered dispersion was diluted in methanol (1:9 v/v for MAD,
1:4 v/v for SLN) and stirred for 3 h in order to extract completely the BC
present. Afterwards, the sample was filtered with filters of 0.45 μm and
analyzed for BC content by high performance liquid chromatography
(HPLC) with the below reported procedure.
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Application of Cubic Phase
For Sedimentation Field Flow Fractionation (SdFFF) and stability
studies, the amount of BC detected by HPLC after filtration was taken as
reference of the total amount of drug.
9.1.2.5.1 Sedimentation Field Flow Fractionation Analysis
A SdFFF system (Model S101, FFFractionation, Inc., Salt Lake City,
UT, USA), described elsewhere [107], was employed to determine the
size distribution of the particles (PSD) by converting the fractograms,
i.e., the graphical results, assuming the particle density is known [125].
The mobile phase was a 0.01% v/v solution of Fl-70 in Milli-Q water
(Millipore S.p.A., Vimodrone, Milan, Italy), flowing at 2.0 ml/min and
the actual flowrate was monitored in each run. The samples were
injected after an appropriate dilution of the original suspensions. The BC
associated with the particles was quantified by HPLC analyses on
several fractions, collected during the separation.
9.1.2.6 HPLC Procedure
The HPLC determinations were performed using a HPLC system
consisting of a two plungers alternative pump (Jasco, Japan), a variable
wavelength UV-detector, operating at 305 nm and a Rheodyne Inc.
injection valve model 7125 with a 50 μl loop. Samples were
chromatographed on a stainless steel C-18 reverse-phase column
(15×0.46 cm) packed with 5 μm particles (Hypersil BDS, Alltech, USA).
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Application of Cubic Phase
Elution was conducted with a mobile phase constituted of ammonium
formate (pH 3, 0.1M) and acetonitrile 55:45 v/v at a flow rate of 0.8
ml/min. The retention time for BC was 5.8 min [104].
9.1.3. Results
9.1.3.1 Production and characterization of lipidic dispersions
Production of MAD was performed by the emulsification-hot
homogenization method described by Esposito et al. [105].
The weight of MAD, calculated while taking into account the loss of
dispersing phase because of water evaporation, was found to be 87 ±
0.01% with respect to water/ MO/ poloxamer weight before production.
Thus, the extent of water loss (calculated by difference) was 13 ± 0.01%.
The weight of the larger particles after filtration and desiccation was 10
± 0.5% with respect to the MO/poloxamer weight before production.
All data were the mean of eight determinations on different batches of
the same type of dispersion.
SLN were produced by the use of sonication method and a tristearin/
tricaprin mixture, as described by Esposito and colleagues [104]
obtaining stable and homogenous dispersions. As previously found, the
highest lost of disperse phase was on the vessel (around 4% w/w with
respect to the weight of lipid phase before dispersion) whilst larger
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Application of Cubic Phase
particles represented less than 1% with respect to the total weight of
disperse phase.
It is worth mentioning that tricaprin is a liquid oil at room temperature.
As previously reported, [104] the use of tricaprin in mixture with solid
lipids leads to the formation of solid carriers with homogenous lipid
nanocompartments (nanostructured lipid carriers, NLC).
Table I summarizes the results of PCS studies conducted to determine
the dimensional distribution of MAD and NLC dispersions, in the
absence and in the presence of BC.
Parameter
MAD dispersion
BC MAD
dispersion*
SLN dispersion
ZAverage (nm)
198.2±1.2
204.8±1.2
196.2±2.4
Analysis by
number (nm)
Peak Area 99.7 %
mean diameter 78
nm
Polidispersity
0.18±0.02
BC SLN dispersion*
195.1±3.3
Peak Area 96.2 %
mean diameter 84.3
nm
Peak Area 99.5 %
Peak Area 98.6%
mean diameter 125.7 nmmean diameter 104.3
nm
Peak Area 3.8%
mean diameter 230.7
nm
Peak Area 1.4%
mean diameter 263.1
nm
0.19±0.01
0.18±0.02
0.19±0.03
Index
*produced in the presence of Bromocriptine
PCS data are means of 5 determinations on different batches of the same type of dispersion
Table I:Mean diameters of MAD and NLC
as determined by PCS
Empty MAD were characterized by an intensity mean diameter of 198.2
nm, expressed as Z Average. The analysis by number revealed that the
most representative amount of nanoparticles/ vesicles (percentage of
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Application of Cubic Phase
peak area, 99.7%) displayed a mean diameter of 78 nm. BC slightly
increased the mean diameter of nanostructures, passing to 204.8 nm in
the presence of the drug. Analyzing in detail the distribution, one can
observe one main peak with a mean diameter of 84.3 nm (percentage of
Peak Area 96.2 %) and another peak with a mean diameter of 230.7 nm
(area 3.8%). The dimensions of the bigger particles separated by
filtration ranged between 25 and 30 μm, as measured by laser diffraction.
With regard to NLC dispersions, empty ones have a mean diameter of
196.2 nm, the presence of the drug doesn’t affect the nanoparticle
diameter but increases the percentage of larger nanoparticles passing
from a monomodal to a bimodal dimensional distribution. Polidispersity
indexes were always low (0.18, 0.19), indicating a narrow dimensional
distribution [106].
Cryo-transmission electron microscopy (Cryo-TEM) analyses were
conducted in order to shed light on the internal structure of the dispersed
particles in MAD and NLC dispersions.
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Application of Cubic Phase
Fig.9.1: Cryo-TEM images of BC containing MAD
Figure 9.1 reports cryo-TEM images of BC containing MAD. Wellshaped particles, with a homogeneous, ordered inner structure, can be
observed. Upon closer inspection, images reveal that two different
internal structures (labeled C and H) characterize the particles, even if
the H internal structural motif appear more rarely compared to the other
(panel A). It should be also observed that the H structural motif is
present in some big multistructural particles (panel B), and that the
bigger particles often show a poly-"crystalline" structure and not single
structures, like smaller "pure" particles. Finally, the coexistence of
particles having an ordered inner structure and vesicles and vesicular
structures attached on their surface, as previously found in other studies
where dispersions were produced using monoolein and poloxamer 407
[108], should be underlined.
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Application of Cubic Phase
Fourier transform (FFT) analysis has been then used to analyze the
internal particle morphology. Indeed, FFT enables one to obtain very
easily an optical diffractogram similar to an electron diffraction pattern.
In this way, periodic or repeatable distances in the mesophase structure
can be easily detected, together with the symmetry of the motif even if
these features are not very clearly observed in the direct image.
According to the different internal morphologies shown in Figure 1, FFT
evidenced two different patterns, the first indicating a two dimensional
(2D) hexagonal symmetry for the inner structure of the H particles (with
2D lattice dimensions corresponding to v = w = 8.2 nm and γ = 120°),
and the second indicating a rectangular symmetry for the inner structure
of the C particles (with 2D lattice parameters v = 6.5 nm, w = 9.8 nm and
γ = 90°). FFT patterns and 2D lattice dimensions and ratios suggested
that the observed motifs correspond to planes normal to the
crystallographic directions [111] and [110] of a cubic lattice,
respectively. Concerning the H motif, it should be recalled that the
projection of a 3D cubic array on 2D is hexagonal when visualized along
the [111] direction, and that the corresponding 2D lattice parameters are
related with the cubic unit cell dimension a by v = w = a/√2. This does
not enable one to identify the space group of the particle internal
structure, nor to differentiate between a hexagonal and a cubic structure,
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Application of Cubic Phase
but the comparison of 2D lattice values with the unit cell dimensions
determined by X-ray diffraction (see below, Table II) strongly suggests
that the H particles are cubosomes with an inner cubic structure
belonging to the Im3m space group (note that only the Pn3m and Im3m
space groups are possible in cubosome dispersions because those are the
only two space groups established in reversed bicontinuous cubic phases
in excess water [69] or in reversed bicontinuous cubic phase dispersions
[109]. Concerning the C-motif, the observed 2D lattice parameters were
consistent with the ideal values for a cubic array (v = w/√2) and appear
to correspond to a cubic unit cell dimension a of 9.8 nm. This value
compares well with the unit cell of the Pn3m cubic phase determined in
the same system by X-ray diffraction (see below, Table II). Therefore, it
can be affirmed that C particles are cubosomes with an inner cubic
structure belonging to the Pn3m space group.
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Table II:. Structure identifications and unit cell dimensions of MAD observed in the
different samples at various temperatures
Table II.:Structure identifications and unit cell dimensions of SLN observed in the different
samples at various temperatures
As a conclusion, cryo-TEM images of BC-MAD dispersed particles give
strong and direct evidence for the presence of cubosome with two
different internal structures within the same dispersion: one with a space
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Application of Cubic Phase
group Pn3m and a lattice parameter of 9.8 nm and another with space
group Im3m and a lattice parameter of 11.6 nm. As a kind of
confirmation, it can be observed that the ratio between the lattice
parameter of the two structures is qualitatively in good agreement with
the theoretical value obtained from the Bonnet transformation (1.27)
between cubic phases having the space group Pn3m and Im3m [110].
Fig. 9.2: Cryo-TEM images of NLC dispersions, prepared in the presence of BC.
Figure 9.2 reports cryo-TEM images of NLC dispersions, prepared in the
presence of BC. Both panels show deformed hexagonal, elongated and
circular platelet-like particles, most likely viewed from the top. In
addition,
“needle”-like
structures
and
hemielliptical
particles,
characterized by inner striations, can be also observed. If the firsts are
probably due to the presence of tricaprin crystals, the second ones
correspond to edge-on view of the NLC particles. In panel B the inset
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Application of Cubic Phase
evidences one NLC particle side viewed, where the distance between the
layers is about 5.0 nm, in full agreement with X-ray diffraction results
obtained in the same system (see below, Table II).
The structure of the two different nanoparticulate systems, both in the
presence and in the absence of BC, was confirmed by X-ray diffraction
experiments. X-ray diffraction profiles, measured in the physiological
temperature range from 20 to 40°C, have been analysed considering
separately the so-called low-angle region, from which information on the
long-range organization of lipids can be derived, and the high-angle
diffraction region, from which the nature of the short-range lipid
conformation can be obtained.
High-angle diffraction profiles are not shown, but a large band centred at
about 4.4 Å, which is typical of the liquid-like conformation of lipid
molecules, characterized MAD. On the other side, a few peaks were
detected in NLC, confirming the gel state of the lipid mixtures [104]. In
all cases, no other peaks due to BC were detected after drug loading,
indicating that the drug is fully dissolved in the nanoparticles.
Low-angle diffraction profiles are shown in Figure 3. Concerning the
MAD, the peak indexing indicated the presence of dispersed cubic phase
particles of Pn3m and Im3m symmetry (see Figure 9.3A).
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Application of Cubic Phase
A
I(Q) (a.u.)
60¡C
20¡C
0.05
0.10
0.15
-1
Q (Å )
0.20
0.25
60¡C
I(Q) (a.u.)
B
20¡C
0.05
0.10
0.15
0.20
0.25
-1
Q (Å )
Fig.9.3:. Low-angle X-ray diffraction profiles observed from the different samples (as
indicated) at different temperatures. Measurements have been performed at 20, 25, 30, 40, 50
and 60°C, and scattering curves are stacked consistently, following the direction of the gray
arrows. For monoolein containing samples, small arrows indicate the peak indexing: upward,
continuous arrow, Im3m phase (the indicated peak sequence is [110], [200], [211]); upward,
dashed arrow, Pn3m phase ([110] and [111]); downward, pointed arrow, H phase ([10] and
[21]).
In other words, the produced cubosomes exhibit the D-surface or the Psurface structures, respectively; according to peak intensities, the Dsurface structure (Pn3m) is however favoured at all the investigated
temperatures. Cubic unit cells are reported in Table II: as expected, unit
- 176 -
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Application of Cubic Phase
cell dimensions decrease on heating, probably due to temperature
induced dehydration and to the decreased hydrocarbon chain order
parameter. Two further points should be noticed: first, at 40°C, a
hexagonal phase starts to form, suggesting that temperature induces
cubosome-to-hexasome phase transition [86]. Second, dispersion
filtration does not change the results, suggesting that small and large
monoolein aggregates show similar structural and thermal behaviour.
Addition of BC determines some modifications in the X-ray diffraction
results (see Figure 3B). At room temperature, cubosomes still exhibit the
Pn3m or Im3m inner structures, but now the most represented cubic
phase shows the P-surface (Im3m) structure. It could be interesting to
remind that both cubic phases are bicontinuous and form at high water
levels, but the P-surface structure only occurs in the monoolein–water
system when a third component is added. As shown in Table II, unit cells
are larger, probably due to an increased hydration of the lipid phases
induced by BC. Thermal effects on cubosome structures are similar to
what has been observed on free-drug formulation, but the transition to
hexagonal phase is prevented, at least up to 40°C.
Low-angle X-ray diffraction results obtained for NLC dispersions are
shown in Figure 3C and 3D.
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Application of Cubic Phase
C
I(Q) (a.u.)
60¡C
20¡C
0.05
0.10
0.15
0.20
0.25
0.15
0.20
0.25
-1
Q (Å )
D
I(Q) (a.u.)
60¡C
20¡C
0.05
0.10
-1
Q (Å )
Fig.9.4: Low-angle X-ray diffraction profiles observed from the different samples (as
indicated) at different temperatures. Measurements have been performed at 20, 25, 30, 40, 50
and 60°C, and scattering curves are stacked consistently, following the direction of the gray
arrows. For monoolein containing samples, small arrows indicate the peak indexing: upward,
continuous arrow, Im3m phase (the indicated peak sequence is [110], [200], [211]); upward,
dashed arrow, Pn3m phase ([110] and [111]); downward, pointed arrow, H phase ([10] and
[21]).
The large peak confirms the lamellar order inside the nanoparticles. As
previously observed [104], addition of BC does not modify NLC
structural properties. Moreover, unit cell dimensions, reported in Table
II, do not show any temperature dependence.
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Application of Cubic Phase
9.1.3.2 Efficiency of BC encapsulation
BC encapsulation yield in the filtered dispersion (Table III) was 70% in
the case of MAD and 84% in the case of NLC dispersions, with respect
to the total amount used for the preparation, as determined by HPLC.
Parameter
MAD dispersion
SLN dispersion
Drug Content (%)a
0.18±0.01
0.38±0.02
Encapsulation yield (%)b
70±0.75
84±0.58
Table III :Bromocriptine content and encapsulation efficiency in MAD and in NLC
dispersions
In the case of MAD, the loss of BC should be attributed to the loss of
disperse phase on the vessel and to the bigger particles separated by
filtration. For NLC, the lost of BC during the preparation was found
consistent with the previously obtained results [104].
SdFFF was employed to obtain information about the drug distribution
in the dispersions. The fractogram obtained by SdFFF can be converted
into a PSD plot, i.e., the amount of material per unit change of diameter,
according to well-proven equations, by transforming the retention time
in diameter of a sphere and the UV signal into a mass frequency function
[107,111]. The collected fractions were analyzed by HPLC to quantify
the amount of drug contained in the different populations of the disperse
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Application of Cubic Phase
phase. Plot shows the PSD plot of a diluted amount of BC-containing
MAD and BC-containing NLC dispersion. The conversion was
performed by assuming a density of 0.9692 g/mL. Also reported in the
same graph is the concentration of BC determined by HPLC. In both
cases (MAD and NLC dispersions) it was found that BC was entirely
associated to particles and not free in the dispersing phase. This result is
in agreement with the low aqueous solubility of BC (n-octanol/water
partition coefficient 4.2).
In panel of MAD, the fraction corresponding to a mean diameter of
about 54 nm contains 25% of the total drug. The highest amount of BC
(46 %) is contained in the most representative amount of
nanoparticles/vesicles (percentage of peak area, 87 %) that is
characterized by particles with a diameter of 98 nm. The remaining 29 %
of BC is associated to a little representative population of particles with
bigger mean diameters. It is in fact known from cryo-TEM and PCS
analyses that MAD are mainly characterized by vesicles, cubosomes
with mean diameter around 90-100 nm, whereas few huge structures
with bigger dimensions are also present.
Also for NLC, whose PSD is reported in panel of SLN, the highest
amount of BC (52 %) is contained in the most representative fraction,
that is characterized by particles with a mean diameter of about 103 nm.
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Application of Cubic Phase
The fraction corresponding to a mean diameter of about 59 nm contain
only 3.5 % of the total drug. While the remaining 44.5 % of BC has been
found into little representative bigger particles.
9.1.4. Discussion
The versatility of formulation, colloidal size, biocompatibility and
sustained release properties of nanosystems have already been accepted
for a wide range of active principles [95]. Moreover the lipid/water
matrix of lipid nanosystems is able to incorporate and release also
insoluble molecules like BC. From several years we are attempting to
develop an approach which will permit to deliver BC in a controlled
fashion [82-84]. Our interest in this molecule arises from its versatility.
BC in fact is a dopamine agonist that is used in the treatment of a
number of pathologies, such as pituitary tumors, Parkinson's disease
(PD), hyperprolactinaemia and neuroleptic malignant syndrome.
Moreover in 2009, BC was approved by the FDA for treatment of type 2
diabetes under the trade name Cycloset (VeroScience). In a previous
study we produced and characterized SLN for BC delivery, based on
different lipidic components, demonstrating that NLC constituted of
tristearin/tricaprin mixture can control BC release [93]. In the present
study we search for an alternative nanotechnology system to deliver BC.
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Application of Cubic Phase
MAD are biocompatible and thermodinamically stable nanosystems able
to incorporate lipid molecules in a molecular sponge consisting of
interprenetating nanochannels filled with water and coated by lipid
bilayers. There is a lot of excitement about the cubic phases due to its
unique microstructure that is biologically compatible and capable to
control release of solubilised active ingredients like drugs and proteins
[84]. Like NLC, also MAD represents an interesting alternative to
liposomes, being delivery systems characterized by a higher viscous
resistance to rupture and a consequent prolonged stability. The
performances of NLC and MAD have been investigated as delivery
system for the same molecule. Cryo-TEM images revealed the different
nanostructures of the disperse phase of MAD and NLC. FFT enabled to
evidence the inner cubic structures of MAD, belonging to the Pn3m and
Im3m space groups. High-angle X-ray diffraction studies evidenced the
typical liquid-like conformation of lipid molecules in the case of MAD
and the gel state of the lipid mixtures for NLC [112]. In both cases, BC
is fully dissolved in the nanoparticles.
Low angle X-ray diffraction studies exhibit in MAD the presence of
dispersed cubic phase particles of Pn3m and Im3m simmetry, in full
agreement with cryo-TEM observations. Interestingly at 40°C a
hexagonal phase forms, indicating a cubosome-to-hexasome phase
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Application of Cubic Phase
transition induced by temperature, as found by other authors [81,83]. The
addition of BC to MAD leads to an increased hydration of the lipid
phases with neither modification of structural properties, either transition
to hexagonal phase. NLC dispersions display a lamellar order inside the
nanoparticles and no modification of NLC structural properties after
addition of BC.
Concerning drug entrapment in MAD and NLC, SdFFF was a precious
method since it gave detailed informations about the size distribution of
the dispersions and about the distribution of BC in the different
nanostructures within dispersions. This method together with HPLC
enabled to evidence that BC can be successfully incorporated both in
MAD and in NLC.
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9.2 X-ray Diffraction Analysis of Nucleotides Effects on MonooleinBased Liquid Crystals
9.2.1 Introduction
Amphiphilic lipid molecules, when dispersed in water, can form a
variety of liquid crystalline phases. Which state predominates depends
on the molecular structure of the lipid, as well as hydration level and
environmental conditions, such as temperature, pressure, pH, ionic
strength and the presence of additives. Monoolein (MO) is a lipid which
forms a wide variety of self-assembly structures when mixed with water.
[10,16] Upon increasing the water content the MO/W binary system
shows a small region of reverse micellar (L2) phase followed by a
lamellar (Lα) phase, and by a CG (Ia3d space group) and a CD (Pn3m
space group) bicontinuous cubic phase. The CG phase evolves towards a
reverse hexagonal (H2) phase at high temperature, whereas the CD phase
can coexist with water excess.
Since the extensive pioneering work of K. Larsson [70] in which the
monoolein (MO) phase behavior in water (W) was clarified, and its
similarity to the physiological lipid membrane organization was found,
monoolein has received great interest for applications in the
pharmaceutical area. [113,115] The ability of encapsulation of
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Application of Cubic Phase
hydrophilic, hydrophobic and amphiphilic additives, [73] together with
the capability to protect and slowly release [114] the entrapped molecule
make monoolein mesophases, and in particular cubic phases, potential
candidates for drug delivery systems. Moreover cubic-like phases are
observed in mitochondria and the endoplasmic reticulum, as intermediate
structures during many common physiological processes such a cell−cell
adhesion and fusion, and during the digestion process in the stomach.
Inverted phases play a role in compartmentalizing (sub)cellular space,
offering a relatively large membrane surface for a given cellular
volume.[134]
The binary system monoolein-water was also investigated for the effects
of changes of temperature, [26] and more recently for phase stability and
the phase transitions related to pressure changes. [27]. Pressure jumps
were used to investigate the cubic-to-cubic phase transition and kinetics
of phase transitions. [115] Pressure was also used to study the interaction
of cytocrome c [72] and α-chymotrypsin [73] with MO/W cubic phase.
Currently, pressure has become a thermodynamic variable of growing
interest because it represents an additional tool for understanding phase
behavior, stability and energetics of amphiphilic molecules. Moreover
pressure only change intermolecular distances and affect conformations
- 185 -
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Application of Cubic Phase
but do not change covalent bond distances or bond angles. Therefore
covalent structure of lipids and small molecules is not perturbed.
Since the work of Briggs, Chung and Caffrey [25], were the MO/H2O
phase diagram as a function of temperature was constructed using the Xray diffraction technique, temperature effects on monoolein mesophases
have deeply been investigated. The pure Ia3d phase is found in samples
ranging from 25 wt % to 38 wt % of water content at 20 °C, and between
9 wt % and 24 wt % at 60 °C. It has an upper temperature limit at ca. 89
°C at 20 wt % of water content. The Pn3m cubic phase is formed
between 40 wt % and 43 wt % of water at 20 °C. At 90 °C this phase is
located between 20 wt % and 25 wt % of water. At higher temperatures a
H2 phase is formed. The Pn3m phase coexists with bulk water between 0
°C and 92 °C at hydration level greater than 48.5 wt % and 25 wt % of
water respectively.
Differently, pressure effect on monoolein liquid crystals has been
investigated in few works. Mariani et al. [69] found that pressure induces
in the MO/H2O = 70/30 Ia3d sample a transition to the lamellar Lα phase
at 800 bar and then to the lamellar crystalline phase at approximately
3000 bar, whereas Czeslik et al. (1995) [116] showed that the Pn3m
cubic phase displays high stability in the presence of hydrostatic
pressure, existing up to 2000 bar at 20 °C.
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Application of Cubic Phase
Structural transitions induced by pressure and temperature usually
display opposite trends: pressure increases the order of the acyl chain
which results in a decrease of molecular wedge shape and in a decrease
of interfacial curvature, an increase of bilayer thickness and the observed
increase of the cubic lattice constant, whereas temperature increases the
molecular wedge shape, favoring reverse curvatures.
In a previous work [117] the long-term stability of monoolein cubic
phase containing 1.5 wt % of nucleotides was studied. It was found that
the various mononucleotides undergo a slow hydrolysis of the sugarphosphate ester bond, induced by specific interactions at the monooleinwater interface. Upon aging, the degradation of the nucleotides induces a
cubic-to-hexagonal phase transition.
The present study is directed towards a better understanding of the effect
of nucleotide inclusion on the stability of cubic mesophases. To obtain
an extended description of stability and phase behavior of cubic liquid
crystalline phases, thermotropic and barotropic effects have here been
investigated. The Ia3d cubic phases with 30 wt % of water and the Pn3m
cubic phases with 40 wt % of water, both containing 1.5 wt % of
nucleotides, were selected for this study. The structural aspects of the
cubic phases were investigated by using the SAXRD and NMR
- 187 -
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Application of Cubic Phase
techniques within a wide range of temperatures (25-85 °C) and
pressures (1–1000 bar).
9.2.2.Materials and methods
9.2.2.1 Materials
(MO, 1-monooleoylglycerol, RYLO MG 90-glycerol monooleate; 98 wt
% monoglyceride, also containing 8 wt % of 2-monooleoylglycerol and
5 wt % of monolinoleoylglycerol as ascertained through a quantitative
13C NMR analysis) was kindly provided by Danisco Ingredients,
Brabrand, Denmark. The mononucleotides AMP, CMP, GMP, UMP
sodium salts were from Sigma. 2H2O, purchased from Cambridge
Laboratory, Inc. with a purity of 99.9% , was used to prepare all
samples.
Sample Preparation. Samples were prepared by weighing the
components into glass tubes that were homogenized by repeated cycles
of centrifuging at 3000 rpm at 25 °C. Homogeneous samples (by visual
inspection) used for the phase diagrams characterization were stored at
25 °C in the dark for three days before any measurement was taken.
9.2.2.2 SAXRD Experiments
The simultaneous detection of small- and wide-angle X-ray diffraction
(SAXRD and WAXRD) of high pressure experiments was recorded with
a S3-MICRO SWAXS camera system (HECUS X-ray Systems, Graz,
- 188 -
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Application of Cubic Phase
Austria). Cu Kα radiation of wavelength 1.542 Å was provided by a
GeniX X-ray generator, operating at 50 kV and 1 mA. A 1D-PSD-50 M
system (HECUS X-ray Systems, Graz, Austria) containing 1024
channels of width 54.0 μm was used for detection of scattered X-rays in
the small-angle region. The working q-range (Å−1) was 0.003 ≤ q ≤ 0.6,
where q = 4π sin(θ)λ−1 is the modulus of the scattering wave vector. The
distance between the sample and detector was 200 mm. The diffraction
patterns were recorded at 25 °C. A few milligrams of the sample were
enclosed in a stainless steel hydrostatic pressure cell with diamond
windows. Scattering patterns were recorded for 1800 s.
The temperature scan experiments were performed at the Austrian
beamline (camera length 100 cm) at the synchrotron light source
ELETTRA (Trieste, Italy), using a Gabriel-type 1D position sensitive
detector containing 2048 channels, which covered a d-range much larger
than that of interest (10-200 Å) at an energy of 8 keV (λ = 1.54 Å).
Experiments were performed using a few milligrams of the sample
enclosed in a stainless steel sample-holder with thin polymer sheet
windows.
Silver behenate (CH3-(CH2)20-COOAg) with a d spacing value of 58.38
Å was used as a standard to calibrate the angular scale of the measured
intensity in both pressure and temperature scans. To minimize scattering
- 189 -
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Application of Cubic Phase
from air, the camera volume was kept under vacuum during the
measurements. The lattice parameters (a) were determined from the
linear fits of the measured peak position q versus Miller indexes, using
the relations q/2π = h/a,q/2π = (h2+k2+l2)1/2/a, and q/2π =
(2/a√3)(h2+k2+hk)1/2 for the lamellar, cubic, and hexagonal phases,
respectively. Here q is the measured peak position, and h, k, and l are the
Miller indexes.
NMR Experiments. 2H and
31
P NMR measurements were carried out
through a Bruker Avance 300 (7.05 T) spectrometer at the operating
frequencies of 46.072 and 121.495 MHz respectively. A standard
variable temperature control unit (with an accuracy of ± 0.5°C) was
used. 1H-decoupling was applied in all 31P NMR experiments. During the
scan in temperature (in the range of 25-80 °C) the sample was allowed
30 min to come to equilibrium.
9.2.3 Results
The barotropic and thermotropic phase behavior of MO/D2O cubic
phases (Ia3d and Pn3m) upon incorporation of 1.5 wt % of nucleotides
(AMP, GMP, CMP and UMP) were investigated through SAXRD and
NMR techniques. Results obtained for nucleotide-containing samples
were compared with the pure lipid system. The
SAXRD pressure-
dependent studies were carried out at 25 °C from 1 to 1000 bar at 200
- 190 -
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Application of Cubic Phase
bar intervals, whereas temperature-dependent studies were performed at
1 bar from 25 to 85 °C. 31P and 2H NMR spectra were acquired at 1 bar
from 25 to 80 °C.
GMO/D O = 70/30
a)
GMO/D O = 60/40
2
180
2
110
c)
170
160
a (Å)
a (Å)
105
150
140
100
130
120
110
95
200
400
600
800
1000
0
200
400
P(bar)
GMO/D O = 70/30
b)
800
1000
GMO/D O = 60/40
2
140
600
P(bar)
d)
Ia3d
Pn3m
H2
120
2
110
Pn3m
H2
100
90
100
a (Å)
a (Å)
80
80
70
60
60
50
40
20
30
40
50
60
70
80
40
90
20
T ( °C)
30
40
50
60
70
80
90
T ( °C)
Fig.9.5:. Lattice parameter pressure and temperature dependence of the cubic phases of the
systems MO/D2O = 70/30 ( a, b) and MO/D2O = 60/40 (c, d). Lines are guides to the eyes
to show the general trends.
Ia3d cubic phase
Temperature and pressure scans were performed on the sample
MO/D2O = 70/30. The sample was used as reference and compared to
those containing nucleotides. Results are shown in figure 1a-b. With
increasing pressure the sample undergoes an increase of unit cell
- 191 -
CHAPTER 9
Application of Cubic Phase
dimension from 133.2 Å (1 bar) to 157.2 Å (1000 bar). No phase
transitions are observed. At 1 bar the effect of temperature is to induce
the formation of a Pn3m phase at about 50 °C. At about 85 °C the system
evolves towards a reverse hexagonal structure.
- 192 -
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Application of Cubic Phase
140
180
a)
b)
160
Ia3d
Pn3m
H2
120
140
100
a (Å)
a (Å)
Ia3d
La
120
100
80
80
60
60
40
0
200
400
600
800
1000
40
20
1200
30
40
P (bar)
60
70
80
90
T (°C)
170
c)
50
d)
140
160
Ia3d
Pn3m
H2
120
150
a (Å)
a (Å)
100
140
80
60
130
40
120
0
200
400
600
800
1000
20
1200
30
40
50
60
70
80
90
T ( °C)
P (bar)
145
e)
140
f)
140
Ia3d
Pn3m
H2
120
a (Å)
a (Å)
135
130
80
125
120
100
60
0
200
400
600
800
1000
40
20
1200
30
40
P (bar)
60
70
80
90
T ( °C)
150
g)
50
150
h)
145
Ia3d
Pn3m
H2
a (Å)
a (Å)
140
135
100
130
125
120
50
0
200
400
600
800
1000
1200
20
P (bar)
30
40
50
60
70
80
90
T ( °C)
Fig.9.6:. Lattice parameter pressure (left) and temperature (right) dependence of the
lamellar, cubic and hexagonal phases of the systems MO/D2O/XMP = 68.9/29.6/1.5 ( a, b)
AMP, (c, d) GMP, (e, f) CMP and (g, h) UMP. Lines are guides to the eyes to show the
general trends.
The lattice parameter pressure and temperature dependence in the Ia3d
systems MO/D2O/XMP = 68.9/29.6/1.5 is shown in figure 9.6.
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Application of Cubic Phase
In the sample containing AMP (Fig.9.6a) pressure induces an increase of
the unit cell dimension from 135.8 Å (1 bar) to 162.7 Å (1000 bar).
Moreover, in the pressure range of 600-1000 bar the cubic phase coexists
with a lamellar phase. Here, it is worth recalling that the wide-angle
profiles can be useful in obtaining information on the packing
arrangement of the acyl chains in a lamellar phase. For instance, a sharp
peak is representative for hexagonally packed hydrocarbons chains in a
lamellar gel (Lβ) phase, while a broad peak is the clear signature of the
liquid-like hydrocarbons chains arrangement in the lamellar fluid Lα
phase.[13] It should be also noted that, differently from lamellar phases
based
on
lipids
particularly
suited
for
WAXS
analysis
like
phospholipids, broadening can make this peak hardly detectable. Since
no peaks were recorded in the wide-angle region, the lamellar phase was
definitely identified as Lα. Its lattice parameter increases from 46.5 Å
(600 bar) to 46.8 Å (1000 bar).
Differently from the sample containing AMP, where a cubic to lamellar
transition is observed, the inclusion of GMP (Fig.9.6c), CMP (Fig.9.6e)
and UMP (Fig.9.6g) preserves the Ia3d cubic structure in the range of
pressure under study. No lamellar phase is formed. Different trends for
the variation of the lattice parameter as a function of pressure are
observed. With GMP the lattice increases from 133.2 Å (1 bar) to 157.2
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Application of Cubic Phase
Å (1000 bar), whereas in presence of CMP from 127.2 Å (1 bar) to 132.4
Å (1000 bar) and with UMP from 132.0 Å (1 bar) to 135.8 Å (1000 bar).
Temperature-dependent SAXRD experiments show a reduction of the
lattice parameter value with increasing temperature. From 25 °C to 45 °C
the Ia3d sample containing AMP (Fig.9.6b) decreases its size from 135.8
Å to 125.1 Å, where a Pn3m cubic phase starts to form. A small biphasic
region of Ia3d and Pn3m is present in the temperature range of 45-50 °C,
after which a pure Pn3m phase is formed. The lattice parameter
decreases from 79.0 Å (45 °C) to 74.2 Å (70 °C). Above 70 °C the Pn3m
cubic phase transforms into a reverse hexagonal phase having a lattice
parameter of 56.9 Å.
The transition to Pn3m cubic phase takes place at different temperatures
depending on the nucleotide included: 40 °C with AMP and UMP
(Fig.9.6h), 30 °C with GMP (Fig.9.6d) and 25 °C with CMP (Fig.9.6f).
After the transition to Pn3m, the evolution to the HII phase takes place at
about 65 °C in samples containing AMP, CMP and UMP, whereas in
the sample containing GMP the formation of the hexagonal phase is
already seen at 55 °C.
- 195 -
CHAPTER 9
Application of Cubic Phase
Pn3m cubic phase
Results obtained from temperature and pressure scans on samples
containing nucleotides and prepared in the Pn3m region of the phase
diagram were compared with the sample MO/D2O = 60/40. Results are
shown in figure 1c-d. With increasing pressure the sample undergoes an
increase of unit cell size from 100.9 Å (1 bar) to 104.1 Å (1000 bar). No
phase transitions are observed. Temperature induces a phase transition
towards a reverse hexagonal phase at about 85 °C.
- 196 -
CHAPTER 9
Application of Cubic Phase
130
a)
110
Pn3m
125
b)
Pn3m
H2
100
120
90
a (Å)
a (Å)
115
110
105
80
70
100
60
95
90
0
200
400
600
800
50
20
1000 1200
30
40
P (bar)
70
80
90
120
d)
160
Pn3m
H2
110
100
140
90
a (Å)
Pn3m
Ia3d
La
120
a (Å)
60
T (°C)
180
c)
50
100
80
70
80
60
60
40
50
0
200
400
600
800
40
20
1000 1200
30
40
P (bar)
60
70
80
90
T (°C)
120
e)
50
100
f)
115
Pn3m
H2
90
110
a (Å)
a (Å)
80
105
70
100
60
95
90
0
200
400
600
800
50
20
1000 1200
30
40
120
g)
60
70
80
90
110
h)
115
Pn3m
H2
100
90
a (Å)
110
a (Å)
50
T (°C)
P (bar)
105
80
70
100
60
95
90
50
0
200
400
600
800
40
20
1000 1200
P (bar)
30
40
50
60
70
80
90
T (°C)
Fig.9.7:Lattice parameter pressure (left) and temperature (right) dependence of the lamellar,
cubic and hexagonal phases of the systems MO/D2O/XMP = 59.1/39.4/1.5( a, b) AMP, (c,
d) GMP, (e, f) CMP and (g, h) UMP. Lines are guides to the eyes to show the general
trends.
- 197 -
CHAPTER 9
Application of Cubic Phase
Figure 9.7 shows the pressure and temperature dependence of the lattice
parameter of the Pn3m systems MO/D2O/XMP = 59.1/39.4/1.5.
In the pressure range under investigation the Pn3m lattice parameter of
the sample containing AMP increase from 101.0 Å (1 bar) to 106.3 Å
(1000 bar). Differently, the Pn3m sample containing GMP undergoes a
transition from the Pn3m phase to the Ia3d (200 bar) and then to the Lα
phase (800 bar). As in case of AMP, samples containing CMP and UMP
preserve their structure in the range of 1-1000 bar. With CMP the lattice
increases from 96.8 Å (1 bar) to 104.1 Å (1000 bar) and with UMP from
96.2 Å (1 bar) to 104.9 Å (1000 bar).
With increasing temperature, as shown in Fig.9.7, in the sample
containing AMP at about 45 °C a reverse hexagonal phase is formed.
Pn3m and H2 phases coexist up till 65 °C. Above 65 °C the system
becomes completely hexagonal. Except for the sample containing AMP,
the Pn3m-to-HII phase transition take place at about 65 °C.
The cubic phases evolution toward hexagonal phase induced by
temperature is clearly shown also by the appearance of 31P CSA and 2H
quadrupolar splitting in 31P and 2H NMR spectra (Fig.9.8). [117]
- 198 -
CHAPTER 9
8
7
Application of Cubic Phase
6
5
4
ppm
3
2
1
0
1000
500
0
-500
-1000
Hz
Fig.9.8:. 31P and 2H NMR spectra of a MO/D2O/AMP = 59.1/39.4/1.5 sample at 65 °C.
Analysis of structural parameters. Assuming distinct lipid and water
regions within the unit cell, the internal structural dimensions of the
lipid-containing phases can be calculated from the measured unit cell
dimensions and the sample concentration. Bicontinuous cubic phases can
be described as lipid bilayers lying on Infinite Periodical Minimal
Surfaces (IPMS). In this model, the surface where the terminal methyl
groups of acyl chains from adjacent lipid monolayers meet defines the
IPMS. Since dealing with inverted mesophases, the cross-sectional area
per lipid molecule is a maximum at the minimal surface and decreases
progressively along the length of the hydrocarbon chain to reach a
minimum value at the glycerol headgroup. The projected cross-sectional
area reduces to zero at the center of the water channel.
For a cubic phase of the type under discussion, [118] have shown that
the molecular cross-sectional area evaluated on a surface parallel to and
- 199 -
CHAPTER 9
Application of Cubic Phase
at a distance ξ from the minimal surface and integrated over one of the
two monolayers within the unit cell, A(ξ), is related to the
experimentally measurable lattice parameter a as follows:
A(ξ ) = σa 2 + 2πχξ 2
(1)
where σ is a unitless quantity that describes the ratio of the minimal
surface in a unit cell to the quantity (unit cell volume)2/3, and χ is the
Euler–Poincarè characteristic of the IPMS geometry (Ia3d, σ = 3.091, χ
= -8; Pn3m, σ = 1.919, χ = -2). Accordingly, the area of the minimal
surface in the unit cell is given by σa2 and defined as A0.
According to Turner and co-workers, [119] in the IPMS model the
monolayer thickness l for a given cubic phase, considered constant
throughout the structure, can be calculated using the lattice parameter a,
determined by SAXRD, the known sample composition (volume fraction
of the lipid φlip), and by using the following relation:
⎛ l ⎞ 4πχ ⎛ l ⎞
φlip = 2σ ⎜ ⎟ +
⎜ ⎟
3 ⎝a⎠
⎝a⎠
3
(2)
The unit cell surface area at the headgroup A, that is, at the lipid-water
interface, which is assumed to be parallel to the minimal surface, can be
obtained using:
A = A0 (1 + K 0 l 2 )
- 200 -
(3)
CHAPTER 9
Application of Cubic Phase
Where <K>0 is the surface averaged Gaussian curvature on the minimal
surface. <K>0 is related to the lattice parameter through the GaussBonnet theorem:
K 0=
2πχ
A0
(4)
Other parameters, that are also necessary to describe a complete
curvature free energy for the lipid layer are the Gaussian <K> and the
Mean <H> curvatures at the lipid-water interface, both averaged over the
unit cell. Their values can be calculated using:
K =
2πχ
A
(5)
H =
2πχ
l
A
(6)
The lipid length in the fluid lamellar Lα phase is given by:
l=
alamφlip
2
(7)
where alam is the lamellar lattice parameter.
Structural parameters, that are lipid length, Gaussian and Mean
curvature, were calculated as a function of pressure and temperature for
the various samples under investigation.
- 201 -
CHAPTER 9
Application of Cubic Phase
24
-6
AMP
GMP
CMP
UMP
MO/D
-7
-8
<K> 10-4 Å-2
l (Å)
20
18
16
AMP
GMP
CMP
UMP
MO/D
-1.6
-9
-1.8
<H> 10-2 Å-1
22
-1.4
AMP
GMP
CMP
UMP
MO/D
-10
-11
-2
-2.2
-12
14
12
-2.4
-13
0
200
400
600
800
1000 1200
-14
0
200
400
P (bar)
600
800
-2.6
1000 1200
0
200
400
P (bar)
600
800
1000 1200
P (bar)
Fig.9.9: Pressure dependence of the structural parameters of the Ia3d cubic phase having
composition MO/D2O/XMP = 68.6/29.9/1.5. l si the thickness of the monolayer, <K> and
<H> are, respectively, the average over the unit cell of the Gaussian and of the mean
curvature, calculated at the water-lipid interface. Lines are guides to the eyes to show the
general trends.
24
-6
AMP
GMP
CMP
UMP
MO/D
<K> 10 Å
-2
-8
16
-9
-10
14
0
200
400
600
800
1000
-11
1200
0
200
400
600
1000
-2
1200
AMP
GMP
CMP
UMP
MO/D
14
12
-1
-10
60
70
80
-14
-18
20
600
800
1000
1200
AMP
GMP
CMP
UMP
MO/D
-1.6
-2
-12
-1.8
-2
-16
T (°C)
400
-1.4
<H> 10 Å
<K> 10 Å
-4
16
50
200
-1.2
-8
-2
18
40
0
P (bar)
-6
AMP
GMP
CMP
UMP
MO/D
20
l (Å)
800
P (bar)
22
30
-1.6
-1.8
P (bar)
10
20
-1.4
-2
-4
l (Å)
18
AMP
GMP
CMP
UMP
MO/D
-1.2
-1
-7
20
12
-1
AMP
GMP
CMP
UMP
MO/D
<H> 10 Å
22
30
40
50
T (°C)
60
70
80
-2.2
20
30
40
50
60
70
T (°C)
Fig.9.10:. Pressure and temperature dependence of the structural parameters of the Pn3m
cubic phase having composition MO/D2O/XMP = 59.1/39.4/1.5. l si the thickness of the
monolayer, <K> and <H> are, respectively, the average over the unit cell of the Gaussian
and of the mean curvature, calculated at the water-lipid interface. Lines are guides to the
eyes to show the general trends.
Figures 9 and 10 shows the effect of pressure on hydrocarbon chains,
Gaussian and mean curvature calculated by equations (2), (5) and (6)
- 202 -
80
CHAPTER 9
Application of Cubic Phase
respectively on samples prepared in the Ia3d and Pn3m regions of the
phase diagram. Experimental results are in good agreement with
previous results . In both kind of cubic phases, the bilayer thickness,
Gaussian and mean curvature increase when pressure increases. The
opposite trend is observed when temperature increase.
Nevertheless the presence purine nucleotides (AMP and GMP) and
pyrimidine nucleotides (CMP and UMP) seems to influence phase
behavior of Ia3d cubic samples differently (Fig.9.9.). The same
dependence is not present in the Pn3m samples and no considerable
differences are visible (Fig.9.10) among the various samples in both
pressure- and temperature-dependent SAXRD measurements.
The effect of pressure and temperature on lipid structures can be
explained with simple molecular packing arguments. [120] The
surfactant packing parameter v/al (v is the hydrophobic chain volume, a
is the head group area and l is the chain length, taken as 80% of the fully
extended chain) is useful to predict which phase can be preferentially
formed by a given surfactant since it connect the molecular properties
with the favored curvatures of the aggregate interface. Moreover
pressure and temperature variations, changes in composition or the
introduction of a new component can strongly affect the molecular
wedge shape of the surfactant and then the packing parameter value. An
- 203 -
CHAPTER 9
Application of Cubic Phase
increased wedge shape of the lipid molecules is a result of increasing
temperature, whereas pressure exert the opposite effect.
In the cubic phase the reduction in chain wedge shape induced by
pressure tends to reduce the magnitude of the (negative) interfacial
curvature, thereby swelling the phase if it is in contact with an excess
water phase [141]. Effects due to pressure increase depend on the nature
of the additive and on the type of cubic phase involved: in the Ia3d cubic
phase, where the interface curvature is higher, pressure more strongly
affects the lattice parameter in comparison to the Pn3m, where the
curvature is smaller.
The nucleotide tendency to adhere to the lipid interface [117] induces an
increase of the surfactant effective packing parameter favoring then the
formation of inverted curvatures. This effect is clearly seen during
temperatures jumps, where the transition to reverse hexagonal phase is
shifted 20 °C toward lower temperatures.
9.3.3.Conclusions
Small-angle X-ray diffraction and NMR spectroscopy were used to study
the interaction of nucleotides with the lipid system monoolein/water. The
influence of nucleotides incorporation on the thermotropic and
barotropic phase behavior of monoolein (MO) cubic phases was
- 204 -
CHAPTER 9
Application of Cubic Phase
investigated. The pressure and temperature dependent studies were
conducted on monoolein/D2O/nucleotide systems at constant D2O
content (30 wt % and 40 wt %) and at fixed nucleotide concentration
(1.5 wt %).Incorporation of nucleotides into the bicontinuous cubic Ia3d
and Pn3m phases doesn't affect the structure and lattice constants
significantly but has pronounced effects on the temperature and pressure
stability of the systems, changing the phase transition temperatures and
pressures. As a general effect, nucleotides tend to increase the monoolein
effective packing parameter, favoring inverse curvatures with increasing
temperature.
- 205 -
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Acknowledgements
E così dopo una serie di soddisfazioni e sacrifici, sono arrivata all’ultima pagina di
questa tesi, che coincide con la fine di questo mio percorso di tre anni, durante il quale
ho avuto modo di ampliare le mie conoscenze didattiche ma anche quelle personali.
Non posso non menzionare il mio relatore, Prof. Paolo Mariani, che mi ha esortato ad
insistere e a proseguire il percorso intrapreso quando ogni possibilità mi sembrava
perduta, ed avrei preferito mollare tutto — facendomi desistere da tale proposito.
Inoltre ringrazio la Prof. Rosangela Itri dell’USP di San Paolo per la massima
disponibilità dimostrata nei miei confronti, per avermi introdotto in una realtà lontana
dalla mia ma ricca di affetto, stima, facendomi sentire a casa nonostante i molti km di
distanza.
Colgo l’occasione per ringraziare il Dipartimento SAIFET che mi ha vista “nascere”
come tesista e poi come dottoranda e nel quale ho avuto modo di instaurare rapporti
lavorativi ed umani importanti per il mio percorso di studi e non solo.
Ringrazio il Dipartimento di Fisica dell’Università di San Paolo, per avermi accolta
con un calore e un affetto unici…i mesi trascorsi con voi li porto nel cuore come ricordi
preziosi.
In particolare vorrei ringraziare il Dott. Leandro Barbosa per il prezioso contibuto
fornitomi nei miei mesi di permanenenza in Brasile, per il supporto morale e lavorativo
per me fondamentali.
Il mio pensiero, ovviamente, va ai miei genitori, a cui dedico questo lavoro: senza il
loro aiuto non avrei mai raggiunto questa meta. Sono davvero grata per tutto il
sostengo economico, ma più di ogni altra cosa di quell’aiuto tacito o esplicito che è
venuto dal loro cuore: a tutte quelle volte che mi hanno incoraggiata vedendomi presa
dal mio lavoro, ma soprattutto per la soddisfazione che hanno saputo donarmi anche
con un solo sguardo.
Mi auguro che tutti i sacrifici spesi siano in questo modo, almeno in parte, ripagati.
Ringrazio Zio Meco e Zia Lella, i nonni, Marco e Luca per avermi dato sempre il
giusto supporto per andare avanti, per credere di più in me stessa e per il loro amore
incondizionato che ci rende “speciali”.
Un pensiero va a Sonia, la mia “America” scoperta solo da poco ma è come se fosse da
sempre, per i silenzi compresi, le risate, per la sua presenza e per l’affetto smisurato e
per la sua capacità di farmi sentire importante.
Desidero ringraziare tutte quelle persone vecchie e nuove con cui ho iniziato e trascorso
il mio dottorato, con cui ho scambiato qualche pensiero, qualche idea, qualche risata:
Sara, Federica, Cristina, Luisa, Marta, Alessio, Alessio,Danilo,Chiara,Mara, Katia..
In diversi modi hanno contribuito nel mio percorso formativo, aiutandomi a credere in
me stessa, suscitando in me nuovi interessi e soprattutto mi hanno suggerito,
direttamente o indirettamente, le modalità per poterli raggiungere.
L’ultimo grazie, ma non per importanza, va ad Enrico, collega di tanti esperimenti poi
trasformatosi in compagno di vita. Grazie per avermi aperto le porte del tuo mondo e
per avermi dedicato la tua presenza e il tuo amore con il desiderio di volerci essere
anche domani.
Chiudo i ringraziamenti dedicando la mia tesi ad un pezzo del mio cuore che non c’è
più: grazie Pietro per l’esempio che mi hai dato e che continui a darmi in silenzio da
lassù.