Thesis for the degree Master of Pharmacy

INFLUENCE OF OSMOTIC STRESS ON
LIPOSOME SIZE AND MORPHOLOGY

By
Helene Moen
May 2008



Supervisors:
Professor Martin Brandl
PhD student Stefan Hupfeld

Department of Pharmaceutics and Biopharmaceutics
Institute of Pharmacy
Faculty of Medicine
University of Tromsø




2


























3

Acknowledgements
This master thesis was carried out at the Department of Pharmaceutics and Biopharmaceutics, Institute of
Pharmacy at the University of Tromsø from September 2007 to May 2008.

First of all I would like to thank my two supervisors, Professor Martin Brandl and PhD student Stefan Hupfeld
for giving me the opportunity to work with this project, but also for inspiration during my work.
Stefan , thank you for your valuable advice and useful input on the writing of this thesis. Thank you for all the
help in the lab, for always being available and for all encouragement.
Martin, thank you for your constructive feedback and good advice in writing of this thesis.
Thanks to everyone in the department for their day-to-day support and conversation. Merete Skar deserves my
thanks for always being helpful and giving me lots of technical advice in the lab. Rahul Haware, thank you so
much for all the help with the viscosity measurements.
I wish also to express my gratitude to dr.scient Roy Lysaa for illustrating two of the figures in my thesis.
Thanks to all my friends for their support throughout these years and for motivating me to achieve my academic
goals. A special thank to all the girls in the master student office, your optimistic spirit and support have been
incredible.
Kent and Linn, thank you for everything you have done for me these last months.
Last but not least, I would like to thank my parents, Kirsten and Nils-Arnt, and my two sisters, Vibeke and
Marie; I could not have done this without you. Thank you for always believing in me and encouraging me
throughout these years. Vibeke deserves an extra appreciation for reading through the thesis and giving me
helpful advice about the writing.

A lot of people deserve my sincere gratitude and thankfulness for giving me the motivation and feedback that
made me able to do this. To all of you that are not mentioned by name; you are not forgotten!

Tromsø, May 2008
Helene Moen




4


























5

CONTENTS

1 ABSTACT ....................................................................................................................................... 7
2 ABBREVIATIONS ......................................................................................................................... 9
3 INTRODUCTION ......................................................................................................................... 11
3.1 Liposomes .............................................................................................................................. 11
3.1.1 Definitions and background ........................................................................................... 11
3.1.2 Classification of liposomes ............................................................................................ 15
3.1.3 Stability of liposomes .................................................................................................... 16
3.1.4 Pharmaceutical use of liposomes ................................................................................... 16
3.1.5 Challenges with liposome formulations......................................................................... 17
3.1.6 Size analysis of liposomes ............................................................................................. 17
3.2 Influence of osmotic stress on liposome size ......................................................................... 18
3.3 Previous studies ..................................................................................................................... 19
4 AIM................................................................................................................................................ 21
5 MATERIAL AND METHODS ..................................................................................................... 23
5.1 Chemicals .............................................................................................................................. 23
5.2 Equipment .............................................................................................................................. 24
5.3 Media and solutions ............................................................................................................... 27
5.4 Preparative methods ............................................................................................................... 29
5.4.1 Preparation of multilamellar vesicles (MLVs) .............................................................. 29
5.4.2 Reduction of lamellarity ................................................................................................ 29
5.4.3 Reduction of liposome size ............................................................................................ 31
5.5 Analytical methods ................................................................................................................ 32
5.5.1 Characterization of particle size by Photon Correlation Spectroscopy .......................... 32
5.5.2 Determination of osmolality in solutions ....................................................................... 38
5.5.3 Determination of viscosity in solutions ......................................................................... 39
5.5.4 Determination of refractive index in solutions .............................................................. 40
5.5.5 Characterization of liposomes, Flow Field-Flow Fractionation .................................... 42







6

6 RESULTS AND DISCUSSION .................................................................................................... 51
6.1 Preliminary experiments ........................................................................................................ 51
6.1.1 Osmolality measurements .............................................................................................. 51
6.1.2 Viscosity measurements ................................................................................................ 52
6.1.3 Refractive index measurements ..................................................................................... 54
6.1.4 Influence of viscosity and refractive index on the accuracy of PCS size measurements
of latex bead standards ................................................................................................... 54
6.1.5 Freeze-thaw experiments ............................................................................................... 56
6.2 Influence of osmotic stress on liposome size, measured by PCS .......................................... 59
6.2.1 Hypertonic osmotic stress experiment with 10 mM NaNO
3
and glucose equivalent to
90 mM NaNO
3
as dilution medium ............................................................................... 59
6.2.2 Hypertonic osmotic stress experiment with 10 mM NaNO
3
and sucrose equivalent to
90 mM NaNO
3
as dilution medium ............................................................................... 62
6.2.3 Hypotonic osmotic stress experiment with 10 mM NaNO
3
as dilution medium ........... 64
6.3 Influence of osmotic stress on liposome size, measured by AF4 .......................................... 65
6.3.1 Hyperosmotic osmotic stress experiment with 10 mM NaNO
3
and sucrose equivalent
to 90 mM NaNO
3
as dilution medium ........................................................................... 67
6.3.2 Hypotonic osmotic stress experiment with 10 mM NaNO
3
as dilution medium ........... 73
7 CONCLUSION .............................................................................................................................. 81
8 REFERENCES .............................................................................................................................. 83
9 APPENDICES ............................................................................................................................... 87
Appendix 1......................................................................................................................................... 87
Appendix 2......................................................................................................................................... 88
Appendix 3......................................................................................................................................... 89
Appendix 4....................................................................................................................................... 110








7

1 ABSTACT
Liposomes are currently being investigated as potential parenterally used drug carriers. The
main factor that influences the in vivo behavior of such liposomes is their vesicle size. A
detailed and reliable knowledge of vesicle size is therefore necessary in order to interpret
results of physical and biological investigations in a correct manner.
It has earlier been discovered that it is feasible to determine the size distribution of vesicle
dispersions in a reliable manner and it appears especially useful to employ the combination of
SEC fractionation, PCS and the enzymatic PC quantitation. A drawback discovered was for
certain liposome dispersions that in some of the dispersions SEC fractionation showed
incomplete recovery of the vesicles. This indicated a demand for a different fractionation
method which does not have the limitations that the SEC method described above has.
To this end flow field-flow fractionation was chosen. In a previous study the influence of
some key factors such as ionic strength of the eluent as well as pore size of the semi-
permeable membrane on liposome fractionation behavior has been evaluated. Neutral
liposomes were found very dependent of the ionic strength when it comes to elution time.
In this study, the intention was to find out if the retention behavior and calculated geometric
radius of liposomes obtained by flow field-flow fractionation in combination with multi-angle
light scattering is affected by the osmotic pressure of the medium used for diluting the
liposomes and/or running the AF4. In order to exclude ionic-strength effects the salt
concentration was kept constant while the osmotic pressure was varied by using mono- and
disaccharides.
In conclusion, this project has demonstrated that a change in osmotic pressure, with constant
ionic strength, affects both elution time and calculated size of liposomes that were prepared
by high pressure filter extrusion. But, osmotic stress was found to affect liposomes of
different sizes in a different manner; liposomes that were smaller than the pore size of the
filter used for extrusion were found to shrink in hyperosmotic medium but stay quite constant
in size in hypo-osmotic medium. In contrast, liposomes that were larger than the pore size of
the filter were found to shrink in hyperosmotic medium and swell in hypo-osmotic medium. A
hypothesis is presented to explain this behavior.



8






















9

2 ABBREVIATIONS

AF4 Asymmetrical flow field-flow fractionation
cP Centipoise
E-80 Unsaturated egg phosphatidyl choline
g Gram
HPLC High performance liquid chromatography
kD KiloDalton
kHz Kilohertz
L Liter
LAF Laminar air flow
LN
2
Liquid nitrogen
LS Light scattering
LUVs Large unilamellar vesicles
MALS Multi-angle light scattering
Min Minutes
MLVs Multi lamellar vesicles
mM Millimolar
mPa·s Milli pascal-second
Mw Molecular weight
NaNO
3
Sodium nitrate
nm Nanometer
No. Number
P.I. Polydispersity index
PC Phosphatidylcholine
PCS Photon correlation spectroscopy
QELS Quasi-elastic light scattering
RI Refractive index
rms Root mean square
R
n
Number-average mean square radius
R
w
Weight-average mean square radius
R
z
z-average mean square radius


10

s Second
SD Standard deviation
SEC Size exclusion chromatography
SUVs Small unilamellar vesicles
t
1/2
Half-life
t
r
Retention time
µm Micron/micrometer
µSec Microseconds
UV Ultraviolet
UV/VIS Ultraviolet-visible
Vs. Versus
w/w Weight ratio
















11

3 INTRODUCTION
3.1 Liposomes
3.1.1 Definitions and background
Liposomes are spherical vesicles which can be thought of as a hollow sphere whose size
ranges from approximately 20 nanometers (nm) to some microns (µm). They are composed of
a bilayer membrane which entraps an aqueous core. The membrane is composed of
phospholipid molecules, the same type of molecules cell membranes are comprised of.
Liposome membranes can be composed of naturally-derived phospholipids with mixed lipid
chains and a variation of head groups or of pure synthetic lipids with defined acyl chains and
head groups.

Liposomes do form spontaneously when phospholipids are mixed with aqueous medium, for
review see (Liposomes: a practical approach Torchilin and Weissig 2003). Phosphatidyl
choline (PC) molecules are not truly soluble in water, i.e. they self-assemble to liquid
crystalline aggregates upon contact with aqueous media. Phospholipids are amphipathic, that
is, part of their structure is hydrophilic and the other part is hydrophobic. Therefore, when
added to water, the hydrophilic part of the phospholipid interacts with the water and the
lipophilic part of the molecule avoids the water. In order to accomplish this, the phospholipids
align themselves side-by-side with their lipophilic heads orienting themselves towards each
other as shown in the middle figure below (Technical Summary - An Introduction to Lipid
Nanoparticles Sciences 2008). This structure is known as a phospholipid bilayer of lamellar
structure as shown to the right in figure 3.1.1.



12


Figure 3.1.1: The formation of liposomes, from phospholipid molecules to a unilamellar vesicle. (Figure
taken with permission from: http://www.encapsula.com/company.html)

The vesicles formed may consist of one or more lamellae. Small liposomes usually consist of
only one bilayer but bigger liposomes can consist of multiple bilayers or several smaller
liposomes can be formed inside the bigger liposome. The thickness of a bilayer is about 4 nm,
reviewed in (Liposomes: from physics to applications Lasic 1993).

Materials can either be entrapped in the aqueous core or incorporated within the membrane
for review see (Liposomes as drug carriers: a technological approach Brandl 2001). Lipophilic
of amphiphilic drug are incorporated into the membrane and hydrophilic drugs are entrapped
in the aqueous core for review see (Liposomal formulations of anticancer drugs: selectivity
and effectiveness Massing and Fuxius 2000).





13

A multi lamellar liposome is shown in figure 3.1.2. We can see that there are many
phospholipid bilayers with water in between the layers. The pink dots are water-soluble drugs
which are entrapped in the core or in the aqueous space between the bilayers. The green rods
are lipid-soluble drugs which are incorporated in the lipid membrane.

Figure 3.1.2: Drug encapsulation in liposomes, the water-soluble drugs (shown in pink) are entrapped in
the aqueous compartments and the lipid-soluble drugs (shown in green) are entrapped within the
membrane. (Figure taken with permission from: http://www.encapsula.com/company.html)

The choice of lipids for liposomal drug carriers depends on the desired stability of the
liposome formulation, and the drug which should be incorporated into the liposomes. The
most common phospholipid used in liposomal drug carriers is phosphatidyl choline. There
exist two sorts of phospholipids, phosphodiglycerides and sphingolipids. PC belongs to the
group of phosphodiglycerides. PC can be derived from natural sources as egg yolk and
soyabeans or be made synthetically (Liposomes: a practical approach Torchilin and Weissig
2003).

PC is amphiphilic and is composed of a hydrophilic head group consisting of the quaternary
ammonium moiety choline linked to the glycerol-backbone via a phosphor-ester and two
lipophilic acyl chains. As the phosphate is negatively charged at physiological pH, PC is
zwitterionic and liposomes made of it have no net charge. A schematic presentation of PC is
shown in figure 3.1.3


14



Figure 3.1.3: A schematic representation of PC (Figure taken with permission from:
http://kvhs.nbed.nb.ca/gallant/biology/biology.html)

PC is hardly ever used alone in liposomal lipid formulations. Blends of PC with other lipids
are used primarily to improve both in-vitro and in-vivo stability of the liposomes (Liposomes
as drug carriers: a technological approach Brandl 2001). When drugs are incorporated into the
liposome one usually wants to prevent leaking and loss of drug through the membrane.

A normal way to prevent leaking is adding cholesterol to the membrane, cholesterol will
induce a tighter packing of the membrane and reduce the fluidity of the membrane, as shown
in figure 3.1.4.




15


Figure 3.1.4: Phospholipid bilayer with cholesterol incorporated in the membrane. (Figure taken with
permission from: http://www.uic.edu/classes/bios/bios100/lecturesf04am/lect08.htm

3.1.2 Classification of liposomes
Liposomes are often classified according to their size. Size and lamellarity of liposomes
formed by spontaneous swelling depend on the type of lipid, composition of the medium and
the mechanical stress exerted during swelling. Lipids with a net charge reduce both size and
number of lamellae of the liposome.

Multi lamellar vesicles (MLVs) are vesicles covering a size range from 100-1000 nm and
consist of five or more lamellae, for review see (Liposomes: a practical approach Torchilin
and Weissig 2003).

Large unilamellar vesicles (LUVs) are vesicles in the same size range as MLVs, from 100-
1000 nm, but they only have one lamella.

Small unilamellar vesicles (SUVs) are defined as the smallest phospholipid vesicles possible
(approximately 20 nm) and up to 50 nm. The size depends on the ionic strength of the
aqueous medium and the lipid composition in the membrane. They usually consist of one
lamella.



16

3.1.3 Stability of liposomes
For phospholipids there are two major degradation reactions known which affect their
chemical stability in aqueous dispersion, hydrolysis and oxidation (Liposomes as drug
carriers: a technological approach Brandl 2001). Physical instability might affect the particle
size of liposomes. Examples of this kind of instability are aggregation and fusion.
Aggregation is the process where liposomes form aggregates. This is a reversible process
which can be resolved by stirring. Fusion is the phenomenon where vesicles fuse together and
make bigger liposomes. The process most often happens to very small liposomes with a
diameter of approximately 20 nm. This is not a reversible process and therefore a much bigger
problem.

3.1.4 Pharmaceutical use of liposomes
In the field of drug delivery, preparations based on submicron particles are emerging as an
important tool for achieving either controlled or targeted delivery of the active compound.
Examples of such drug carriers are polymeric and solid lipid nanoparticles as well as
liposomes. The objective is to achieve selective localization of active drug in disease sites as
tumors and inflammation sites. A potential field that is very interesting is cancer therapy. The
systemic environment does not recognize the drug when it is incorporated into a liposome. It
recognizes only the liposome and the intrinsic pharmacokinetics of the drug is masked by the
pharmacokinetic behavior of the liposomal vesicle, hence it protects the drug from premature
recognition, excretion and degradation in the blood stream. The liposomes are also able to
accumulate in tumors because of the enhanced permeability and retention effect, EPR-effect,
reviewed in (Liposomal formulations of anticancer drugs: selectivity and effectiveness
Massing and Fuxius 2000). Blood vessels in tumors are leakier than normal blood vessels
because of their fast growth. In addition, the cells in tumors are often not as closely packed as
in healthy tissue.

Most of the cancer drugs on the marked now have dose-limiting toxicity problems and thus
relatively low efficacy. The liposomal preparations may have the potential to change this with
time. The physical properties of the liposomes, such as size and size distribution, play an
important role in the work of developing successful drug formulations. Hence, there is a great
demand on detailed and reliable information about this subject.


17

3.1.5 Challenges with liposome formulations
Intravenous injection is regarded as the most promising route of administration for liposomal
drug delivery. The role of a liposomal drug carrier is to circulate in the blood pool and reach
the desired organ or tissue. The pharmacokinetics and biodistribution of the carrier primarily
depend on the size and surface characteristics of the liposome.

Upon entering the blood pool, liposomes should avoid to be taken up by macrophages. Big
liposomes (diameter >200 nm) are quite rapidly taken up and disappear from the circulation.
Liposomes with a diameter between 70 and 200 nm tend to circulate long enough in the blood
stream to reach the desired organ. Smaller liposomes with a diameter under 70 nm show
shorter circulation time due to extravasation through the capillary walls of the liver reviewed
in (Liposomes as drug carriers: a technological approach Brandl 2001). The lipid composition
and lamellarity is also important since it together with the physiochemical properties of the
drug determines the retention of the active ingredient within the liposome reviewed in
(Liposomal formulations of anticancer drugs: selectivity and effectiveness Massing and
Fuxius 2000). Due to these facts, there is clearly a need to develop methods that are not only
able to measure the size and the size distribution of liposome dispersions, but in a next step
generates liposomes of defined size.

3.1.6 Size analysis of liposomes
The main factor that influences the in vivo behavior is the size of the liposomes. Because of
that fact it is important to have methods for determining the size and size distributions in a
reliable manner, and in a reproducible manner. Some of the techniques which have been used
are various electron microscopic methods, photon correlation spectroscopy and methods
based on fractionation of liposomes according to size such as size exclusion chromatography
(SEC), ultracentrifugation and flow field-flow fractionation (AF4).

Preferable are methods which are able to give a qualitative and quantitative overview over the
full size range, which unfortunately is difficult to achieve. Some of the methods mentioned
above such as SEC and ultracentrifugation are rather time consuming, and when developing a
method for routine size analysis of liposomes, use of time should be evaluated.


The method should be able to quantify the amount of large particles and eventually aggregates
in comparison to the amount of SUVs
size exclusion chromatography and phot

3.2 Influence of osmotic stress on liposome size
It is likely that liposomes diluted in a hypertonic medium will shrink and become smaller than
they originally were. Liposomes, which exhibit similar permeab
membranes represents a convenient model system to study osmotic stress
and water permeability of phospholipid liquid crystals Bangham 1967)
seen as a cell with a semi-permeable membran
contact with a hypertonic environment it is
impermeable solute than exists
and outside of the membrane causes
shrink (Tonicity Wikipedia 2008)
movement of water in to the cell, causing it to swell. Both of these pri
figure 3.2.1.

Figure 3.2.1: Effect of hypertonic and hypotonic solutions on blood cells. (Figure taken with permission
from: http://en.wikipedia.org/wiki/Tonicity#I

The method should be able to quantify the amount of large particles and eventually aggregates
in comparison to the amount of SUVs (Size analysis of submicron particles and liposomes by
size exclusion chromatography and photon correlation spectroscopy Ingebrigtsen 2001)
Influence of osmotic stress on liposome size
It is likely that liposomes diluted in a hypertonic medium will shrink and become smaller than
they originally were. Liposomes, which exhibit similar permeability properties to biological
membranes represents a convenient model system to study osmotic stress (Osmotic properties
and water permeability of phospholipid liquid crystals Bangham 1967). A liposome can be
permeable membrane and an aqueous core. When a cell comes in
contact with a hypertonic environment it is surrounded by a higher concentration of
meable solute than exists inside of the cell. The difference in osmotic pressure inside
causes a net movement of water out of the cell, causing it to
(Tonicity Wikipedia 2008). In the opposite case, a hypotonic environment causes a net
movement of water in to the cell, causing it to swell. Both of these principles are shown in
Figure 3.2.1: Effect of hypertonic and hypotonic solutions on blood cells. (Figure taken with permission
http://en.wikipedia.org/wiki/Tonicity#Isotonicity)

18
The method should be able to quantify the amount of large particles and eventually aggregates
(Size analysis of submicron particles and liposomes by
on correlation spectroscopy Ingebrigtsen 2001).
It is likely that liposomes diluted in a hypertonic medium will shrink and become smaller than
ility properties to biological
(Osmotic properties
. A liposome can be
e and an aqueous core. When a cell comes in
surrounded by a higher concentration of
The difference in osmotic pressure inside
a net movement of water out of the cell, causing it to
In the opposite case, a hypotonic environment causes a net
nciples are shown in

Figure 3.2.1: Effect of hypertonic and hypotonic solutions on blood cells. (Figure taken with permission


19

3.3 Previous studies
Lars Ingebrigtsen and Christer Bakke Frantzen investigated in their master theses
accomplished in December 2001 and May 2003 respectively, whether a combination of size
exclusion chromatography with subsequent size analysis performed by photon correlation
spectroscopy combined with a quantitative assay achieved a total qualitative as well as
quantitative insight into the size distribution of liposome dispersions and thus was suited as a
routine analysis method. Previous studies of the size distribution of liposomes gained
qualitative results of various qualities, but none of them provided quantitative information.
Ingebrigtsen checked how reliable results photon correlation spectroscopy (PCS) can give
when it is used for routine particle analysis of latex beads of submicron sizes. He found out
that PCS was able to resolve bimodal size distributions of the heterogeneous samples within
certain limits, i.e. for certain ratios and for certain sizes. But it was obvious that PCS is
inappropriate to resolve bimodal size distributions with a broader overlap or tri- or
multimodal size distributions. In the second part of his study he employed SEC, PCS and a
quantitative enzymatic PC assay. He found that it was feasible to determine the size
distribution of vesicle dispersions in a reliable manner and it appears especially useful to
employ the combination of SEC fractionation, PCS and the enzymatic PC quantitation. A
drawback discovered was for certain liposome dispersions that in some of the dispersions
SEC fractionation showed incomplete recovery of the vesicles. It can be explained in terms of
aggregation of these rather small vesicles which subsequently got stuck on the SEC column.
In addition, the described method is very time consuming.

This clearly indicated a demand for different fractionation method which does not have the
limitations that the SEC method described above has.

Dominik Albert Ausbacher investigated in his diploma thesis accomplished in October 2007,
if asymmetrical flow field-flow fractionation was convenient for fractionation of liposomes.
He tried to evaluate the influence of some key factors such as ionic strength of the eluent as
well as pore size of the semi-permeable membrane on liposome fractionation behavior.
Neutral liposomes were found very dependent of the ionic strength when it comes to elution
time. He saw a shift of the liposome peak to later retention times when an eluent with a higher


20

ionic strength was used, but he was not able to say whether the change in retention time of
liposomes in higher ionic strength media was due to osmotic stress or zeta-potential.
Nevertheless, his master project can only be seen as a first step in paving on the way towards
routine AF4-multi-angle light scattering (MALS) liposome analysis.

These previously executed master projects form the basis of this master project.






















21

4 AIM
The purpose if this study was to investigate how a change in osmotic pressure, with constant
ion strength, affects both elution time and calculated size of liposomes.
In more detail, our aim was to find out if the retention behavior and calculated geometric
radius of liposomes obtained by flow field-flow fractionation in combination with multi-angle
light scattering is affected by the osmotic pressure of the medium used for diluting the
liposomes and/or running the AF4. In order to execute ionic-strength effects the salt
concentration was kept constant while the osmotic pressure was varied by using mono- and
disaccharides.

























22



















23

5 MATERIAL AND METHODS
5.1 Chemicals

Table 5.1.1: Lipid
Name of lipid Batch numbers Manufacturer
Unsaturated egg phosphatidyl choline
Lipoid E-80
1031492-9/904,
1031492-11/902,
1031492-11/904
Lipoid GmbH, Ludwigshafen,
Germany


Table 5.1.2: Latex bead
Name of latex bead Mean diameter Batch number Manufacturer
Nanosphere
TM
size
standards
102 nm ± 3 nm 28570 Duke Scientific
Corporation, Freemont,
CA, USA


Table 5.1.3: Chemicals
Chemical Quality Batch number Manufacturer
Ethanol 96 % N/A Arcus Kjemi AS, Vestby,
Norway
Glucose For parenteral use 1A102/4 Norsk Medisinaldepot, Oslo,
Norway
Liquid nitrogen N/A N/A AGA AS, Trondheim,
Norway
Purified Water N/A N/A Prepared in-house by
Millipore water purification
system



24

Sodium chloride
solution,
400 mOsm/kg
N/A 5484C41 Dr. Ing. Herbert Knauer
GmbH, Berlin, Germany
Sodium nitrate p.a. A571737 519 Merck KGaA, Darmstadt,
Germany
Sucrose Ph Eur K341881187 544

K33825286 524
Merck KGaA, Darmstadt,
Germany
VWR International Ltd,
Poole, England


5.2 Equipment

Table 5.2.1: Equipment

Equipment
Type Manufacturer
Filtration device, 142 mm
diameter
SM 16275 Sartorius AG, Göttingen,
Germany
Freezer (-80 °C) Forma Laboratory Freezer,
model 738
ThermoQuest/
Forma Scientific Division,
Marietta, OH, USA
Glass tubes for PCS analysis Borosilicate glass disposable
culture tubes, 6 x 50 mm
VWR International AB,
Karlskoga, Sweden
HPLC variable-wavelength-
UV/VIS-detector
G1314A, 1100 series Agilent Technologies
Europe, Santa Rosa, CA,
USA
LAF (laminar air flow) bench Holten maxisafe 2000 Heto Holten A/S, Allerød,
Denmark
MALS-detector Dawn EOS Wyatt Tech. Corp. Europe,
Dernbach, Germany



25

Osmometer Knauer Semi-Micro
Osmometer, Type ML,
No. A0299
Wissenschaftliche Gerätebau
Dr. Ing. Herbert Knauer
GmbH, Berlin, Germany
Photon Correlation
Spectrometer- PCS
Submicron Particle Sizer
Model 380
Nicomp Particle Sizing
Systems, Santa Barbara, CA,
USA
Prototype filter extruder Continuous consisting of
Lewa diaphragh pump type
LDB 1 and
Millipore 47 mm high
pressure filter holder
Custom made,
Lewa GmbH, Leonberg,
Germany
Millipore S.A. Molsheim,
France
Pycnometer Specific gravity bottle,
25 cm
3
Brand GMBH + CO KG,
Wertheim, Germany
RI-detector Optilab rEX Wyatt, Tech. Corp. Europe.
Dernbach, Germany
Stainless steel filtration
vessel, 5 liter
SM 1753 Sartorius AG, Göttingen,
Germany
Ultrasonic bath Branson Ultrasonic Cleaner
1510E-MT
Branson Ultrasonic
Corporation, Danbury, CT,
USA
Viscometer Capillary viscometer,
capillary type 0c
Ubbelohde viscometers,
Schott-Geräte, Hofheim,
Germany
Water bath Büchi Waterbath B-480 Büchi Labortechnik AG,
Flawil, Switzerland
Water purification system Millipore water purification
system
Millipore S.A., Molsheim,
France






26

Filters for:
Filtration device, 142 mm



Syringe filter




Liposome filter extruder








Milli-Q water system;
Millipak 20 Express

Milli-Q Synthesis Quantum
EX Ultrapure organic

Nitrocellulose VCWP
0.1 µm filter,
batch no: H5JN02152

Acrodisc syringe filter,
0.2 µm filter,
batch no: 21182


Millipore Isopore Membrane
filters:
0.4 µm filter;
batch no: R5SN28296
0.2 µm filter;
batch no: R8MM92556
0.1 µm filter;
batch no: R8NM25306


0.22 µm filter;
batch no: MPGP02001
Cartridge;
batch no: F5HN65923

Millipore Corporation,
Billerica, MA, USA


Pall Corporation, Ann Arbor,
MI, USA



Millipore Ireland B.V., Cork,
Ireland








Millipore S.A., Molsheim,
France
Millipore S.A., Molsheim,
France








27

5.3 Media and solutions
For all the solutions the composition is given per 1 liter:
Table 5.3.1: Media and solutions
Name of medium Content Application
10 mM sodium nitrate solution Sodium nitrate 0.8499 g +
Purified water ad 1000.0 ml
Hydration medium in
MLV production and as
diluting agent before PCS
measurements
Also used for diluting
liposome dispersion 1:10
prior to investigation of
size changes as a result of
osmotic stress and as
mobile phase in the AF4
experiments
20 mM sodium nitrate solution Sodium nitrate 1.6998 g +
Purified water ad 1000.0 ml
Osmolality measurements
50 mM sodium nitrate solution Sodium nitrate 4.2495 g +
Purified water ad 1000.0 ml
Osmolality measurements
10 mM sodium nitrate and
19.8 mM glucose solution
Sodium nitrate 0.8499 g +
Glucose 3.5590 g +
Purified water ad 1000.0 ml
Osmolality measurements
10 mM sodium nitrate and
16.6 mM sucrose solution
Sodium nitrate 0.8499 g +
Sucrose 5.6940 g +
Purified water ad 1000.0 ml
Osmolality measurements
10 mM sodium nitrate and
79 mM glucose solution
Sodium nitrate 0.8499 g +
Glucose 14.2358 g +
Purified water ad 1000.0 ml
Osmolality measurements
10 mM sodium nitrate and
66.4 mM sucrose solution
Sodium nitrate 0.8499 g +
Sucrose 22.7780 g +
Purified water ad 1000.0 ml
Osmolality measurements



28

10 mM sodium nitrate and
177.6 mM glucose solution
Sodium nitrate 0.8499 g +
Glucose 32.0306 g +
Purified water ad 1000.0 ml
Viscosity measurements.
Also used for diluting
liposome dispersion 1:10
prior to investigation of
size changes as a result of
osmotic stress and as
diluting agent before PCS
measurements
10 mM sodium nitrate and
149.3 mM sucrose solution
Sodium nitrate 0.8499 g +
Sucrose 51.2490 g +
Purified water ad 1000.0 ml
Hydration medium in
MLV production and as
diluting agent before PCS
measurements
Also used for diluting
liposome dispersion 1:10
prior to investigation of
size changes as a result of
osmotic stress and as
mobile phase in the AF4
experiments
100 mM sodium nitrate
solution
Sodium nitrate 8.499 g +
Purified water ad 1000.0 ml
Mobile phase in the AF4
experiments

All the different solutions were prepared according to the same procedure; the solid
components were weighted in and transferred to a volumetric flask where they were dissolved
with some water. The concentrated solution was then diluted by adding water up to the
desired volume (1 liter) in a volumetric flask. All the solutions were filtered through a 0.1 µm
nitrocellulose filter.






29

5.4 Preparative methods
5.4.1 Preparation of multilamellar vesicles (MLVs)
Theory:
MLVs form spontaneously when phospholipids are blended with excess aqueous medium
(Diffusion of univalent ions across the lamellae of swollen phospholipids Bangham, Standish
1965).

Experiment:
MLVs were prepared according to the hand-shaken method:
Hand-shaken method:
10 % w/w E-80 (unsaturated egg phosphatidyl choline) in different aqueous media
1. E-80 5 g
2. Aqueous medium 45 g

E-80 and the aqueous medium were weighed in directly in a round bottom flask. The
components were stirred using a magnetic stirrer until E-80 was finely dispersed, which takes
approximately 45 minutes.

5.4.2 Reduction of lamellarity
Theory:
To increase the proportion of unilamellar vesicles in preparations it is a common practice to
subject MLVs to freeze-thaw cycles prior to extrusion. (Osmotic properties of large
unilamellar vesicles prepared by extrusion Mui, Cullis 1993). The freezing and thawing
cycles cause the MLVs to rupture and re-assemble such as the aqueous layers between
concentric lamellae increase in thickness; this probably reduces the number of closely
associated bilayers. (Liposome technology Mui and Hope 2006)



30


Experiment:
In order to find out which freeze-thawing method would give the best result, four preliminary
experiments were executed.

1. Freeze-thawing was performed before extrusion. The liposome dispersion was frozen
in a -80°C freezer for 1 hour and then thawed on a 50°C water bath. The freeze-thaw
cycle was repeated three times.

2. Freeze-thawing was performed before extrusion. The liposome dispersion was frozen
in liquid nitrogen (LN
2
) and then thawed on a 50°C water bath. The freeze-thaw cycle
was repeated three times.

3. Freeze-thawing was performed between extrusion through 400 nm filer and 200 nm
filter. The liposome dispersion was frozen for one hour in a -80°C freezer and thawed
on a 50°C water bath. The freeze-thaw cycle was repeated three times.

4. Freeze-thawing was performed after extrusion. The liposome dispersion was frozen in
a -80°C freezer for one hour and thawed on a 50°C water bath. The freeze-thaw cycle
was repeated three times.







31

5.4.3 Reduction of liposome size
Theory:
MLVs have a broad particle size distribution and have multiple internal compartments. Due to
this fact; unprocessed MLVs have limited use in medical research. In order to achieve
liposomes with homogeneous size, filter extrusion was accomplished. The extrusion was
performed on a custom made extruder, as shown in figure 5.4.1. Filter extrusion involves the
process of forcing the liposome preparations through pores of membrane filters with defined
pore sizes. The preferred filter type for reducing the size of liposomes is made of
polycarbonate with straight-through, cylindrical pores. The pores have been formed by
chemical etching along ion tracks. When the MLV preparation is squeezed through the filter
pore under pressure a process of membrane rupture and resealing occurs, this process
generates large to small unilamellar vesicles with a mean vesicle diameter usually slightly
larger than the pore size of the polycarbonate membranes. After about 10 cycles through
filters with 100-nm pores a homogeneous population of vesicles with a mean diameter of
approximately 100 to 120 nm is obtained. (Liposome technology Mui and Hope 2006)

Experiment:
The MLV dispersion obtained by the hand-shaken method was extruded using filters with
decreasing pore sizes of 400 nm, 200 nm and 100 nm. The transfer of liposomes through the
filter was repeated 10 times, as recommended by (Liposome technology Mui and Hope 2006).
The pump of the extruder was running at the same speed every time.








32


Figure 5.4.1: The custom made extruder

5.5 Analytical methods
5.5.1 Characterization of particle size by Photon Correlation Spectroscopy
Theory:
PCS is an analytical tool to determine the size distribution of submicron particles suspended
in an aqueous medium. The technique has proven to be especially powerful in measuring
particles with a diameter of approximately 20-200 nm. A laser light beam (typically 5 mW
Helium and Neon laser) is focused on a glass tube containing a diluted suspension of
particles. Each of the particles scatters light in all directions and the intensity of scattered light
varies with the particles molecular weight, size and shape. The difference in refractive indices
of the particle and the surrounding medium also play an important role (Windows based
software, Dynamic light scattering theory User Manual 1997).




33

PCS measures the fluctuations in the scattered light intensity. Scattered light intensity
fluctuates with time because many individual waves add coherently. This is the physical
phenomenon known as interference. All the different waves interfere at a distant slit on the
face of a photomultiplier detector, which measures the net scattering intensity at a 90 degrees
scattering angle. The suspended particles move around randomly in the medium by Brownian
motion. As a consequence of these motions, the phase of each of the scattered light waves that
is arriving at the detector will fluctuate randomly in time due to the random positions of the
particles (Size analysis of submicron particles and liposomes by size exclusion
chromatography and photon correlation spectroscopy Ingebrigtsen 2001). The fluctuation of
light intensity is dependent on the size of the particle. Small particles will move around faster
and give rapid fluctuation of the light intensity.

The next step is to determine the diffusion coefficient, D, of the particles from the raw data.
From D it is possible to calculate the particle diameter using the Stokes-Einstein equation.

Equation 1: ˖ = ˫ˠ¡ ŷnnˤ
s


k = Boltzmann´s constant (1.38 X 10
-16
erg K
-1
)
T = temperature (°K, = °C + 273)
η = shear viscosity of the solvent
d
s
= Stokes particle diameter

From equation 1 we can see that the diffusion coefficient, D, of particles increases with
increasing temperature, T. This is primarily due to the temperature dependent viscosity of the
solvent, η.




34

Autocorrelation is the mathematical process of extracting quantitative information as the size
of the particles and their size distribution in a sample from the fluctuation of the intensity of
the scattered laser light (Size analysis of submicron particles and liposomes by size exclusion
chromatography and photon correlation spectroscopy Ingebrigtsen 2001). The autocorrelation
function is used to study the similarity between the value of I
s
(light intensity)

at a given time
and the value of I
s
at an earlier time, t-t´. Such comparisons are carried out for many values of
(t) in order to get a statistical meaningful average value for C (t´).

The correlation function can be expressed:

Equation 2: ˕ (ˮ´) = X Is (ˮ) × Is (ˮ -ˮ´)

One can describe the autocorrelation function as an exponential function that gradually
decreases as the value of (t´) increases. As expressed below in equation 3.


Equation 3: ˕ (ˮ´) = ˓ ˥˲p (-ˮ´¡¡) + ˔

A = Σ I
s
2
(t) - Σ I
s
(t)
2

B = Σ I
s
(t)
2


Variable τ is the characteristic decay time constant of the exponential function. The value of τ
describes the duration of a major fluctuation in the scattered intensity I
s
. Hence, the larger the
particles, the slower fluctuations in I
s
and the longer the decay constant τ. We are able to
predict the diffusion coefficient of the particles from the decay constant τ.



35


Equation 4: ˖ = (ŵ¡Ŷ KŶ) (ŵ¡¡)

K = scattering wavevector (A constant which depends on the laser wavelength in the solvent
and the angle between the laser beam and where the detector is placed.)

Fitting and interpretation of the results:
The PCS software fits the raw data, collected by the detector, using either the NICOMP model
or the monomodal Gaussian model. NICOMP is used for bi- or multimodal size distribution
and Gaussian is used for a unimodal size distribution. The Gaussian model states how good a
fit is approaching a normal distribution.

The Gaussian analysis is restricted to simple, unimodal particle size distributions which are
the case in this thesis, and NICOMP distribution analysis will consequently not be further
explained.

Gaussian distribution:
The PCS software will indicate how well the measured results fit with the normal distribution
or the Gaussian model. The quality of this fit is stated by the statistical value Chi squared.
Any value close to or below one indicates an exceptionally good fit, but any value under three
is regarded well enough. If the value of Chi squared is over three, the PCS software suggests
that the Gaussian model is inappropriate, and the NICOMP model should be used instead.

The value for baselines adjust is indicating an adjustment needed to obtain a low value of Chi
squared. The ideal value is zero. A higher baseline adjust value is indicative for large particles
or aggregates in the sample.



36

Polydispersity index (P.I.) is stating how broad the distribution is around the mean particle
size. A low P.I. value thus indicates a homogenous size distribution. A P.I. value close to zero
is therefore most desirable. For highly polydisperse samples the P.I. approaches one
(Windows based software, Dynamic light scattering theory User Manual 1997).

Experiment:
Measurements were carried out as described by (Determination of the size distribution of
liposomes by SEC fractionation, and PCS analysis and enzymatic assay of lipid content
Ingebrigtsen and Brandl 2002). In brief, the test tubes used for PCS measurements were
sonicated for 10 minutes and then rinsed with the dilution medium. Samples were diluted
using particle free medium, until the intensity was between 250 and 350 kilohertz (kHz). They
were diluted with the same medium as used for producing the liposomes. It is very important
that the intensity level is correct because the correlator´s input counter must not receive more
photons than it can count in a single sample time otherwise the correlation function will be
distorted (Particle size analysis in pharmaceutics and other industries: theory and practice
Washington 1992). To avoid particle contamination the dilution medium was filtrated through
a sterile filter with 0.2 µm pore size. All the preparative work was done in a laminar airflow
bench to avoid particle contamination.

Before any measurement was carried out the instrument parameters were set according to the
values listed in table 5.5.1. For statistical accuracy, a cycle of 5 minutes was run for each
sample in order to calculate how long a sample need to be run to ensure count rates above
1000 K (1 million) in channel no. 1.








37

Table 5.5.1: PCS parameters
Parameter Value
Channel width Auto set
Temperature Room temperature, usually 23-25 °C
Liquid viscosity If the liquid only contained sodium nitrate, values of viscosity of water
was used. The values were obtained from a table in the PCS manual.
(E.g. if the temperature was 23 °C then the viscosity would be 0.9325
cP.) The viscosity values for the solutions that contained glucose or
sucrose were measured with a capillary viscometer.
Liquid index of
refraction
1.333 is the literature value of water, and the same value was used
when the solution only contained water and sodium nitrate. Values for
the solutions that contained sucrose and glucose were measured with
the Optilab rEX refractive index detector
Intensity setpoint 300 ± 50 kHz
Laser wavelength 632.8 nm
Scattering angle 90 °



Figure 5.5.1: Block diagram of the PCS (NICOMP Model 380 submicron particle sizer)



38

5.5.2 Determination of osmolality in solutions
Theory:
Osmolality is a measure of the osmoles of solute, per kilogram of solvent. An osmole is the
amount of substance that yields, in ideal solutions, that number of particles that would reduce
the freezing point of the solvent by 1.86 °C. E.g., when one mole of non-ionic solute is added
to one kilogram of water, the freezing point goes down 1.86 °C. When one mole ionic solute
e.g., NaNO
3
is dissolved in a kilogram of water it will yield almost twice as many particles
since NaNO
3
dissociates almost completely into one mole Na
+
and one mole NO
3
-
ions
(Refractive index 2008).

The osmotic strength of a solution can be measured by an osmometer. Currently available
osmometers use the colligative properties of freezing point depression or vapour pressure
depression.

The equation to determine the osmolality of a solution is shown in equation 5:

Equation 5: ˛s˭oˬoˬ˩ˮ˳ = u × n × ˭oˬoˬ˩ˮ˳

Ф = osmotic coefficient, which accounts for the degree of non-ideality of the solution. Ф is
between 0 and 1, 1 means that 100 % dissociates.
n = number of particles into which the molecule can dissociate (e.g., 1 for sucrose, 2 for
NaNO
3
)
The unit of osmolality is Osm/kg (osmole per kilogram).





39

Experiment:
Calculations of the osmolalities were performed according to the calculation method
described in appendix 1. Measurements were carried out by Knauer semi-micro osmometer to
ensure that the calculations were correct. E.g., one solution with 20 mM NaNO
3
was
compared to a solution with 10 mM NaNO
3
and an amount of glucose equivalent to 10 mM
NaNO
3
. Three parallels were measured for every solution. If the measured values were equal
to each other, it would prove that the calculated amount of glucose was correct. The
measurement was executed on an osmometer which measured the freezing point depression of
the solutions. Figure 5.52 shows the osmometer used for these experiments.

Figure 5.5.2: Knauer semi-micron osmometer

5.5.3 Determination of viscosity in solutions
Theory:
Viscosity is a measure of the fluids resistance to flow. Viscosity can be measured by various
types of viscometers. One of the most common and most accurate instruments for measuring
kinematic viscosity of Newtonian fluid´s is the glass capillary viscometer. Dynamic viscosity
coefficient is calculated from kinematic viscosity by multiplying the dynamic viscosity by the
density of the Newtonian fluid (Viscosity Wikipedia 2008).




40

The equation to calculate dynamic viscosity from kinematic viscosity is shown in equation 6:
Equation 6: n = v × µ

η = dynamic viscosity
ν = kinematic viscosity
ρ = density
The unit of dynamic viscosity is mPa·s (pascal-second).

Experiment:
Measurements of viscosity were carried out because the knowledge of the accurate viscosity
is crucial when PCS analysis is performed. Every solution that was used as dilution medium
in PCS was measured, except those who only contained NaNO
3
because it was assumed that
the viscosity would not change noticeably. The viscosity of both the solutions that contained
sucrose and NaNO
3
, or glucose and NaNO
3
were measured. The kinematic viscosity was
measured using a glass capillary viscometer. To calculate the dynamic viscosity the density of
the solution is needed. The density of the solutions was measured with a pycnometer. Four
parallels for every solution were measured, and the average value was used in the PCS
software.

5.5.4 Determination of refractive index in solutions
Theory:
Refractive index is the other parameter that needs to be determined to get reliable results from
the PCS measurements, besides viscosity. The refractive index of a solution is a measure of
how much the speed of light is reduced inside the medium or the bending of a ray of light
when passing from one medium into another. The refractive index of vacuum is by definition
1, and the refractive index of water is 1.333.


41

A refractive index of 1.333 means that light travels at 1 / 1.333 = 0.75 times the speed in
vacuum (Refractive index Wikipedia 2008).
The refractive index can be defined by equation 7:


Equation 7: n =
c
¡
µ


n = the refractive index
c = phase velocity of a wave
v
p
= phase velocity of the medium itself

Experiment:
The refractive indices were measures using the Optilab rEX on-line refractive index detector.
All solutions used during this project were measured. For the measurements the respective
solution was injected into the instrument with a syringe. It was necessary to set the Optilab
rEX into purge mode, which means that both the glass cells in the instrument are flushed with
medium and the absolute refractive index is measured. To prevent dilution of the medium that
should be measured, the instrument was flushed until the value of refractive index did not
change anymore.


42


Figure 5.5.3: Picture of the Optilab rEX refractive index detector (Figure used with permission from:
http://www.wyatt.com/solutions/hardware/Refractive_Index_Detector-OptilabrEx.cfm)

5.5.5 Characterization of liposomes, Flow Field-Flow Fractionation
Theory:
Asymmetrical flow field-flow fractionation is a one-phase chromatography technique which
allows separation of heterogeneous samples and is able to perform fractionation ranging from
the 1 nm up to 10 microns (Changes in Liposome Morphology Induced by Actin
Polymerization in Submicrometer Liposomes Nickels 2003). The instrumental Set-Up of an
AF4 system is comparable to a HPLC (high performance liquid chromatography) system.
However, the fractionation of samples takes place in a separation channel instead of a
separation column. Particles are separated by flow in aqueous media. This is done by the
application of field force generated by the transverse movement of carrier liquid (cross flow)
across the channel. AF4 is fractionating particles according to their size and determining size
distribution of polydisperse particle samples from an observed retention profile (Size
characterization of liposomes by flow field-flow fractionation and photon correlation
spectroscopy Effect of ionic strength and pH of carrier solutions Moon, Park 1998).



43

The channel consists of a lower block which contains the cross flow outlet, the permeable frit,
the membrane and the spacer. The spacer foil has a typical thickness of 100 to 500 µm. The
thickness and the form of the spacer foil are defining the dimensions of the actual channel.
The upper block contains the channel inlet, the injection port and the channel outlet as shown
in figure 5.5.4.
The upper channel plate is impermeable, but the bottom channel plate, on the other hand, is
permeable. An ultra filtration membrane with a typical size barrier of 10 kD, covers the
bottom plate to prevent the sample from penetrating the channel (How Asymmetric Field
Flow Fractionation (AFFF) Theory Works Technology 2008).
Figure 5.5.4: Channel setup, different flows and forces during A4F (Figure used with permission from
Dominik Ausbacher (A4F/MALS-Analysis of Liposomes Influence of Key Factors on Fractionation
Behavior and Evaluation of MALS Fit Routines Ausbacher 2007))

Upon injection into the AF4 channel particles are driven toward the bottom of the channel
wall by the cross flow. After injection the sample is focused on a small band near the injection
point by applying an inverse flow through the channel outlet.
Equilibrium positions are established away from the accumulation wall, due to the particles
diffusive transport. The Brownian motion of the particles or vesicles leads them to be
differentially distributed over the accumulation wall according to their size; large particles
have a small diffusion coefficient and are therefore driven closer to the accumulation wall.
The small particles will move around faster and float further from the accumulation wall
hence they are displaced by the fast flow stream and are eluted earlier than the larger ones, as
can be seen in figure 5.5.5 (A4F/MALS-Analysis of Liposomes Influence of Key Factors on
Fractionation Behavior and Evaluation of MALS Fit Routines Ausbacher 2007).
back-diffusion
force by flow
field = generated by cross flow
channel out
membrane + frit
eluent eluent
injection
port
channel in
channel
height
provided
by spacer
Cross flow out
upper block
lower block
sample
(focusing)
back-diffusion
force by flow
field = generated by cross flow
channel out
membrane + frit
eluent eluent
injection
port
channel in
channel
height
provided
by spacer
Cross flow out
upper block
lower block
sample
(focusing)


44



Figure 5.5.5: Side view of particle migration according to the size in the AF4 channel

The separation based on AF4 technology, is divided into four steps. These are injection,
relaxation, focusing and elution. The first three steps injection, relaxation and focusing are
quite simultaneous and are followed by the elution. In the first step, the channel flow is split
and introduced both at the inlet and at the outlet of the channel (Asymmetric Flow Field-Flow
Fractionation Analytics 2008). After focusing, the next step is the experiment is the so-called
elution mode. In elution mode both the cross flow and channel flow active and fractionation
can take place. In AF4 is retention time (t
r
) of a particle given by equation 8.

Equation 8: ˮ
¡
=
w
2

×
v
x
v


V = channel flow
V
x
= cross flow
w = channel thickness
D = diffusion coefficient



45

As we can see in equation 8, the retention time is directly proportional to the square of the
channel thickness, and inversely proportional to the diffusion coefficient. The diffusion
coefficient can be used for calculating the molecular dimensions in the form of the Stokes
diameter (On-line coupling of flow field-flow fractionation and multi-angle laser light
scattering Roessner and Kulicke 1994). The mathematical basis for this is provided by the
Stokes-Einstein equation shown in equation 1. If we link equation 1 and equation 8 we get
equation 9, which gives the dependence of the retention time on the material and experimental
parameters.

Equation 9: ˮ
¡
=
nnw
2
2k1
×
v
x
v
× ˤ
s


Vesicle diameter can readily be calculated from experimental retention time (t
r
) when the
experimental parameters are known. A4F can give a direct measure of liposome size since
separation is based on the difference in hydrodynamic radius of the particles (Size
characterization of liposomes by flow field-flow fractionation and photon correlation
spectroscopy Effect of ionic strength and pH of carrier solutions Moon, Park 1998). However
a direct determination of hydrodynamic radius was not performed in this work because the
applied method requires more complex mathematics for calculating the hydrodynamic radius
which is not available at the time.

Theory:
Instead of determining the hydrodynamic radius from the retention time of particles in an AF4
run, the particles size is measured by MALS. In a MALS detector several photo diodes are
arranged in a circle around a glass cell with a bore where the sample runs through. When light
from a polarized laser light beam hits a sample molecule, LS (light scattering) will occur in all
directions as demonstrated in figure 5.5.6. The resulting scattered light will then be detected
by the photo diodes at the different angles from 10° to 160°. The wavelength of the laser light
used to illuminate the solution containing the sample is 690 nm (Wyatt Technology
Corporation User Manual 2007).


46


Figure 5.5.6: Laser light scattering. (Figure taken with permission from: Wyatt Technology (Introduction
to Light Scattering, Light Scattering University Technology 2006))

One big advantage with light scattering experiments is that the solute can be measured in
solution in a non-invasive manner. The symbol used to describe angle-dependent light
scattering is R
θ
, called the excess Rayleigh ratio. R
θ
is defined in equation 10. The excess
Rayleigh ratio is a ratio of the scattered light and incident light intensities that take into
account different factors which are shown in equation 10. It is called the excess ratio because
it is for scattered light in excess of scattered light from the solvent, for the solute or particle
alone (Wyatt Technology Corporation User Manual 2007).


Equation 10: ˞
0
=
(I
0
-I
0,scl¡cnt

2
I
0
v


I
θ
= scattered intensity
I
θ, solvent
= scattered intensity of the solvent
I
0
= intensity of the beam
V = volume of the scattering medium
r = distance between the scattering volume and the detector


47

If we know R
θ
at a number of different angles it leads directly to the weight average molar
mass and mean square size of the solute molecules. This makes R
θ
the most important
measured quantity in light scattering (Wyatt Technology Corporation User Manual 2007).

The intensity carries information about the molar mass, while the angular dependency carries
information about the size of the macromolecule. It can hence give information about both the
particle size and the molar mass of the particle. The calculations given from the MALS
detector software are based on equation 11.

Equation 11:
R
0
Kc
= H˜(0) - Ŷ˓
2
cH
2
˜
2
(0)

R
θ
= excess Rayleigh ratio (cm
-1
)
K = optical constant (=4π
2
n
0
2
(dn/dc)
2
λ
0
-4
N
A
-1
), where n
0
is the refractive index of the solution,
λ
0
is the radiation wavelength in vacuum expressed in nanometers, N
A
is Avogadro´s number
and dn/dc is the differential refractive index of the solvent-solute concentration
c = concentration
M = molar mass (g/mol)

P(θ) = theoretically-derived form factor
A
2
= second virial coefficient (mol mL/g
2
)

Astra is the software which processes the MALS data. Astra calculates an rms (root mean
square) radius moments for each peak. The different rms radius´s measured are number-
average mean square radius (equation 12), z-average mean square radius (equation 13) and
weight-average mean square radius (equation 14). All summaries are taken over one peak
(Wyatt Technology Corporation User Manual 2007).


48

Equation 12: < r
2
>
n
=
l|
c
i
M
i

2
>
i
1
l
c
i
M
i


Equation 13: < r
2
>
z
=
l(c
i
M
i

2
>
i
)
l (c
i
M
i
)


Equation 14: < r
2
>
w
=
l(c
i

2
>
i
)
lc
i


c
i
= mass concentration
M
i
= molar mass
< r
2
> = mean square radius of the i
th
slice

Experiment:
AF4 experiments were performed using the Eclipse 2 instrument system from Wyatt
Technology Europe. The flow field-flow fractionation is coupled on-line with a Dawn EOS
18 angle light scattering, a single wavelength UV detector and an Optilab rEX differential
refractive index detector (RI-detector). A 250-µm spacer was applied and a main flow of 1.0
ml/min. A cross flow gradient was applied; the cross flow was reduced from 1.0 to 0.15
ml/min. All the samples were diluted 1:10 with the mobile phase prior to the measurements.
The injection volume was the same in every experiment, 10 µl.

The liposomes are assumed to be hollow spheres in which each of the lipid molecules acts as
an isotropic scattering element. The angular dependence of the scattering is expressed by the
so-called form factor or shape-factor, P(θ).


49

The form factor is assuming a shell thickness of 3.7 nm for phosphatidylcholine vesicles, as
has been measured using X-ray diffraction (Characterization of vesicles by classical light
scattering Van Zanten and Monbouquette 1991).
The form factor is the mathematical relationship describing the angular variation of the
scattered intensity as a function of particle size, shape and structure. It is also called the
particle scattering function (Introduction to Light Scattering, Light Scattering University
Technology 2006). For processing the received data the ASTRA (version) 5.1.5 and Eclipse
software from Wyatt Technology were used.

























50

















51

6 RESULTS AND DISCUSSION
6.1 Preliminary experiments
6.1.1 Osmolality measurements
In order to expose the liposomes to osmotic stress it was necessary to prepare solutions of
distinct osmolalities. In addition to calculating the amount of solute that is needed for a
solution with a given osmolality it was decided to measure the osmolality of different
solutions to check the calculated values and to check that sodium nitrate dissociates
completely. We calculated how much glucose or sucrose is needed to make a solution with
the same osmolality as a solution with a known sodium nitrate concentration, as describes in
appendix 1. The calculated amounts are given in table 6.1.1.

Table 6.1.1: Amount of sucrose, glucose and sodium nitrate needed to make solutions of various given
osmolalities
Amount solid (given in gram per liter) Concentration of the solution
3.5592 g glucose + 0.8499 g NaNO
3
Equivalent to 20 mM sodium nitrate
14.2363 g glucose + 0.8499 g NaNO
3
Equivalent to 50 mM sodium nitrate
0.5694 g sucrose + 0.8499 g NaNO
3
Equivalent to 20 mM sodium nitrate
2.2778 g sucrose + 0.8499 g NaNO
3
Equivalent to 50 mM sodium nitrate
0.8499 g NaNO
3
20 mM sodium nitrate
4.2495 g NaNO
3
50 mM sodium nitrate


We prepared two different solutions, one with 20 mM sodium nitrate and one with 50 mM
sodium nitrate. Corresponding solutions that contained 10 mM sodium nitrate and an amount
of glucose or sucrose (equivalent to either 10 mM sodium nitrate or 40 mM sodium nitrate)
that made the osmolality equal to the corresponding sodium nitrate solution were also
prepared. Both corresponding solutions contained the same amount of salt because we wanted
to have the same ionic strength in all the solutions.


52

The freezing points of all the solutions were measured using an osmometer, against a sodium
chloride calibration solution of known osmolality. The results, expressed in mOsm/kg, are
summarized in table 6.1.2.

Table 6.1.2: Results from the osmolality measurements
Solution Measured value (average ± SD of three parallels)
20 mM sodium nitrate 40.0 mOsm/kg ± 0 mOsm/kg
10 mM sodium nitrate and glucose
equivalent to 10 mM sodium nitrate
42.0 mOsm/kg ± 0 mOsm/kg
10 mM sodium nitrate and sucrose
equivalent of 10 mM sodium nitrate
39.0 mOsm/kg ± 1 mOsm/kg
50 mM sodium nitrate 95.3 mOsm/kg ± 0.58 mOsm/kg
10 mM sodium nitrate and glucose
equivalent to 40 mM sodium nitrate
101.0 mOsm/kg ± 1 mOsm/kg
10 mM sodium nitrate and sucrose
equivalent to 40 mM sodium nitrate
98.0 mOsm/kg ± 0 mOsm/kg


The results show that the measured osmolalities of the salt solutions and corresponding
combined salt and sugar solutions were within 5 % variability. The calculated values of sugar
needed to prepare a solution with a distinct osmolality were thus confirmed. It was decided
that when other solutions with different osmolality were to be made, it would be adequate
only to calculate the values and not measure every solution with the osmometer.

6.1.2 Viscosity measurements
It is also important to determine the exact viscosity of the various dispersion media used for
PCS measurements. The reason why viscosity is so important can be described with the
Stokes-Einstein equation expressed in equation 15:



53

Equation 15: ˖ = ˫ˠ¡źnn˞

From D (the diffusion coefficient) in the Stokes-Einstein equation it is easy to calculate the
particle radius as described in section 5.5.1. η in equation 15 is liquid viscosity and it is
affecting the size calculation.
If an incorrect viscosity value is entered in the PCS software the calculation will be wrong,
resulting in an incorrect mean Stokes diameter.

Both media that contained sugar (glucose or sucrose) were measured. For the solution just
containing sodium nitrate the viscosity was assumed to be the same as for water. In table 5.3.1
we can see that the solution with 10 mM sodium nitrate contained 0.8499 g/L or 0.8499 %
sodium nitrate. The literature value for viscosity of an aqueous solution with 0.5 % sodium
nitrate is 1.0016 cP (20°C), and for a solution with 1.0 % the viscosity is 1.0050 cP (20°C). If
we compare those values to the viscosity of water 1.0020 cP (20°C) we can see that the
amount of sodium nitrate in 10 mM sodium nitrate solution would not affect the viscosity
markedly (CRC Handbook of Chemistry and Physics Lide 2008). The measurements were
executed as described in section 5.5.3, with a glass capillary viscometer. The results are given
in table 6.1.3. A calculation example is given in appendix 2.

Table 6.1.3: Results from the viscosity measurements
Solution Measured viscosity
10 mM sodium nitrate and glucose equivalent
to 90 mM sodium nitrate
1.001 cP (mPa·s)
10 mM sodium nitrate and sucrose equivalent
to 90 mM sodium nitrate
0.9898 cP (mPa·s)




54

6.1.3 Refractive index measurements
The intensity of light scattered by a single, isolated particle depends on its molecular weight
and overall size and shape, but also on the difference in refractive indices of the particle and
the surrounding solvent. Therefore it is of great significance to know the exact refractive
index of the dilution medium when PCS measurements are executed.
The refractive indices of media were measured with a RI (refractive index)-detector in the
batch mode as described in section 5.5.4. The results are summarized in table 6.1.4.

Table 6.1.4: Results from refractive index measurements
Solution Measured refractive index
10 mM sodium nitrate 1.333
10 mM sodium nitrate and glucose equivalent
to 90 mM sodium nitrate
1.336
10 mM sodium nitrate and sucrose equivalent
to 90 mM sodium nitrate
1.339


6.1.4 Influence of viscosity and refractive index on the accuracy of PCS size
measurements of latex bead standards
Latex particles with a specified size of 102 ± 3 nm were used. Latex bead standards were
diluted in all the different media used to dilute liposomes for PCS measurements. They were
diluted in 10 mM sodium nitrate medium, in 10 mM sodium nitrate and glucose medium and
in 10 mM sodium nitrate and sucrose medium. Firstly the viscosity and refractive index
values of water were used in the PCS software. The PCS results are given in table 6.1.5.







55

Table 6.1.5: PCS mean diameters of latex beads calculated on the basis of viscosity and refractive index
values of water. The number of valid parallels used for calculations of mean vesicle sizes and standard
deviations are given in table 9.13-9.15 in appendix 3
Sample Mean vesicle size ± SD (from PCS measurements)
Intensity weighting Volume weighting Number weighting
Latex bead standards diluted
with 10 mM sodium nitrate
105.6 nm ± 0.21 nm 103.6 nm ± 1.52 nm 101.3 nm ± 1.97 nm
Latex bead standards diluted
with 10 mM sodium nitrate
and glucose equivalent to 90
mM sodium nitrate
113.5 nm ± 0.41 nm 110.7 nm ± 1.88 nm 107.6 nm ± 4.20 nm
Latex bead standards diluted
with 10 mM sodium nitrate
and sucrose equivalent to 90
mM sodium nitrate
117.9 nm ± 0.39 nm 116.6 nm ± 0.86 nm 115.1 nm ± 1.97 nm


From the table above it is possible to see that the viscosity and refractive index values play a
significant role in the size measurements executed by PCS. As we can see the mean diameter
is around 15 nm larger than the defined diameter when sucrose is used as medium with the
viscosity and refractive index values of water.
In the next step the PCS results were recalculated size using the measured values for both
refractive index and viscosity of the sugar solutions. The PCS results are given in table 6.1.6.









56

Table 6.1.6: PCS mean diameters of latex beads calculated on the basis of measured viscosity and
refractive index values. The number of valid parallels used for calculations of mean vesicle sizes and
standard deviations are given in table 9.16-9.17 in appendix 3
Sample Mean vesicle size ± SD (from PCS measurements)
Intensity weighting Volume weighting Number weighting
Latex bead standards diluted
with 10 mM sodium nitrate
and glucose equivalent to
90 mM sodium nitrate
106.2 nm ± 0.40 nm 103.6 nm ± 1.74 nm 100.7 nm ± 3.86 nm
Latex bead standards diluted
with 10 mM sodium nitrate
and sucrose equivalent to
90 mM sodium nitrate
104.6 nm ± 0.34 nm 103.8 nm ± 0.64 nm 102.8 nm ± 1.52 nm


As we can see in table 6.1.6 the measured viscosity and refractive indices yield diameters
closer to the value specified by the manufacturer. The mean diameter of the latex particles is
smaller as compared to the data based on the viscosity and refractive index value of water,
and is close to 102±3 nm as specified by the producer.

6.1.5 Freeze-thaw experiments
Liposomes were prepared by the hand-shaken method according to section 5.4.1, with
subsequent extrusion of the raw MLV dispersion through polycarbonate filters. Extrusions
were carried out following the method described in section 5.4.3, continuous filter extrusion.
It is a common practice to increase the proportion of unilamellar vesicles in preparations by
subjecting MLVs to freeze-thaw cycles prior to extrusion (Liposome technology Mui and
Hope 2006). Experiments were executed to investigate how different freezing methods
affected the size distributions of liposomes.




57

-80°C freezer and liquid nitrogen were used for freezing the liposome dispersions. In addition
we investigated whether it would make a difference if the freeze-thawing cycles were done
before, after or during the extrusion process. All the different liposome preparations were
thawed on a 50°C water bath for approximately 20 minutes, until the liposome dispersion was
completely thawed. In total, four preliminary freeze-thaw experiments were executed as
described in section 5.4.2. The results from the PCS measurements are given in table 6.1.7.

Table 6.1.7: PCS mean diameter ± standard deviation and distribution width ± standard deviation from
freeze-thawing experiments. The number of valid parallels used for calculations of mean vesicle sizes,
distribution widths and standard deviations are given in table 9.18-9.23 in appendix 3
Experiment description Mean vesicle size ± standard deviation
Intensity weighting Volume weighting Number weighting
1. Liposome dispersion frozen
in -80°C freezer before
extrusion
116.6 nm ± 0.35 nm 116.5 nm ± 0.40 nm 89.9 nm ± 2.81 nm
2. Liposome dispersion frozen
in LN
2
before extrusion
112.4 nm ± 0.38 nm 111.7 nm ± 0.45 nm 86.1 nm ± 2.17 nm
3. Liposome dispersion frozen
in -80°C freezer during
extrusion
114.5 nm ± 0.25 nm 114.1 nm ± 0.28 nm 91.3 nm ± 2.70 nm
4. Liposome dispersion frozen
in -80°C freezer after extrusion
Not possible, the liposomes were broken and formed much bigger
aggregates. It was not possible to measure the liposomes by PCS.
Experiment description Distribution width ± standard deviation
Intensity weighting Volume weighting Number weighting
1. Liposome dispersion frozen
in -80°C freezer before
extrusion
32.1 nm ± 1.81 nm 32.1 nm ± 1.83 nm 24.7 nm ± 0.62 nm
2. Liposome dispersion frozen
in LN
2
before extrusion
31.3 nm ± 1.17 nm 31.1 nm ± 1.17 nm 23.9 nm ± 0.38 nm
3. Liposome dispersion frozen
in -80°C freezer during
extrusion
29.5 nm ± 1.62 nm 29.4 nm ± 1.62 nm 23.5 nm ± 0.67 nm


58

As the results in table 6.1.7 show, there is no major difference in the measured mean sizes for
the first three preparation methods. It seems that it does not make a big difference whether the
freeze-thawing cycles are done before extrusion or during extrusion. It might help to look at
the distribution width (expressed as standard deviation), because ideally the liposomes should
be more homogenous in size after freeze-thawing cycles and then the standard deviation
would decrease. There is a minor difference, but it is too small to say whether one method
should be preferred above another.

It was decided to perform a two sample t-test to see if there was a significant difference
between the different freeze-thawing methods. All the t-tests were executed with a
significance level of 0.05. It was discovered that there was a significant difference between
the liposome dispersion frozen in a -80° freezer before extrusion and the liposome dispersion
frozen in a -80° freezer during extrusion. When the other liposomes dispersions were
compared it was not discovered a statistical significant difference.

There is not a large size difference from the liposomes frozen in liquid nitrogen to the
liposomes frozen in -80°C freezer. Again only small differences could be seen when looking
at the standard deviations of the different methods. The third setup/experiment was rather
impractical because the extrusion process had to be stopped. This doubled the loss of
dispersion and the extruder had to be cleaned twice. For being the most time saving method
the first procedure was chosen for further preparations.

All liposomes show a slightly larger mean diameter than the pore size of the filter used for the
extrusions. This can be explained by the flexibility of the liposomal membranes. Lipoid E-80
forms very flexible liposomes that are assumed to alter their shapes while squeezing through
the filter pores (Liposome technology Gregoriadis 2006). Liposomes that are substantially
bigger then 100 nm will either break and form smaller liposomes that are able to pass through
the polycarbonate filter, or they can alter their shape and hence pass through the filter even
though the diameter is bigger than 100 nm.



59

6.2 Influence of osmotic stress on liposome size, measured by PCS
All the liposome dispersions made throughout this project were prepared as the liposomes in
experiment 1 in section 5.4.2. Every liposome dispersion was made as a 10 % lipid dispersion,
produced by the hand-shaken method, described in section 5.4.1 with subsequent filter
extrusion, as described in section 5.4.3. The dispersion media varied between sodium nitrate
solution, sodium nitrate and glucose solution or sodium nitrate and sucrose solution.
We could have used more sodium nitrate to adjust the osmolality, but it would also influence
the ionic strength and thus potentially the retention behavior in the flow field-flow
fractionation experiments (A4F/MALS-Analysis of Liposomes Influence of Key Factors on
Fractionation Behavior and Evaluation of MALS Fit Routines Ausbacher 2007).

6.2.1 Hypertonic osmotic stress experiment with 10 mM NaNO
3
and glucose equivalent
to 90 mM NaNO
3
as dilution medium
The first experiment was executed with liposomes prepared in 10 mM sodium nitrate
medium. The liposome dispersion was freeze-thawed as described in section 6.1.5 before
extrusion, and the mean diameter was measured by PCS right after preparation. The liposome
dispersion was diluted 1:10 with 10 mM sodium nitrate and glucose equivalent to 90 mM
sodium nitrate (a hyperosmolal medium). The liposome size was measured by PCS 1 hour, 24
hours and 48 hours after dilution, to see if the osmotic stress affected their size. The results
from the PCS measurements are given in table 6.2.1.











60

Table 6.2.1: The liposomes mean diameters during osmotic stress experiments (hyperosmolal dilution)
with glucose. Valid parallels used for calculations of mean vesicle sizes and standard deviations are given
in table 9.1-9.4 in appendix 3
Experiment description Mean vesicle size ± SD (from PCS measurements)
Intensity weighting Volume weighting Number weighting
Liposome dispersion prepared
with 10 mM sodium nitrate
119.7 nm ± 0.73 nm 120.4 nm ± 0.91 nm 86.8 nm ± 1.77 nm
Liposome dispersion 1 hour
after dilution with 10 mM
sodium nitrate and glucose
equivalent to 90 mM sodium
nitrate
110. 8 nm ± 0.93 nm 93.5 nm ± 2.02 nm 76.4 nm ± 3.11 nm
Liposome dispersion 24 hours
after dilution with 10 mM
sodium nitrate and glucose
equivalent to 90 mM sodium
nitrate
113.9 nm ± 0.44 nm 96.3 nm ± 2.36 nm 78.8 nm ± 4.32 nm
Liposome dispersion 48 hours
after dilution with 10 mM
sodium nitrate and glucose
equivalent to 90 mM sodium
nitrate
113.8 nm ± 0.35 nm 95.7 nm ± 0.96 nm 77.7 nm ± 1.60 nm


Obviously the mean sizes after dilution with hyperosmotic medium changed. The mean
diameter has reduced by almost 6 nm with intensity weighting, almost 27 nm with volume
weighting and over 10 nm with number weighting. Intensity weighting reflects the relative
intensity of scattered light vs. diameter for a sample run. Volume weighting reflects the
relative particle diameter vs. diameter; the value of the volume-weighted particle size
distribution assumes that the particles are spheres of uniform density. Volume weighting and
mass weighting are equal terms. Number weighting displays the relative number of particles
in a sample run vs. diameter, and they are also calculated assuming that the particles are
spheres.


61

The value of the mean diameter can vary significantly with the choice of weighting,
depending on the width of the gaussian-like distribution (Windows based software, Dynamic
light scattering theory User Manual 1997). One big particle will hence affect the mean size
distribution in volume weighting more than number weighting. This may explain why the
measured size difference is so deviating when looking at the different weightings.

But, irrespective of weighting a clear change of size can be seen when the liposomes were
diluted with the hyperosmolal solution. As we could expect it does look like the liposomes
have shrunk. When the osmotic pressure inside the liposomes is smaller than the osmotic
pressure outside, water will diffuse out of the liposomes, in order to equal the concentration
inside and outside the liposomes. The biggest mean size difference can be seen 1 hour after
dilution. The mean size does not change much from 1 hour to 24 hours, there is a small
difference and it could well be that the glucose starts to penetrate the membrane and hence
reverse the shrinking to some extent.

It was reported that glucose is slowly membrane permeable, half-life (t
1/2
)

= 1 hour, at 45°C.
According to (Osmotic properties of large unilamellar vesicles prepared by extrusion Mui,
Cullis 1993) glucose will penetrate the liposome membrane. . (Osmotic properties of large
unilamellar vesicles prepared by extrusion Mui, Cullis 1993) used another liposome
membrane, their membrane contained cholesterol which will make the membrane stiffer and
less permeable. Since Lipoid E-80 forms a less stiff membrane one could assume that glucose
will penetrate the membrane even faster. Since we did not know if that could occur in our
experiment it was decided to perform a similar experiment with sucrose instead of glucose. If
glucose to some extent penetrates the membrane the osmotic stress would not be as large as it
was supposed to be. Sucrose is a bigger molecule (almost twice as big as glucose) and should
thus penetrate the liposome membrane to an even lower extent than glucose. The molecular
weight (Mw) of glucose is 180.16 g/mol while the sucrose has an Mw of 342.30 g/mol. Since
we were not sure how fast glucose was able to penetrate through our liposome membrane it
was decided to also perform the next experiment with sucrose.




62

6.2.2 Hypertonic osmotic stress experiment with 10 mM NaNO
3
and sucrose equivalent
to 90 mM NaNO
3
as dilution medium
Liposomes again were prepared according to the hand-shaken method as described before,
with 10 mM sodium nitrate as dispersion medium. The size was measured by PCS right after
preparation, and the liposome dispersion was diluted 1:10 with 10 mM sodium nitrate and
sucrose equivalent to 90 mM sodium nitrate (a hyperosmolal medium). It is the same
experiment as the one described in section 6.2.1, the only difference is that glucose is replaced
by sucrose. The liposome size was measured by PCS 1 hour, 24 hours and 48 hours after
dilution. This was done to see if a size difference from the original undiluted sample could be
detected, and how fast it happened. Results from the PCS measurements are given in table
6.2.2.



















63

Table 6.2.2: Mean diameters during osmotic stress experiment (hyperosmolal dilution) with sucrose. Valid
parallels used for calculations of mean vesicle sizes ± SD are given in table 9.5-9.8 in appendix 3
Experiment description Mean vesicle size ± SD (from PCS measurements)
Intensity weighting Volume weighting Number weighting
Liposome dispersion
prepared with 10 mM
sodium nitrate
118.3 nm ± 0.61 nm 118.6 nm ± 0.67 nm 90.5 nm ± 3.34 nm
Liposome dispersion 1
hour after dilution with
10 mM sodium nitrate
and sucrose equivalent to
90 mM sodium nitrate
114.3 nm ± 0.29 nm 93.7 nm ± 1.71 nm 73.8 nm ± 2.73 nm
Liposome dispersion 24
hours after dilution with
10 mM sodium nitrate
and sucrose equivalent to
90 mM sodium nitrate
116.5 nm ± 0.46 nm 97.0 nm ± 3.04 nm 77.8 nm ± 4.97 nm
Liposome dispersion 48
hours after dilution with
10 mM sodium nitrate
and sucrose equivalent to
90 mM sodium nitrate
116.6 nm ± 0.29 nm 95.1 nm ± 2.10 nm 74.4 nm ± 3.54 nm


Again a difference in the sizes before and after dilution with the sucrose medium is seen. The
size difference in this experiment is almost 17 nm (number weighting) and almost 25 nm
(volume weighting). If we compare these results with the results when glucose was used, a
somewhat bigger size difference can be seen here.



64

6.2.3 Hypotonic osmotic stress experiment with 10 mM NaNO
3
as dilution medium
After it could be seen that is was possible to discover a size difference when liposomes were
diluted with a hyperosmolal solution, the liposome dispersion was prepared with 10 mM
sodium nitrate and sucrose equivalent to 90 mM sodium nitrate. The exact viscosity and
refractive index of the sucrose medium was already determined, so these values were used
when the size was measured by PCS. The liposomes were then diluted with 10 mM sodium
nitrate (hypoosmolal medium). And the sizes were measured by PCS after one hour, 24 hours
and 48 hours. The results from the PCS measurements are given in table 6.3.2.

Table 6.2.3: Liposome sizes during osmotic stress experiment (hypoosmolal dilution) with sucrose. Valid
parallels used for calculations of mean vesicle sizes ± SD are given in table 9.9-9.12 in appendix 3
Experiment description Mean vesicle size ± SD (from PCS measurements)
Intensity weighting Volume weighting Number weighting
Liposome dispersion prepared
with 10 mM NaNO
3
and
sucrose equivalent to 90 mM
NaNO
3

121.6 nm ± 0.36 nm 103.8 nm ± 2.20 nm 85.4 nm ± 3.86 nm
Liposome dispersion 1 hour
after dilution with 10 mM
NaNO
3

118.4 nm ± 0.36 nm 118.5 nm ± 0.40 nm 96.1 nm ± 4.49 nm
Liposome dispersion 24 hours
after dilution with 10 mM
NaNO
3

118.3 nm ± 0.26 nm 118.4 nm ± 0.25 nm 94.1 nm ± 2.34 nm
Liposome dispersion 48 hours
after dilution with 10 mM
NaNO
3

118.4 nm ± 0.35 nm 118.5 nm ± 0.37 nm 95.1 nm ± 2.28 nm

From the table above we can see that the mean size of the liposomes increases after they are
diluted in a hypoosmolal medium at least when looking at volume weighting and number
weighting. The intensity weighting shows only little change and it looks like the liposomes
have shrunk because the mean diameter is smaller after the dilution.


65

But the change in diameter is only 3.2 nm and it is not considered as a change due to the
osmotic stress the liposomes have been exposed to. When we look at the size changes with
volume weighting we can see a much bigger effect, the mean diameter increases with almost
15 nm. As one could predict, it seems that the liposomes have swollen when they were diluted
with the hypoosmolal medium. The concentration is bigger inside, than outside of the
liposome and as a consequence water will diffuse from the surrounding liquid into the
liposome.

6.3 Influence of osmotic stress on liposome size, measured by AF4
The liposomes used for these following experiments were prepared in the same manner as
those described in section 6.2. Again was chosen to adjust different osmotic pressures by
adding sucrose. After the previous experiments with both 10 mM sodium nitrate and glucose
solution and 10 mM sodium nitrate and sucrose solution as dilution agent it was decided to
only use the sucrose solution in the following experiments, because glucose might penetrate
the membrane as discussed earlier is section 6.2.1. Increasing the sodium nitrate concentration
is at the same time influencing the ionic strength which however could influence retention
time to an unknown degree (A4F/MALS-Analysis of Liposomes Influence of Key Factors on
Fractionation Behavior and Evaluation of MALS Fit Routines Ausbacher 2007).

Two different liposome batches were prepared, one with 10 mM sodium nitrate and one with
10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate as dispersion medium.
Both of the two batches were then diluted 1:10 with either the same medium as the one used
for preparing the liposome or the other medium giving four different experiment, as shown in
table 6.3.1. Every sample was analyzed by AF4 one hour after dilution. We had already
learned, from the PCS measurements, that the change of particle size by osmotic pressure
occurs within the first hour. The mobile phase in the AF4 experiments was always the same
medium as the one used as dilution medium.





66


Table 6.3.1: The four different liposome preparations used for analysis by the flow field-flow fractionation
Liposome sample Dilution medium and mobile phase
100 nm liposomes prepared in 10 mM sodium nitrate 10 mM sodium nitrate
100 nm liposomes prepared in 10 mm sodium nitrate 10 mM sodium nitrate and sucrose
equivalent to 90 mM sodium nitrate
100 nm liposomes prepared in 10 mM sodium nitrate
and sucrose equivalent to 90 mM sodium nitrate
10 mM sodium nitrate
100 nm liposomes prepared in 10 mM sodium nitrate
and sucrose equivalent to 90 mM sodium nitrate
10 mM sodium nitrate and sucrose
equivalent to 90 mM sodium nitrate


It was decided to compare the liposome samples prepared in the same medium, because they
were from the same batch and hence we knew that they had the same size before dilution and
that they generally had the same physical properties. We could not know if the liposomes
made in 10 mM sodium nitrate and the liposomes made in 10 mM sodium nitrate and sucrose
equivalent to 90 mM sodium nitrate had the same size after preparation. The extrusion of the
two different batches was performed in the same manner, but it is unknown how parameters
like different viscosity of the dispersion medium were influencing the size distribution and the
properties of the liposomes.

If we look at table 6.2.2, we can see the mean size distribution of the liposomes prepared in 10
mM sodium nitrate and we can compare them to table 6.2.3 where we can see that the mean
size distribution of the liposomes prepared in 10 mM sodium nitrate and sucrose equivalent to
90 mM sodium nitrate. The liposomes prepared in 10 mM sodium nitrate have an intensity
weighted mean diameter of 118.3 nm, a volume weighted mean diameter of 118.6 nm and a
number weighted mean diameter of 90.5 nm. The liposomes prepared in 10 mM sodium
nitrate and sucrose equivalent to 90 mM sodium nitrate have an intensity weighted mean
diameter of 121.6 nm, a volume weighted mean diameter of 103.8 nm and a number weighted
mean diameter of 85.4 nm. We can see that the mean diameter of the liposomes is not the
equal when the liposomes are prepared in the two different media.


67

And it is unknown if the liposomes prepared in different media would react differently when
they are diluted in a hypo-/hyperosmolal solution.

6.3.1 Hyperosmotic osmotic stress experiment with 10 mM NaNO
3
and sucrose
equivalent to 90 mM NaNO
3
as dilution medium
First we looked at the two samples prepared in 10 mM sodium nitrate, diluted in either 10
mM sodium nitrate or 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate.
(The first two experiments listed in table 6.3.1.)

Each of the samples was run in five parallels and both; the UV/VIS-absorbance and the
Rayleigh ratio were recorded over time. UV/VIS-detection is common in flow field-flow
fractionation. The first plot, shown in figure 6.3.1 presents the absorbance vs. time.


Figure 6.3.1: UV/VIS absorbance vs. time for the two liposomes samples prepared in 10 mM sodium
nitrate, diluted in either 10 mM sodium nitrate or 10 mM sodium nitrate and sucrose equivalent to 90 mM
sodium nitrate (five parallels each)
time (min)
15.0 20.0 25.0 30.0 35.0 40.0
r
e
l
a
t
i
v
e

s
c
a
l
e
0.0
0.2
0.4
0.6
0.8
1.0
10 mM sodium nitrate and sucrose
equivalent of 90 mM sodium nitrate as
dilution medium and mobile phase
10 mM sodium nitrate
as dilution medium
and mobile phase
U
V
/
V
I
S

a
b
s
o
r
b
a
n
c
e



68

The Rayleigh ratio is a quantity used to characterize the scattered intensity as a function of
scattering angle and is defined in section 5.5.5. Figure 6.3.2 show a plot of Rayleigh ratio vs.
time.


Figure 6.3.2: Plot of Rayleigh ratio vs. time for the two liposome samples prepared in 10 mM sodium
nitrate, diluted in either 10 mM sodium nitrate or 10 mM sodium nitrate and sucrose equivalent to 90 mM
sodium nitrate (five parallels each)

As we can see are the two plots very similar. The parallels for the same sample are very close
to each other, whereas the two samples behave markedly different: The curves of the
liposome dispersion diluted in 10 mM sodium nitrate and sucrose equivalent to 90 mM
sodium nitrate appear shifted left in both plots. This indicates that the liposomes prepared in
10 mM sodium nitrate solution and diluted in 10 mM sodium nitrate and sucrose equivalent to
90 mM sodium nitrate (hypertonic) solution elutes before the liposomes prepared and diluted
in 10 mM sodium nitrate solution. According to the AF4 theory; smaller particles will elute
earlier from the channel as described in section 5.5.5.
time (min)
15.0 20.0 25.0 30.0 35.0 40.0
r
e
l
a
t
i
v
e

s
c
a
l
e
0.0
0.2
0.4
0.6
0.8
1.0
10 mM sodium nitrate
as dilution medium
and mobile phase
10 mM sodium nitrate and sucrose
equivalent of 90 mM sodium nitrate as
dilution medium and mobile phase


69

When we are comparing these two samples we can see that the sample diluted in the
hypertonic medium elutes before the sample diluted in the isotonic medium. We can assume
thus that the liposomes have become smaller when they were blended with the hypertonic
medium since they eluted earlier.

Next, from the angular dependence of the light scattering signal the geometric radii were
calculated for all slices over the whole elution. Figure 6.3.3 shows the resulting size,
geometric radius vs. elution type. Since we used a sphere model for the sample is the radius
presented as geometric radius (Wyatt Technology Corporation User Manual 2007). Figure
6.3.3 shows the same samples as figure 6.3.1 and 6.3.2. We can see in figure 6.3.3 that both
the samples show very similar sizes over time. The geometric radii reach from approximately
25 nm to 70 nm for both, the liposome sample diluted in 10 mM sodium nitrate and the
liposome samples diluted in 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium
nitrate.

The lower part of figure 6.3.3 shows the same plot as the one above, but here the area between
20 and 25 min is enlarged to show the difference between the two samples clearer. Here it is
possible to see that there is a slight shift to smaller sizes for a given elution time for the
liposomes in hypertonic medium.



70




Figure 6.3.3: Geometric radius of the two liposome samples prepared in 10 mM sodium nitrate, diluted in
either 10 mM sodium nitrate or 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate
(five parallels of each). The lower picture shows an enlarged section of the upper plot

time (min)
15.0 20.0 25.0 30.0 35.0
g
e
o
m
e
t
r
i
c

r
a
d
i
u
s

(
n
m
)
0.0
20.0
40.0
60.0
80.0
time (min)
20.0 21.0 22.0 23.0 24.0 25.0
g
e
o
m
e
t
r
i
c

r
a
d
i
u
s

(
n
m
)
36.0
38.0
40.0
42.0
44.0
10 mM sodium nitrate as dilution
medium and mobile phase
10 mM sodium nitrate and sucrose
equivalent of 90 mM sodium nitrate as
dilution medium and mobile phase


71


Figure 6.3.4: Parametric plot of the two liposome samples prepared in 10 mM sodium nitrate, diluted in
either 10 mM sodium nitrate or 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate

Finally a plot was constructed where the measured UV absorbance was plotted over the
calculated geometric radii, a parametric plot. A parametric plot is a plot that generates a new
data set for two different types of x-y data that share the same x-axis (Wyatt Technology
Corporation User Manual 2007). For example, we can make a plot of absorbance vs.
geometric radius, as shown in figure 6.3.4. In figure 6.3.4 only one parallel of each sample is
shown. This is done because it gives a more perspicuous plot. The five parallels of each
sample are shown in appendix 3; figure 9.5 and figure 9.6. The size distributions obtained this
way are clearly different from each other. The size distribution of the liposomes in the
hypertonic medium appears shifted towards smaller sizes as compared to liposomes in
isotonic medium. This confirms that the shift in elution time seen before in figure 6.3.1 and
figure 6.3.2 is due to a difference in the liposome size after the sample has been diluted in a
hypertonic medium.

0
0,001
0,002
0,003
0,004
0,005
0,006
0,007
0,008
0,009
0,01
20 30 40 50 60 70 80
A
b
s
o
r
b
a
n
c
e

(
A
U
)
Geometric radius (nm)
10 mM sodium nitrate as
dilution medium and
mobile phase
10 mM sodium nitrate and sucrose
equivalent of 90 mM sodium nitrate as
dilution medium and mobile phase


72

In the parametric plot we can see that all the liposomes from the lower end of the distribution
(approximately 30 nm radius) to the upper end of the distribution (approximately 75 nm
radius) have been equally affected by the osmotic stress they have been exposed to. The
parallel shift of the whole curve to the left in figure 6.3.4 indicates that all the different sized
liposomes are the sample is almost equally affected by the osmotic stress.

Table 6.3.2: Mean radii and standard deviation of the liposome samples prepared in 10 mM sodium
nitrate, diluted in either 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate or 10 mM
sodium nitrate, from table 9.26 in appendix 3
Sample R
n
± SD R
w
± SD R
z
± SD
Liposomes prepared in 10
mM sodium nitrate, diluted
in 10 mM sodium nitrate
and sucrose equivalent to
90 mM sodium nitrate
46.8 nm ± 0.49 nm 51.8 nm ± 0.26 nm 55.5 nm ± 0.23 nm
Liposomes prepared in 10
mM sodium nitrate, diluted
in 10 mM sodium nitrate
53.1 nm ± 0.35 nm 57.0 nm ± 0.08 nm 59.8 nm ± 0.32 nm

Table 6.3.2 shows the mean radius of two liposome samples prepared in 10 mM sodium
nitrate as calculated from AF4 analysis. The liposomes diluted in the hypertonic medium are
in mean smaller than the liposomes diluted in the isotonic medium. This corresponds with the
result as we have seen from the plots in figure 6.3.1- figure 6.3.4. Here we can see that the
number-average mean square radius decrease after hypertonic dilution by 6.3 nm, the weight-
average mean square radius decrease by 5.2 nm and the z-average mean square radius
decrease by 4.3 nm.

Table 6.2.2 shows the mean diameter, measured by PCS, of the same two liposome samples
as the liposome samples in table 6.3.2.



73

The same hyperosmolal experiment are performed in both cases, the only difference is that the
liposome sizes are measured with two different techniques. As we can see from the results,
the same principles are constituted; the liposome shrink and the size decrease when it is
diluted in a hypertonic medium.

6.3.2 Hypotonic osmotic stress experiment with 10 mM NaNO
3
as dilution medium
Secondly we looked at the liposome samples that were prepared in 10 mM sodium nitrate and
sucrose equivalent to 90 mM sodium nitrate, diluted in either the same medium or in 10 mM
sodium nitrate (hypotonic solution), the last two experiments in table 6.3.1. Here we would
expect that the liposomes diluted in the hypoosmolal medium would expand and become
larger. In figure 6.3.1 and 6.3.2 we saw that the liposomes shrunk when they were diluted in
the hyperosmolal medium. This is the exact opposite experiment where the liposomes have a
greater concentration inside than outside. If they became larger as we predicted they would
elute last.

Figure 6.3.5: UV/VIS plot of absorbance vs. time for the two liposomes samples prepared in 10 mM
sodium nitrate and sucrose equivalent to 90 mM sodium nitrate, diluted in either 10 mM sodium nitrate
or 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate (five parallels each)

time (min)
15.0 20.0 25.0 30.0 35.0 40.0
r
e
l
a
t
i
v
e

s
c
a
l
e
0.0
0.2
0.4
0.6
0.8
1.0
10 mM sodium nitrate and sucrose
equivalent of 90 mM sodium nitrate as
dilution medium and mobile phase
10 mM sodium nitrate
as dilution medium
and mobile phase
U
V
/
V
I
S

a
b
s
o
r
b
a
n
c
e



74

Both the plots in figure 6.3.5 and figure 6.3.6 shows the same liposome samples which were
prepared in 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate. In figure
6.3.5 the absorbance is plotted against elution time. It looks like both the liposome samples
elute roughly at the same time.

Figure 6.3.6: Plot of Rayleigh ratio vs. time for the two liposome samples prepared in 10 mM sodium
nitrate and sucrose equivalent to 90 mM sodium nitrate, diluted in either 10 mM sodium nitrate or 10 mM
sodium nitrate and sucrose equivalent to 90 mM sodium nitrate (five parallels each)

In figure 6.3.6 Rayleigh ratio is plotted against elution time. As in figure 6.3.5 both of the
liposome samples elute at almost the same time. We can see it is no major difference in the
two graphs.

time (min)
15.0 20.0 25.0 30.0 35.0 40.0
r
e
l
a
t
i
v
e

s
c
a
l
e
0.0
0.2
0.4
0.6
0.8
1.0
10 mM sodium nitrate
as dilution medium
and mobile phase
10 mM sodium nitrate and sucrose
equivalent of 90 mM sodium nitrate as
dilution medium and mobile phase


75




Figure 6.3.7: Geometric radius of the two liposome samples prepared in 10 mM sodium nitrate and
sucrose equivalent to 90 mM sodium nitrate, diluted in either 10 mM sodium nitrate or 10 mM sodium
nitrate and sucrose equivalent of 90 mM sodium nitrate (five parallels of each). The lower picture shows
an enlarged section of the upper plot
time (min)
15.0 20.0 25.0 30.0 35.0
g
e
o
m
e
t
r
i
c

r
a
d
i
u
s

(
n
m
)
0.0
20.0
40.0
60.0
80.0
time (min)
20.0 21.0 22.0 23.0 24.0 25.0
g
e
o
m
e
t
r
i
c

r
a
d
i
u
s

(
n
m
)
36.0
38.0
40.0
42.0
44.0
10 mM sodium nitrate as dilution
medium and mobile phase
10 mM sodium nitrate
and sucrose equivalent
of 90 mM sodium nitrate
as dilution medium and
mobile phase


76

Figure 6.3.7 shows a plot of sizes over time of the samples prepared in 10 mM sodium nitrate
and sucrose equivalent to 90 mM sodium nitrate. It shows the same two samples as figure
6.3.5 and 6.3.6. When we compare these two samples it is possible to see a clear difference in
sizes. The liposomes that were diluted in a hypotonic medium appear to have become larger.
Furthermore the size-over-time lines are not fully parallel. When the concentration is higher
inside of the liposomes than outside of the liposome, water will diffuse through the membrane
into the core of the liposomes to equal the osmotic pressure. As a consequence the liposome
will swell and become bigger. The geometric radius of the liposomes diluted in the hypotonic
medium ranges from approximately 20 nm to 65 nm. The liposomes diluted in the isotonic
medium ranges from approximately 20 nm to a little less than 60 nm. The lower part of figure
6.3.7 shows the same plot as the upper plot, but here the area between 20 and 25 min is
enlarged to show the difference between the two samples clearer.


Figure 6.3.8: Parametric plot of the two liposome samples prepared in 10 mM sodium nitrate and sucrose
equivalent to 90 mM sodium nitrate, diluted in either 10 mM sodium nitrate or 10 mM sodium nitrate and
sucrose equivalent to 90 mM sodium nitrate

0
0,001
0,002
0,003
0,004
0,005
0,006
0,007
0,008
0,009
0,01
20 30 40 50 60 70 80
A
b
s
o
r
b
a
n
c
e

(
A
U
)
Geometric radius (nm)
10 mM sodium nitrate
and sucrose equivalent
of 90 mM sodium nitrate
as dilution medium and
mobile phase
10 mM sodium nitrate as dilution
medium and mobile phase


77

As we have seen before there is a difference in the liposome size after the sample has been
diluted in a hypotonic medium. In the parametric plot in figure 6.3.8 we can see that not all
the liposomes have been affected to the same extend when they are diluted in the hypotonic
medium. Only one parallel of each sample is shown in figure 6.3.8, but all the five parallels
are shown in appendix 3; figure 9.7 and figure 9.8. The outer curve in figure 6.3.8 shows the
liposomes that have been diluted in the hypoosmolal medium; inner curve is the liposomes
diluted in the isotonic medium. On the right side of the curve we can see that the liposomes
that have been diluted in the hypoosmolal solution have become bigger. This is the same
information we received from the geometric radius plots. And it is agreeing with the theory.

There will always be some liposomes that are smaller then the rest when they are prepared by
extrusion, these liposomes is shown on the left side of the curve. On that side it is not possible
to see a size difference between the two liposome samples. This information is also shown in
figure 6.3.8, where the liposomes diluted in the hypotonic solution has almost the same
minimum diameter as the liposomes diluted in the isotonic solution.
We could see from the plot that both of the liposome samples had geometric radius reaching
from approximately 20 nm. The difference between the two samples was in the maximum
size; there we could clearly see that the liposomes diluted in the hypotonic medium were
bigger than the liposomes diluted in the isotonic medium. The two curves are crossing when
the geometric radius is approximately 25 nm.

A conceivable explanation can be that the smaller liposomes have a different shape than the
bigger liposomes and hence can change less in size. The liposomes that are smaller then 100
nm will pass through the filter without problem, and they will maintain their original
conformation. But the liposomes that are larger than 100 nm need to either break and form
smaller spheres or change their conformation when they are squeezing through the pores on
the filter. In figure 6.3.9 and figure 6.3.10 the phenomenon is illustrated which we think might
explain the size difference we saw in the parametric plot.

 
 
78 
 

Figure 6.3.9: A liposome bigger than 100 nm squeezing itself through a 100 nm filter by altering its
conformation, then it is diluted in either hypo- or hypertonic medium
 
 
79 
 

Figure 6.3.10: A liposome smaller than 100 nm passing through a 100 nm filter, then it is diluted in either
hypo- or hypertonic medium












80

Table 6.3.3: Mean radius of the liposome samples prepared in 10 mM sodium nitrate and sucrose
equivalent to 90 mM sodium nitrate, diluted in either 10 mM sodium nitrate and sucrose equivalent to 90
mM sodium nitrate or 10 mM sodium nitrate, from table 9.26 in appendix 3
Sample R
n
R
w
R
z
Liposomes prepared in 10 mM
sodium nitrate and sucrose
equivalent of 90 mM sodium
nitrate, diluted in 10 mM sodium
nitrate and sucrose equivalent to
90 mM sodium nitrate
44.1 nm ± 0.15 nm 48.1 nm ± 0.47 nm 50.9 nm ± 0.70 nm
Liposomes prepared in 10 mM
sodium nitrate and sucrose
equivalent to 90 mM sodium
nitrate, diluted in 10 mM sodium
nitrate
45.8 nm ± 0.09 nm 50.1 nm ± 0.10 nm 53.1 nm ± 0.18 nm

Table 6.3.3 shows the mean radius of the liposomes prepared in 10 mM sodium nitrate and
sucrose equivalent to 90 mM sodium nitrate. The liposomes diluted in 10 mM sodium nitrate
(hypotonic medium) are slightly bigger than the liposomes diluted in the isotonic medium.
The number-average mean square radius increase with 1.7 nm, the weight-average mean
square radius increase with 2 nm and the z-average mean square diameter increase with 2.2
nm.

Table 6.2.3 shows the mean diameter of the same liposomes as the liposomes shown in table
6.3.3; the only difference is that the sizes are measured with different techniques. First was
the liposomes measured by PCS and then by AF4. Both methods show the same principle, the
liposome swell and the size increase after the liposome has been diluted in the hypoosmolal
medium.




81

7 CONCLUSION
In conclusion, this project has demonstrated that a change in osmotic pressure, with constant
ionic strength, affects both elution time and calculated size of liposomes. But, osmotic stress
was found to affect liposomes of different sizes in a different manner; liposomes that were
smaller than the pore size of the filter used for extrusion were found to shrink in hyperosmotic
medium but stay quite constant in size in hypo-osmotic medium. In contrast, liposomes that
were larger than the pore size of the filter were found to shrink in hyperosmotic medium and
swell in hypo-osmotic medium. A hypothesis is presented to explain this behavior.
It was found that the retention behavior and calculated geometric radius of liposomes obtained
by flow field-flow fractionation in combination with multi-angle light scattering is affected by
the osmotic pressure of the medium used for diluting the liposomes and/or running the AF4.

Ideally, thus aqueous media with the same osmotic pressure as the liposome dispersion should
be used as dilution medium/mobile phase when liposomes are fractionated by AF4. This gives
some limitations with the AF4 method. Sugar should be added to adjust the osmotic pressure
of the mobile phase to avoid osmotic stress artifacts and to avoid changes in ionic strength,
which could alter the elution behavior.
















82




































83

8 REFERENCES

Asymmetric Flow Field-Flow Fractionation. 2008 [updated 2008]; Available from:
http://www.postnova.com/content/english/theory/theory_fff.htm.

Ausbacher DA. A4F/MALS-Analysis of Liposomes Influence of Key Factors on
Fractionation Behavior and Evaluation of MALS Fit Routines. 2007.

Bangham AD. Osmotic properties and water permeability of phospholipid liquid crystals.
Chemistry and physics of lipids. 1967;1:225.

Bangham AD, Standish MM, Watkins JC. Diffusion of univalent ions across the lamellae of
swollen phospholipids. Journal of molecular biology. 1965;13(1):238-52.

Brandl M. Liposomes as drug carriers: a technological approach. Biotechnol Annu Rev.
2001;7:59-85.

Gregoriadis G. Liposome technology. 3rd ed. New York: Informa Healthcare; 2006.

How Asymmetric Field Flow Fractionation (AFFF) Theory Works. 2008 [updated 2008];
Available from: http://www.wyatt.com/theory/fieldflowfractionation/.

Ingebrigtsen L. Size analysis of submicron particles and liposomes by size exclusion
chromatography and photon correlation spectroscopy. Tromsø: University of Tromsø,
Institute of Pharmacy; 2001.

Ingebrigtsen L, Brandl M. Determination of the size distribution of liposomes by SEC
fractionation, and PCS analysis and enzymatic assay of lipid content. AAPS PharmSciTech.
2002;3(2).

Lasic D. Liposomes: from physics to applications. Amsterdam: Elsevier; 1993.



84

Lide DR. CRC Handbook of Chemistry and Physics. Taylor and Francis Group, LLC; 2008.
Available from: http://www.hbcpnetbase.com/.

Massing U, Fuxius S. Liposomal formulations of anticancer drugs: selectivity and
effectiveness. Drug resistance updates. 2000;3(3):171-7.

Moon MH, Park I, Kim Y. Size characterization of liposomes by flow field-flow fractionation
and photon correlation spectroscopy Effect of ionic strength and pH of carrier solutions.
Journal of chromatography. 1998;813(1):91.

Mui BL, Cullis PR, Evans EA, Madden TD. Osmotic properties of large unilamellar vesicles
prepared by extrusion. Biophysical Journal. 1993;64(2):443-53.

Mui BL, Hope MJ. Liposome technology. In: Gregoriadis G, editor. 3rd ed. New York:
Informa Healthcare; 2006. p. 324.

Nickels JCH. Changes in Liposome Morphology Induced by Actin Polymerization in
Submicrometer Liposomes. Langmuir. 2003;19(25):10581.

Refractive index. 2008 [updated 2008]; Available from:
http://en.wikipedia.org/wiki/Refractive_index.

Roessner D, Kulicke W-M. On-line coupling of flow field-flow fractionation and multi-angle
laser light scattering. Journal of chromatography. 1994;687(2):249.

Technical Summary - An Introduction to Lipid Nanoparticles. 2008 [updated 2008];
Available from: http://www.encapsula.com/company.html.

Tonicity. 2008 [updated 2008]; Available from:
http://en.wikipedia.org/wiki/Tonicity#Isotonicity.

Torchilin VP, Weissig V, editors. Liposomes: a practical approach. 2nd ed. Oxford: Oxford
University Press; 2003.



85

User Manual ASTRA V User´s Guide. Wyatt Technology Corporation. 2007. 300 p.

User Manual Nicomp Model 380. Windows based software, Dynamic light scattering theory.
1997.

Van Zanten JH, Monbouquette HG. Characterization of vesicles by classical light scattering.
Journal of colloid and interface science. 1991;146(2):330.

Viscosity. 2008 [updated 2008]; Available from: http://en.wikipedia.org/wiki/Viscosity.

Washington C. Particle size analysis in pharmaceutics and other industries: theory and
practice. New York: Ellis Horwood; 1992.

Wyatt Technology Introduction to Light Scattering, Light Scattering University. 2006.

















86

























87

9 APPENDICES
Appendix 1
Calculation of osmolality in solutions

Example:
E-value NaNO
3
= 0.67
E-value glucose = 0.16
If we should make a solution with 10 mM NaNO
3
and glucose equivalent to 90 mM NaNO
3


Amount NaNO
3
in a 100 mM solution: 84.99 g/mol × 0.1 mol/l = 8.499 g/l
Amount after correction with the E-value (NaNO
3
): 8.499 g × 0.67 = 5.694 g
Amount NaNO
3
in 10 mM solution: 84.99 g/mol × 0.01 mol/l = 0.8499 g/l
Amount after correction with the E-value (NaNO
3
): 0.8499 g × 0.67 = 0.569 g
The amount needed from glucose: 5.694 g – 0.569 g = 5.125 g
Amount glucose needed (when corrected with the E-value of glucose): 0.16 × X = 5.125 g
32.031 g









88

Appendix 2
Calculation of dynamic viscosity of solutions
Kinematic viscosity (u):
˯ = K × (ˮ -0)

ϑ = Hagenbach correction for average flow time (s) (from manual)
K = constant (mm
2
/s
2
) (from manual)
t = time (seconds)

Example:
u = 0.003 mm
2
/s
2
× (331.39 s – 0.61 s)
u = 0.9923 mm
2
/s


Dynamic viscosity (η):
n = ˯ × µ

ρ = density

Example:
η = 0.9923 mm
2
/s × 1.0092 g/cm
3

η = 1.0014 mPa × s



89

Appendix 3
PCS results
All the results marked red in appendix 3 are excluded from the summary because they did not
fulfill the requirements that chi squared should be < 3, and baseline adjust should be ≤ 0.05 %.

All results are from Gaussian distributions.
Table 9.1: PCS measurements of 100 nm liposomes, prepared with 10 mM NaNO
3

Intensity
weighting
Volume
weighting
Number
weighting
Channel
width
Chi
squared
Baseline
adjust
119.0 nm 119.6 nm 84.6 nm 13.0 µSec 0.50 0.00 %
119.2 nm 119.6 nm 90.7 nm 13.0 µSec 0.35 0.08 %
119.2 nm 119.7 nm 87.2 nm 13.0 µSec 0.33 0.00 %
120.1 nm 120.8 nm 86.8 nm 13.0 µSec 0.33 0.00 %
120.6 nm 121.6 nm 85.8 nm 13.0 µSec 0.47 0.00 %
121.0 nm 122.0 nm 85.4 nm 13.0 µSec 0.99 0.00 %
119.2 nm 119.8 nm 86.0 nm 13.0 µSec 0.54 0.00 %
119.1 nm 119.5 nm 88.2 nm 13.0 µSec 0.22 0.03 %
119.3 nm 120.0 nm 86.5 nm 13.0 µSec 0.32 0.00 %
119.9 nm 120.4 nm 90.6 nm 13.0 µSec 1.92 0.00 %











90

Table 9.2: PCS measurements of 100 nm liposomes prepared with 10 mM NaNO
3,
1 hour after dilution
with 10 mM NaNO
3
and glucose equivalent of 90 mM NaNO
3

Intensity
weighting
Volume
weighting,
Number
weighting
Channel
width
Chi squared Baseline
adjust
109.7 nm 91.8 nm 74.3 nm 13.0 µSec 2.80 0.00 %
110.0 nm 92.8 nm 75.8 nm 13.0 µSec 2.51 0.00 %
109.7 nm 91.4 nm 73.7 nm 13.0 µSec 0.44 0.00 %
110.4 nm 92.7 nm 75.3 nm 13.0 µSec 1.71 0.00 %
110.6 nm 94.4 nm 78.3 nm 13.0 µSec 0.59 0.00 %
111.0 nm 94.6 nm 78.3 nm 13.0 µSec 1.15 0.00 %
111.0 nm 91.5 nm 72.7 nm 13.0 µSec 0.68 0.00 %
111.8 nm 93.4 nm 75.4 nm 13.0 µSec 0.94 0.00 %
111.7 nm 94.0 nm 76.6 nm 13.0 µSec 1.23 0.00 %
112.4 nm 98.2 nm 83.6 nm 13.0 µSec 2.52 0.00 %


Table 9.3: PCS measurements of 100 nm liposomes, prepared with 10 mM NaNO
3
, 24 hours after dilution
with 10 mM NaNO
3
and glucose equivalent of 90 mM NaNO
3
Intensity
weighting
Volume
weighting
Number
weighting
Channel
width
Chi squared Baseline
adjust
113.4 nm 100.5 nm 87.1 nm 15.0 µSec 1.03 0.05 %
113.3 nm 93.8 nm 74.8 nm 15.0 µSec 2.80 0.00 %
114.6 nm 99.0 nm 83.0 nm 15.0 µSec 6.32 0.00 %
113.9 nm 97.5 nm 81.0 nm 15.0 µSec 1.23 0.00 %
113.5 nm 94.1 nm 75.3 nm 15.0 µSec 0.38 0.00 %
114.5 nm 97.6 nm 80.4 nm 15.0 µSec 1.82 0.00 %
113.9 nm 98.7 nm 83.1 nm 15.0 µSec 0.62 0.02 %
114.3 nm 95.0 nm 76.1 nm 15.0 µSec 0.53 0.00 %
113.8 nm 94.7 nm 76.0 nm 15.0 µSec 0.44 0.01 %
114.4 nm 94.7 nm 75.5 nm 15.0 µSec 1.10 0.00 %




91

Table 9.4: PCS measurements of 100 nm liposomes, prepared with 10 mM NaNO
3
, 48 hours after dilution
with 10 mM NaNO
3
and glucose equivalent of 90 mM NaNO
3

Intensity
weighting
Volume
weighting
Number
weighting
Channel
width
Chi squared Baseline
adjust
113.2 nm 94.8 nm 76.8 nm 14.0 µSec 0.37 0.00 %
113.6 nm 95.1 nm 76.9 nm 14.0 µSec 1.08 0.00 %
113.9 nm 96.4 nm 78.9 nm 14.0 µSec 0.87 0.00 %
113.7 nm 94.8 nm 76.2 nm 14.0 µSec 0.91 0.00 %
113.6 nm 96.8 nm 79.8 nm 14.0 µSec 0.37 0.00 %
113.8 nm 96.2 nm 78.7 nm 14.0 µSec 1.35 0.00 %
113.9 nm 94.2 nm 75.0 nm 14.0 µSec 0.92 0.00 %
114.3 nm 98.0 nm 81.4 nm 14.0 µSec 3.32 0.00%
114.3 nm 95.9 nm 77.7 nm 14.0 µSec 1.76 0.00 %
114.3 nm 96.8 nm 79.3 nm 14.0 µSec 0.96 0.00 %


Table 9.5: PCS measurements of 100 nm liposomes, prepared with 10 mM NaNO
3

Intensity
weighting
Volume
weighting
Number
weighting
Channel
width
Chi squared Baseline
adjust
118.9 nm 119.4 nm 86.4 nm 14.0 µSec 0.69 0.00 %
118.3 nm 118.5 nm 93.9 nm 14.0 µSec 2.20 0.00 %
118.1 nm 118.3 nm 91.2 nm 14.0 µSec 0.32 0.08 %
118.4 nm 118.7 nm 90.8 nm 14.0 µSec 0.90 0.00 %
118.7 nm 119.1 nm 89.7 nm 14.0 µSec 1.06 0.00 %
117.6 nm 117.8 nm 88.6 nm 14.0 µSec 0.28 0.03 %
117.2 nm 117.4 nm 86.4 nm 14.0 µSec 0.36 0.03 %
118.7 nm 118.9 nm 92.6 nm 14.0 µSec 1.15 0.00 %
119.2 nm 119.3 nm 98.2 nm 14.0 µSec 3.98 0.00 %
118.8 nm 118.9 nm 95.4 nm 14.0 µSec 1.06 0.00 %




92

Table 9.6: PCS measurements of 100 nm liposomes, prepared with 10 mM NaNO
3
, 1 hour after dilution
with 10 mM NaNO
3
and sucrose equivalent of 90 mM NaNO
3

Intensity
weighting
Volume
weighting
Number
weighting
Channel
width
Chi squared Baseline
adjust
114.6 nm 95.5 nm 76.6 nm 14.0 µSec 1.69 0.00 %
113.7 nm 90.2 nm 68.3 nm 14.0 µSec 0.59 0.00 %
114.3 nm 94.2 nm 74.6 nm 14.0 µSec 1.04 0.00 %
114.4 nm 95.5 nm 76.9 nm 14.0 µSec 1.27 0.00 %
114.4 nm 94.2 nm 74.5 nm 14.0 µSec 1.26 0.00 %
115.0 nm 97.6 nm 80.0 nm 14.0 µSec 4.32 0.00 %
114.6 nm 94.5 nm 74.9 nm 14.0 µSec 1.63 0.00 %
114.2 nm 93.6 nm 73.6 nm 14.0 µSec 0.48 0.00 %
114.0 nm 93.7 nm 74.0 nm 14.0 µSec 0.69 0.00 %
114.1 nm 91.8 nm 70.7 nm 14.0 µSec 0.38 0.00 %


Table 9.7: PCS measurements of 100 nm liposomes, prepared with 10 mM NaNO
3
, 24 hours after dilution
with 10 mM NaNO
3
and sucrose equivalent of 90 mM NaNO
3
Intensity
weighting
Volume
weighting
Number
weighting
Channel
width
Chi squared Baseline
adjust
116.1 nm 93.7 nm 72.3 nm 15.0 µSec 0.35 0.00 %
116.6 nm 97.3 nm 78.2 nm 15.0 µSec 2.63 0.00 %
115.6 nm 91.9 nm 69.7 nm 15.0 µSec 0.30 0.01 %
117.0 nm 100.9 nm 84.2 nm 15.0 µSec 2.34 0.00 %
116.4 nm 95.6 nm 75.4 nm 15.0 µSec 0.54 0.00 %
117.2 nm 101.9 nm 85.9 nm 15.0 µSec 2.05 0.00 %
116.3 nm 96.4 nm 76.9 nm 15.0 µSec 1.13 0.00 %
116.7 nm 96.8 nm 77.1 nm 15.0 µSec 1.79 0.00 %
116.7 nm 99.1 nm 81.3 nm 15.0 µSec 1.59 0.00 %
116.2 nm 96.5 nm 77.2 nm 15.0 µSec 1.32 0.00 %



93

Table 9.8: PCS measurements of 100 nm liposomes, prepared with 10 mM NaNO
3
, 48 hours after dilution
with 10 mM NaNO
3
and sucrose equivalent of 90 mM NaNO
3
Intensity
weighting
Volume
weighting
Number
weighting
Channel
width
Chi squared Baseline
adjust
117.0 nm 98.2 nm 79.4 nm 14.0 µSec 1.46 0.00 %
116.7 nm 93.9 nm 72.0 nm 14.0 µSec 0.41 0.00 %
117.1 nm 99.8 nm 82.1 nm 14.0 µSec 4.91 0.00 %
117.2 nm 98.7 nm 80.1 nm 14.0 µSec 3.14 0.00 %
116.8 nm 96.1 nm 75.8 nm 14.0 µSec 1.95 0.00 %
117.6 nm 99.9 nm 82.0 nm 14,0 µSec 4.65 0.00 %
116.2 nm 93.1 nm 71.1 nm 14.0 µSec 0.30 0.00 %
116.4 nm 97.2 nm 78.1 nm 14.0 µSec 1.34 0.00 %
116.5 nm 92.7 nm 70.2 nm 14.0 µSec 0.33 0.00 %
116.3 nm 94.7 nm 73.9 nm 14.0 µSec 0.39 0.00 %


Table 9.9: PCS measurements of 100 nm liposomes, prepared with 10 mM NaNO
3
and sucrose equivalent
of 90 mM NaNO
3

Intensity
weighting
Volume
weighting
Number
weighting
Channel
width
Chi squared Baseline
adjust
121.5 nm 104.6 nm 87.0 nm 15.0 µSec 0.51 0.03 %
120.9 nm 101.6 nm 82.0 nm 15.0 µSec 0.23 0.00 %
122.0 nm 107.9 nm 92.7 nm 15.0 µSec 2.28 0.00 %
121.3 nm 103.2 nm 84.5 nm 15.0 µSec 0.46 0.01 %
121.5 nm 104. 7 nm 87.1 nm 15.0 µSec 1.90 0.00 %
121.3 nm 101.6 nm 81.7 nm 15.0 µSec 0.44 0.05 %
121.6 nm 102.2 nm 82.4 nm 15.0 µSec 1.30 0.00 %
121.7 nm 102.1 nm 82.1 nm 15.0 µSec 0.57 0.00 %
121.9 nm 102.9 nm 83.5 nm 15.0 µSec 0.93 0.00 %
122.1 nm 106.8 nm 90.5 nm 15.0 µSec 1.65 0.00 %



94

Table 9.10: PCS measurements of 100 nm liposomes, prepared with 10 mM NaNO
3
and sucrose equivalent
of 90 mM NaNO
3
, 1 hour after dilution with 10 mM NaNO
3

Intensity
weighting
Volume
weighting
Number
weighting
Channel
width
Chi squared Baseline
adjust
118.7 nm 118.9 nm 95.8 nm 13.0 µSec 3.34 0.00 %
118.2 nm 118.3 nm 95.7 nm 13.0 µSec 0.51 0.00 %
118.1 nm 118.3 nm 90.3 nm 13.0 µSec 0.20 0.00 %
118.4 nm 118.5 nm 98.1 nm 13.0 µSec 0.54 0.00 %
117.8 nm 117.8 nm 94.5 nm 13.0 µSec 0.30 0.05 %
118.7 nm 118.9 nm 92.3 nm 13.0 µSec 0.60 0.00 %
119.0 nm 119.1 nm 97.6 nm 13.0 µSec 1.74 0.00 %
118.7 nm 118.7 nm 98.1 nm 13.0 µSec 0.54 0.00 %
118.6 nm 118.7 nm 95.0 nm 13.0 µSec 0.68 0.00 %
118.5 nm 118.7 nm 92.8 nm 13.0 µSec 0.36 0.00 %


Table 9.11 PCS measurements of 100 nm liposomes, prepared with 10 mM NaNO
3
and sucrose equivalent
of 90 mM NaNO
3
, 24 hours after dilution with 10 mM NaNO
3

Intensity
weighting
Volume
weighting
Number
weighting
Channel
width
Chi squared Baseline
adjust
118.1 nm 118.2 nm 92.1 nm 12.0 µSec 0.37 0.00 %
118.0 nm 118.2 nm 91.9 nm 12.0 µSec 0.72 0.00 %
118.3 nm 118.4 nm 96.2 nm 12.0 µSec 0.75 0.00 %
118.8 nm 118.9 nm 97.1 nm 12.0 µSec 2.97 0.00 %
118.0 nm 118.1 nm 92.9 nm 12.0 µSec 0.57 0.00 %
118.3 nm 118.5 nm 93.9 nm 12.0 µSec 1.14 0.00 %
118.5 nm 118.6 nm 96.9 nm 12.0 µSec 2.17 0.00 %
118.1 nm 118.2 nm 97.7 nm 12.0 µSec 0.44 0.07 %
118.3 nm 118.5 nm 90.8 nm 12.0 µSec 0.35 0.00 %
118.5 nm 118.6 nm 95.2 nm 12.0 µSec 0.34 0.00 %




95

Table 9.12: PCS measurements of 100 nm liposomes, prepared with 10 mM NaNO
3
and sucrose equivalent
of 90 mM NaNO
3
, 48 hours after dilution with 10 mM NaNO
3
Intensity
weighting
Volume
weighting
Number
weighting
Channel
width
Chi squared Baseline
adjust
117.9 nm 118.0 nm 94.1 nm 13.0 µSec 1.08 0.00 %
118.4 nm 118.4 nm 98.0 nm 13.0 µSec 1.26 0.00 %
117.6 nm 117.7 nm 92.3 nm 13.0 µSec 0.25 0.07 %
118.5 nm 118.5 nm 96.7 nm 13.0 µSec 2.43 0.00 %
118.0 nm 118.0 nm 94.4 nm 13.0 µSec 0.45 0.05 %
118.9 nm 119.1 nm 95.5 nm 13.0 µSec 2.77 0.00 %
118.0 nm 118.2 nm 90.2 nm 13.0 µSec 0.26 0.00 %
118.7 nm 118.9 nm 95.9 nm 13.0 µSec 1.73 0.00 %
118.2 nm 118.4 nm 94.3 nm 13.0 µSec 0.32 0.00 %
118.6 nm 118.6 nm 97.0 nm 13.0 µSec 0.60 0.00 %


Table 9.13: Latex bead standards, 100 nm, diluted with 10 mM NaNO
3.
PCS mean sizes calculated on the
basis of viscosity and refractive index values of water
Intensity
weighting
Volume
weighting
Number
weighting
Channel
width
Chi squared Baseline
adjust
105.4 nm 100.4 nm 94.9 nm 12.0 µSec 0.44 0.01 %
105.7 nm 104.6 nm 103.3 nm 12.0 µSec 0.41 0.00 %
105.6 nm 104.5 nm 103.2 nm 12.0 µSec 1.06 0.00 %
105.7 nm 104.4 nm 102.8 nm 12.0 µSec 0.26 0.02 %
105. 6 nm 104.6 nm 103.3 nm 12.0 µSec 0.49 0.05 %
105.3 nm 105.3 nm 105.3 nm 12.0 µSec 0.38 0.07 %
105.6 nm 103.4 nm 100.8 nm 12.0 µSec 0.81 0.00 %
105.9 nm 104.3 nm 102.4 nm 12.0 µSec 0.58 0.00 %
105.3 nm 102.3 nm 98.9 nm 12.0 µSec 0.36 0.01 %
105.3 nm 104.7 nm 104.0 nm 12.0 µSec 0.39 0.00 %




96

Table 9.14: Latex bead standards, 100 nm, diluted with 10 mM NaNO
3
and sucrose equivalent to 90 mM
NaNO
3
.

PCS mean sizes calculated on the basis of the viscosity and refractive index values of water
Intensity
weighting
Volume
weighting
Number
weighting
Channel
width
Chi squared Baseline
adjust
117.7 nm 116.9 nm 115.9 nm 13.0 µSec 0.40 0.02 %
117.5 nm 115.9 nm 114.0 nm 13.0 µSec 0.26 0.00 %
117.8 nm 116.3 nm 114.5 nm 13.0 µSec 0.74 0.00 %
117.6 nm 117.6 nm 117.6 nm 13.0 µSec 0.56 0.00 %
117.7 nm 117.2 nm 116.5 nm 13.0 µSec 0.31 0.02 %
117.6 nm 117.4 nm 117.1 nm 13.0 µSec 0.42 0.05 %
118.7 nm 116.4 nm 113.6 nm 13.0 µSec 2.01 0.00 %
117.9 nm 117.5 nm 116.8 nm 13.0 µSec 0.87 0.00 %
117.8 nm 115.9 nm 113.6 nm 13.0 µSec 0.42 0.07 %
117.9 nm 115.1 nm 111.7 nm 13.0 µSec 0.31 0.02 %


Table 9.15: Latex bead standards, 100 nm, diluted with 10 mM NaNO
3
and glucose equivalent of 90 mM
NaNO
3
. PCS mean sizes calculated on the basis of viscosity and refractive index values of water
Intensity
weighting
Volume
weighting
Number
weighting
Channel
width
Chi squared Baseline
adjust
112.9 nm 111.3 nm 109.5 nm 13.0 µSec 0.57 0.04 %
113.0 nm 110.9 nm 108.4 nm 13.0 µSec 0.50 0.00 %
113.4 nm 109.5 nm 105.0 nm 13.0 µSec 0.40 0.13 %
113.7 nm 112.1 nm 110.1 nm 13.0 µSec 0.34 0.04 %
113.8 nm 111.0 nm 107.8 nm 13.0 µSec 0.80 0.00 %
113.3 nm 111.8 nm 110.0 nm 13.0 µSec 0.39 0.00 %
113.3 nm 111.1 nm 108.5 nm 13.0 µSec 0.67 0.00 %
114.1 nm 106.2 nm 97.4 nm 13.0 µSec 0.33 0.03 %
113.5 nm 111.5 nm 109.1 nm 13.0 µSec 0.38 0.00 %
112.8 nm 109.7 nm 106.1 nm 13.0 µSec 0.30 0.10 %



97

Table 9.16: Latex bead standards, 100 nm, diluted with 10 mM NaNO
3
and sucrose equivalent of 90 mM
NaNO
3
.

PCS mean sizes calculated on the basis of measured viscosity and refractive index values


Intensity
weighting
Volume
weighting
Number
weighting
Channel
width
Chi squared Baseline
adjust
104.5 nm 104.2 nm 103.8 nm 13.0 µSec 0.41 0.02 %
104.3 nm 102.7 nm 100.7 nm 13.0 µSec 0.26 0.00 %
104.6 nm 103.3 nm 101.8 nm 13.0 µSec 0.73 0.00 %
104.4 nm 104.4 nm 104.4 nm 13.0 µSec 0.56 0.00 %
104.6 nm 104.6 nm 104.6 nm 13.0 µSec 0.31 0.03 %
104.4 nm 104.1 nm 103.8 nm 13.0 µSec 0.41 0.05 %
105.4 nm 103.4 nm 101.1 nm 13.0 µSec 2.01 0.00 %
104.7 nm 103.7 nm 102.4 nm 13.0 µSec 0.78 0.00 %
104.6 nm 102.0 nm 99.1 nm 13.0 µSec 0.43 0.07 %
104.7 nm 102.1 nm 99.2 nm 13.0 µSec 0.31 0.02 %


Table 9.17: Latex bead standards, 100 nm, diluted with 10 mM NaNO
3
and glucose equivalent of 90 mM
NaNO
3
. PCS mean sizes calculated on the basis of measured viscosity and refractive index values


Intensity
weighting
Volume
weighting
Number
weighting
Channel
width
Chi squared Baseline
adjust
105.7 nm 104.1 nm 102.2 nm 13.0 µSec 0.55 0.04 %
105.8 nm 104.1 nm 102.2 nm 13.0 µSec 0.50 0.00 %
106.1 nm 102.4 nm 98.2 nm 13.0 µSec 0.40 0.13 %
106.5 nm 105.2 nm 103.8 nm 13.0 µSec 0.34 0.04 %
106.5 nm 103.8 nm 100.6 nm 13.0 µSec 0.80 0.00 %
106.1 nm 104.5 nm 102.7 nm 13.0 µSec 0.40 0.00 %
106.0 nm 103.6 nm 100.9 nm 13.0 µSec 0.68 0.00 %
106.9 nm 99.5 nm 91.5 nm 13.0 µSec 0.33 0.03 %
106.2 nm 104.3 nm 102.0 nm 13.0 µSec 0.37 0.00 %
105.6 nm 103.2 nm 100.5 nm 13.0 µSec 0.28 0.10 %




98

Table 9.18: Liposomes 100 nm, freeze-thawed (3 cycles) before extrusion. Frozen in -80 °C freezer, thawed
on a 50°C water bath
Intensity
weighting
Volume
weighting
Number
weighting
Channel
width
Chi squared Baseline
adjust
116.5 nm 116.4 nm 90.7 nm 14.0 µSec 0.31 0.05 %
116.8 nm 116.8 nm 92.1 nm 14.0 µSec 4.06 0.00 %
116.6 nm 116.5 nm 94.5 nm 14.0 µSec 2.67 0.00 %
116.2 nm 116.1 nm 89.9 nm 14.0 µSec 0.53 0.00 %
116.0 nm 115.9 nm 88.7 nm 14.0 µSec 0.49 0.00 %
116.3 nm 116.2 nm 90.9 nm 14.0 µSec 0.31 0.05 %
116.7 nm 116.8 nm 86.4 nm 14.0 µSec 0.36 0.03 %
117.1 nm 117.0 nm 92.6 nm 14.0 µSec 0.55 0.00 %
116.8 nm 116.9 nm 85.5 nm 14.0 µSec 0.62 0.00 %
116.9 nm 116.9 nm 90.1 nm 14.0 µSec 0.55 0.00 %


Table 9.19: PCS measurements of 100 nm liposomes, freeze-thawed (3 cycles) during extrusion, after 400
nm filter, before 200 nm filter. Frozen in -80 °C freezer, thawed on a 50°C water bath
Intensity
weighting
Volume
weighting
Number
weighting
Channel
width
Chi squared Baseline
adjust
114.4 nm 114.0 nm 92.9 nm 12.0 µSec 0.90 0.00 %
114.8 nm 114.4 nm 95.0 nm 12.0 µSec 1.81 0.00 %
114.8 nm 114.5 nm 92.6 nm 12.0 µSec 1.35 0.00 %
114.3 nm 113.8 nm 89.3 nm 12.0 µSec 0.30 0.00 %
114.2 nm 113.8 nm 88.9 nm 12.0 µSec 1.22 0.00 %
114.4 nm 114.0 nm 89.4 nm 12.0 µSec 0.61 0.00 %
114.0 nm 113.6 nm 92.6 nm 12.0 µSec 0.27 0.07 %
114.9 nm 114.5 nm 94.7 nm 12.0 µSec 2.42 0.00 %
114.4 nm 114.0 nm 91.6 nm 12.0 µSec 0.94 0.00 %
114.4 nm 114.1 nm 87.4 nm 12.0 µSec 0.32 0.03 %




99

Table 9.20: PCS measurements of 100 nm liposomes, freeze-thawed (3 cycles) before extrusion. Frozen in
liquid nitrogen (LN
2
), thawed on a 50°C water bath
Intensity
weighting
Volume
weighting
Number
weighting
Channel
width
Chi squared Baseline
adjust
112.5 nm 111.8 nm 89.0 nm 13.0 µSec 1.26 0.00 %
112.2 nm 111.5 nm 84.4 nm 13.0 µSec 2.72 0.00 %
112.2 nm 111.5 nm 85.1 nm 13.0 µSec 0.73 0.00 %
112.3 nm 111.6 nm 83.8 nm 13.0 µSec 0.36 0.04 %
112.8 nm 112.2 nm 87.1 nm 13.0 µSec 2.72 0.00 %
112.3 nm 111.6 nm 85.0 nm 13.0 µSec 0.38 0.00 %
112.2 nm 111.5 nm 83.1 nm 13.0 µSec 0.30 0.01 %
112.4 nm 111.8 nm 89.3 nm 13.0 µSec 4.61 0.00 %
111.7 nm 110.9 nm 86.9 nm 13.0 µSec 0.58 0.01 %
113.1 nm 112.6 nm 87.7 nm 13.0 µSec 2.22 0.00 %


Table 9.21: Distribution width from PCS measurements of 100 nm liposomes, freeze-thawed (3 cycles)
before extrusion. Frozen in -80 °C freezer, thawed on a 50°C water bath
Intensity weighting Volume weighting Number weighting
31.6 nm 31.6 nm 24.6 nm
31.0 nm 30.9 nm 24.4 nm
29.2 nm 29.1 nm 23.6 nm
31.8 nm 31.8 nm 24.6 nm
32.5 nm 32.5 nm 24.8 nm
34.4 nm 34.4 nm 25.5 nm
30.8 nm 30.8 nm 24.4 nm
35.2 nm 35.2 nm 25.7 nm
32.3 nm 32.3 nm 24.9 nm
31.3 nm 31.3 nm 24.4 nm




100

Table 9.22: Distribution width from PCS measurements of 100 nm liposomes, freeze-thawed (3 cycles)
during extrusion, after 400 nm filter, before 200 nm filter. Frozen in -80 °C freezer, thawed on a 50°C
water bath
Intensity weighting Volume weighting Number weighting
28.4 nm 28.3 nm 23.0 nm
27.2 nm 27.1 nm 22.5 nm
28.9 nm 28.8 nm 23.3 nm
30.6 nm 30.5 nm 23.9 nm
30.8 nm 30.7 nm 24.0 nm
30.7 nm 30.6 nm 24.0 nm
28.2 nm 28.0 nm 22.9 nm
27.6 nm 27.5 nm 22.7 nm
29.3 nm 29.2 nm 23.4 nm
32.0 nm 31.9 nm 24.5 nm


Table 9.23: Distribution width from PCS measurement of 100 nm liposomes, freeze-thawed (3 cycles)
before extrusion. Frozen in liquid nitrogen (LN
2
), thawed on a 50°C water bath
Intensity weighting Volume weighting Number weighting
29.4 nm 29.2 nm 23.2 nm
32.1 nm 31.9 nm 24.1 nm
31.6 nm 31.4 nm 24.0 nm
32.6 nm 32.4 nm 24.3 nm
30.9 nm 30.7 nm 23.9 nm
31.8 nm 31.6 nm 24.1 nm
32.9 nm 32.7 nm 24.3 nm
29.1 nm 28.9 nm 23.1 nm
30.0 nm 29.8 nm 23.4 nm
30.8 nm 30.6 nm 23.8 nm




101

Table 9.24: A summary of all the results presented in the tables above. Mean diameters and standard
deviations for all the valid results. The results that had a too high value for chi squared or baseline adjust
are not included.
Sample Intensity weighting Volume weighting Number weighting
From table 9.1,
liposomes prepared
with 10 mM NaNO
3

119.7 nm ± 0.73 nm 120.4 nm ± 0.91 nm 86.8 nm ± 1.77 nm
From table 9.2,
liposomes 1 hour after
dilution with 10 mM
NaNO
3
and glucose
110. 8 nm ± 0.93 nm 93.5 nm ± 2.02 nm 76.4 nm ± 3.11 nm
From table 9.3,
liposomes 24 hours
after dilution with 10
mM NaNO
3
and
glucose
113.9 nm ± 0.44 nm 96.3 nm ± 2.36 nm 78.8 nm ± 4.32 nm
From table 9.4,
liposomes 48 hours
after dilution with 10
mM NaNO
3
and
glucose
113.8 nm ± 0.35 nm 95.7 nm ± 0.96 nm 77.7 nm ± 1.60 nm
From table 9.5,
liposomes prepared
with 10 mM NaNO
3

118.3 nm ± 0.61 nm 118.6 nm ± 0.67 nm 90.5 nm ±3.34 nm
From table 9.6,
liposomes 1 hour after
dilution with 10 mM
NaNO
3
and sucrose
114.3 nm ± 0.29 93.7 nm ± 1.71 73.8 nm ± 2.73
From table 9.7,
liposomes 24 hours
after dilution with 10
mM NaNO
3
and
sucrose
116.5 nm ± 0.46 nm 97.0 nm ± 3.04 nm 77.8 nm ± 4.97 nm


102


From table 9.8,
liposomes 48 hours
after dilution with 10
mM NaNO
3
and
sucrose
116.6 nm ± 0.29 nm 95.1 nm ± 2.10 nm 74.4 nm ± 3.54 nm
From table 9.9,
liposomes prepared
with 10 mM NaNO
3

and sucrose equivalent
to 90 mM NaNO
3

121.6 nm ± 0.36 nm 103.8 nm ± 2.20 nm 85.4 nm ± 3.86 nm
From table 9.10,
liposomes 1 hour after
dilution 10 mM NaNO
3

118.4 nm ± 0.36 nm 118.5 nm ± 0.40 nm 96.1 nm ± 4.49 nm
From table 9.11,
liposomes 24 hours
after dilution with 10
mM NaNO
3

118.3 nm ± 0.26 nm 118.4 nm ± 0.25 nm 94.1 nm ± 2.34 nm
From table 9.12,
liposomes 48 hours
after dilution with 10
mM NaNO
3

118.4 nm ± 0.35 nm 118.5 nm ± 0.37 nm 95.1 nm ± 2.28 nm
From table 9.13, Duke
size standards diluted
with 10 mM NaNO
3

105.6 nm ± 0.21 nm 103.6 nm ± 1.52 nm 101.3 nm ± 1.97
nm
From table 9.14, Duke
size standards diluted
with 10 mM NaNO
3

and sucrose
117.9 nm ± 0.39 nm 116.6 nm ± 0.86 nm 115.1 nm ± 1.97
nm
From table 9.15, Duke
size standards diluted
with 10 mM NaNO
3

and glucose
113.5 nm ± 0.41 nm 110.7 nm ± 1.88 nm 107.6 nm ± 4.20
nm


103


From table 9.16, Duke
size standards diluted
with 10 mM NaNO
3

and sucrose
104.6 nm ± 0.34 nm 103.8 nm ± 0.64 nm 102.8 nm ± 1.52 nm
From table 9.17, Duke
size standards diluted
with 10 mM NaNO
3

and glucose
106.2 nm ± 0.40 nm 103.6 nm ± 1.74 nm 100.7 nm ± 3.86 nm
From table 9.18,
liposomes frozen in
-80°C freezer and
thawed before
extrusion
116.6 nm ± 0.35 nm 116.5 nm ± 0.40 nm 89.9 nm ± 2.81 nm
From table 9.19,
liposomes frozen in
-80°C freezer and
thawed during
extrusion
114.5 nm ± 0.25 nm 114.1 nm ± 0.28 nm 91.3 nm ± 2.70 nm
From table 10.20,
liposomes frozen in
liquid nitrogen and
thawed before
extrusion
112.4 nm ± 0.38 nm 111.7 nm ± 0.45 nm 86.1 nm ± 2.17 nm








104

Table 9.25: Mean distribution width and standard deviations for all the valid results. The results that had
a too high value for chi squared or baseline adjust are not included.
Sample Intensity weighting Volume weighting Number weighting
From table 9.21,
liposomes frozen in
-80 °C freezer and
thawed before
extrusion
32.1 nm ± 1.81 nm 32.1 nm ± 1.83 nm 24.7 nm ± 0.62 nm
From table 9.22,
liposomes frozen in
-80°C freezer and
thawed during
extrusion
29.5 nm ± 1.62 nm 29.4 nm ± 1.62 nm 23.5 nm ± 0.67 nm
From table 9.23,
liposomes frozen in
liquid nitrogen and
thawed before
extrusion
31.3 nm ± 1.17 nm 31.1 nm ± 1.17 nm 23.9 nm ± 0.38 nm














105

Table 9.26: The different liposome sizes (radius) gained from the AF4
Sample R
n
(nm) R
w
(nm) R
z
(nm)
1. Liposomes prepared in 10 mM sodium
nitrate and diluted in 10 mM sodium nitrate
and sucrose equivalent of 90 mM sodium
nitrate
47.6 (1 %) 52.2 (0.8 %) 55.8 (0.8 %)
46.7 (0.6 % ) 51.7 (0.5 %) 55.3 (0.5 %)
46.6 (0.7 %) 51.7 (0.6 %) 55.5 (0.6 %)
46.7 (0.7 %) 51.7 (0.6 %) 55.5 (0.5 %)
46.3 (0.8 %) 51.5 (0.6 %) 55.2 (0.6 %)
2. Liposomes prepared in 10 mM sodium
nitrate and diluted in 10 mM sodium nitrate
53.6 (0.6 %) 57.1 (0.5 %) 59.3 (0.5%)
53.0 (0.5 %) 56.9 (0.5 %) 59.7 (0.5 %)
52.9 (0.6 %) 57.0 (0.5 %) 59.9 (0.5 %)
52.9 (0.7%) 57.1 (0.7 %) 60.1 (0.7 %)
52.9 (0.6 %) 57.0 (0.6 %) 60.0 (0.5 %)
3. Liposomes prepared in 10 mM sodium
nitrate and sucrose equivalent of 90 mM
sodium nitrate, diluted in 10 mM sodium
nitrate and sucrose equivalent of 90 mM
sodium nitrate
44.1 (1 %) 48.8 (0.7 %) 52.0 (0.6 %)
44.3 (2 %) 48.3 (1 %) 51.2 (1 %)
44.2 (2 %) 48.0 (2 %) 50.7 (2%)
43.9 (3%) 47.6 (3 %) 50.3 (2 %)
44.1 (4 %) 47.8 (3 %) 50.4 (3 %)
4. Liposomes prepared in 10 mM sodium
nitrate and sucrose equivalent of 90 mM
sodium nitrate, diluted in 10 mM sodium
nitrate
45.7 (0.6 %) 50.0 (0.5 %) 52.9 (0.5 %)
45.7 (0.6 %) 50.0 (0.5 %) 53.0 (0.5 %)
45.9 (0.8 %) 50.2 (0.7 %) 53.3 (0.7 %)
45.7 (0.7 %) 50.1 (0.6 %) 53.2 (0.6 %)
45.8 (0.7 %) 50.2 (0.6 %) 53.3 (0.6 %)

Table 9.27: Mean radii and standard deviations for all samples in table 9.26
Sample R
n
(nm) R
w
(nm) R
z
(nm)
1.from table 9.26 46.8 nm ± 0.49 nm 51.8 nm ± 0.26 nm 55.5 nm ± 0.23 nm
2. from table 9.26 53.1 nm ± 0.35 nm 57.0 nm ± 0.08 nm 59.8 nm ± 0.32 nm
3. from table 9.26 44.1 nm ± 0.15 nm 48.1 nm ± 0.47 nm 50.9 nm ± 0.70 nm
4. from table 9.26 45.8 nm ± 0.09 nm 50.1 nm ± 0.10 nm 53.1 nm ± 0.18 nm



106

Figure 9.1: The five parallels of liposomes produced in 10 mM sodium nitrate, diluted 1:10 in 10 mM
sodium nitrate and sucrose equivalent of 90 mM sodium nitrate. 10 mM sodium nitrate and sucrose
equivalent of 90 mM sodium nitrate as mobile phase

Figure 9.2: The five parallels of liposomes produced in 10 mM sodium nitrate and sucrose equivalent of 90
mM sodium nitrate, diluted 1:10 in 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium
nitrate. 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate as mobile phase
2.1
gfedcb
2.2
gfedcb
2.3
gfedcb
2.4
gfedcb
2.5
gfedcb
time (min)
10.0 15.0 20.0 25.0 30.0 35.0 40.0
g
e
o
m
e
t
r
i
c

r
a
d
i
u
s

(
n
m
)
0.0
50.0
100.0
150.0
4.1
gfedcb
4.2
gfedcb
4.3
gfedcb
4.4
gfedcb
4.5
gfedcb
time (min)
10.0 15.0 20.0 25.0 30.0 35.0 40.0
g
e
o
m
e
t
r
i
c

r
a
d
i
u
s

(
n
m
)
0.0
50.0
100.0
150.0


107


Figure 9.3: The five parallels of liposomes produced in 10 mM sodium nitrate, diluted 1:10 in 10 mM
sodium nitrate. 10 mM sodium nitrate as mobile phase

Figure 9.4: The five parallels of liposomes produced in 10 mM sodium nitrate and sucrose equivalent of 90
mM sodium nitrate, diluted 1:10 in 10 mM sodium nitrate. 10 mM sodium nitrate as mobile phase
5.1
gfedcb
5.2
gfedcb
5.3
gfedcb
5.4
gfedcb
5.5
gfedcb
time (min)
10.0 15.0 20.0 25.0 30.0 35.0 40.0
g
e
o
m
e
t
r
i
c

r
a
d
i
u
s

(
n
m
)
0.0
50.0
100.0
150.0
7.1
gfedcb
7.2
gfedcb
7.3
gfedcb
7.4
gfedcb
7.5
gfedcb
time (min)
10.0 15.0 20.0 25.0 30.0 35.0 40.0
g
e
o
m
e
t
r
i
c

r
a
d
i
u
s

(
n
m
)
0.0
50.0
100.0
150.0


108


Figure 9.5: Parametric plot of the liposome sample prepared in 10 mM sodium nitrate solution, 10 mM
sodium nitrate and sucrose equivalent of 90 mM sodium nitrate solution used as dilution medium and as
mobile phase (five parallels)


Figure 9.6: Parametric plot of the liposome sample prepared in 10 mM sodium nitrate and sucrose
equivalent of 90 mM sodium nitrate solution, 10 mM sodium nitrate and sucrose equivalent of 90 mM
sodium nitrate solution used as dilution medium and as mobile phase (five parallels)

0
0,002
0,004
0,006
0,008
0,01
0,012
20 30 40 50 60 70 80
A
b
s
o
r
b
a
n
c
e

(
A
U
)
Geometric radius (nm)
1 2
3 4
5
0
0,001
0,002
0,003
0,004
0,005
0,006
0,007
0,008
20 30 40 50 60 70
A
b
s
o
r
b
a
n
c
e

(
A
U
)
Geometric radius (nm)
1 2
3 4
5


109


Figure 9.7: Parametric plot of the liposome sample prepared in 10 mM sodium nitrate solution, 10 mM
sodium nitrate solution used as dilution medium and as mobile phase (five parallels)


Figure 9.8: The five parallels of the liposome sample prepared in 10 mM sodium nitrate and sucrose
equivalent of 90 mM sodium nitrate solution, 10 mM sodium nitrate


0
0,001
0,002
0,003
0,004
0,005
0,006
0,007
0,008
0,009
0,01
20 30 40 50 60 70 80
A
b
s
o
r
b
a
n
c
e

(
A
U
)
Geometric radius (nm)
1 2
3 4
5
0
0,001
0,002
0,003
0,004
0,005
0,006
0,007
0,008
0,009
0,01
20 30 40 50 60 70 80
A
b
s
o
r
b
a
n
c
e

(
A
U
)
Geometric radius (nm)
1 2
3 4
5


110

Appendix 4
The liposome samples which also were analyzed by AF4, but not were included in the thesis.

Table 9.28: Summary of the liposome samples which were analyzed by AF4, but not included in the thesis
Liposome dispersion medium Dilution medium Mobile phase
10 mM sodium nitrate 10 mM sodium nitrate 10 mM sodium nitrate and
sucrose equivalent to 90
mM sodium nitrate
10 mM sodium nitrate 10 mM sodium nitrate and
sucrose equivalent to 90
mM sodium nitrate
10 mM sodium nitrate
10 mM sodium nitrate 10 mM sodium nitrate 100 mM sodium nitrate
10 mM sodium nitrate 10 mM sodium nitrate and
sucrose equivalent to 90
mM sodium nitrate
100 mM sodium nitrate
10 mM sodium nitrate and
sucrose equivalent to 90 mM
sodium nitrate
10 mM sodium nitrate 10 mM sodium nitrate and
sucrose equivalent to 90
mM sodium nitrate
10 mM sodium nitrate and
sucrose equivalent to 90 mM
sodium nitrate
10 mM sodium nitrate 10 mM sodium nitrate
10 mM sodium nitrate and
sucrose equivalent to 90 mM
sodium nitrate
10 mM sodium nitrate 100 mM sodium nitrate
10 mM sodium nitrate and
sucrose equivalent to 90 mM
sodium nitrate
10 mM sodium nitrate and
sucrose equivalent to 90
mM sodium nitrate
100 mM sodium nitrate




111

Figure 9.9: The five parallels of liposomes produced in 10 mM sodium nitrate, diluted 1:10 in 10 mM
sodium nitrate. 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate as mobile phase


Figure 9.10: The five parallels of liposomes produced in 10 mM sodium nitrate and sucrose equivalent of
90 mM sodium nitrate, diluted 1:10 in 10 mM sodium nitrate. 10 mM sodium nitrate and sucrose
equivalent of 90 mM sodium nitrate as mobile phase
1.1
gfedcb
1.2
gfedcb
1.3
gfedcb
1.4
gfedcb
1.5
gfedcb
time (min)
10.0 15.0 20.0 25.0 30.0 35.0 40.0
g
e
o
m
e
t
r
i
c

r
a
d
i
u
s

(
n
m
)
0.0
50.0
100.0
150.0
3.1
gfedcb
3.2
gfedcb
3.3
gfedcb
3.4
gfedcb
3.5
gfedcb
time (min)
10.0 15.0 20.0 25.0 30.0 35.0 40.0
g
e
o
m
e
t
r
i
c

r
a
d
i
u
s

(
n
m
)
0.0
50.0
100.0
150.0


112


Figure 9.11: The five parallels of liposomes produced in 10 mM sodium nitrate, diluted 1:10 in 10 mM
sodium nitrate and sucrose equivalent of 90 mM sodium nitrate. 10 mM sodium nitrate as mobile phase

Figure 9.12: The five parallels of liposomes produced in 10 mM sodium nitrate and sucrose equivalent of
90 mM sodium nitrate, diluted 1:10 in 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium
nitrate. 10 mM sodium nitrate as mobile phase
6.1
gfedcb
6.2
gfedcb
6.3
gfedcb
6.4
gfedcb
6.5
gfedcb
time (min)
10.0 15.0 20.0 25.0 30.0 35.0 40.0
g
e
o
m
e
t
r
i
c

r
a
d
i
u
s

(
n
m
)
0.0
50.0
100.0
150.0
8.1
gfedcb
8.2
gfedcb
8.3
gfedcb
8.4
gfedcb
8.5
gfedcb
time (min)
10.0 15.0 20.0 25.0 30.0 35.0 40.0
g
e
o
m
e
t
r
i
c

r
a
d
i
u
s

(
n
m
)
0.0
50.0
100.0
150.0


113


Figure 9.13: The five parallels of liposomes produced in 10 mM sodium nitrate, diluted 1:10 in 10 mM
sodium nitrate. 100 mM sodium nitrate as mobile phase

Figure 9.14: The five parallels of liposomes produced in 10 mM sodium nitrate, diluted 1:10 in 10 mM
sodium nitrate and sucrose equivalent of 90 mM sodium nitrate. 100 mM sodium nitrate as mobile phase
9.1
gfedcb
9.2
gfedcb
9.3
gfedcb
9.4
gfedcb
9.5
gfedcb
time (min)
10.0 15.0 20.0 25.0 30.0 35.0 40.0
g
e
o
m
e
t
r
i
c

r
a
d
i
u
s

(
n
m
)
0.0
50.0
100.0
150.0
10.1
gfedcb
10.2
gfedcb
10.3
gfedcb
10.4
gfedcb
10.5
gfedcb
time (min)
10.0 15.0 20.0 25.0 30.0 35.0 40.0
g
e
o
m
e
t
r
i
c

r
a
d
i
u
s

(
n
m
)
0.0
50.0
100.0
150.0


114


Figure 9.15: The five parallels of liposomes produced in 10 mM sodium nitrate and sucrose equivalent of
90 mM sodium nitrate, diluted 1:10 in 10 mM sodium nitrate. 100 mM sodium nitrate as mobile phase

Figure 9.16: The five parallels of liposomes produced in 10 mM sodium nitrate and sucrose equivalent of
90 mM sodium nitrate, diluted 1:10 in 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium
nitrate. 10 mM sodium nitrate as mobile phase
11.1
gfedcb
11.2
gfedcb
11.3
gfedcb
11.4
gfedcb
11.5
gfedcb
time (min)
10.0 15.0 20.0 25.0 30.0 35.0 40.0
g
e
o
m
e
t
r
i
c

r
a
d
i
u
s

(
n
m
)
0.0
50.0
100.0
150.0
12.1
gfedcb
12.2
gfedcb
12.3
gfedcb
12.4
gfedcb
12.5
gfedcb
time (min)
10.0 15.0 20.0 25.0 30.0 35.0 40.0
g
e
o
m
e
t
r
i
c

r
a
d
i
u
s

(
n
m
)
0.0
50.0
100.0
150.0


115


Figure 9.17: All the four liposome preparations with 100 mM sodium nitrate as mobile phase. Only one
parallel of each sample shown



Figure 9.18: All the four liposome preparations with 10 mM sodium nitrate as mobile phase. Only one
parallel of each sample shown
E-80 liposomes produced with 10 mMsodiumnitrate, diluted 1:10 with 10 mMsodiumnitrate
gfedcb
E-80 liposomes produced with 10 mMsodiumnitrate, diluted 1:10 with 10 mMsodiumnitrate and sucrose equivalent of 90 mMsodiumnitrate
gfedcb
E-80 liposomes produced with 10 mMsodiumnitrate and sucrose equivalent of 90 mMsodiumnitrate, diluted 1:10 with 10 mMsodiumnitrate
gfedcb
E-80 liposomes produced with 10 mMsodiumnitrate and sucrose equivalent of 90 mMsodiumnitrate, diluted 1:10 with 10 mMsodiumnitrate and sucrose equivalent of 90 mMsodiumnitrate
gfedcb
time (min)
10.0 15.0 20.0 25.0 30.0 35.0 40.0
g
e
o
m
e
t
r
i
c

r
a
d
i
u
s

(
n
m
)
0.0
50.0
100.0
150.0
E-80 liposomes produced with 10 mMsodiumnitrate, diluted 1:10 with 10 mMsodiumnitrate
gfedcb
E-80 liposomes produced with 10 mMsodiumnitrate, diluted 1:10 with 10 mMsodiumnitrate and sucrose equivalent of 90 mMsodiumnitrate
gfedcb
E-80 liposomes produced with 10 mMsodiumnitrate and sucrose equivalent of 90 mMsodiumnitrate, diluted 1:10 with 10 mMsodiumnitrate
gfedcb
E-80 liposomes produced with 10 mMsodiumnitrate and sucrose equivalent of 90 mMsodiumnitrate, diluted 1:10 with 10 mMsodiumnitrate and sucrose equivalent of 90 mMsodiumnitrate
gfedcb
time (min)
10.0 15.0 20.0 25.0 30.0 35.0 40.0
g
e
o
m
e
t
r
i
c

r
a
d
i
u
s

(
n
m
)
0.0
50.0
100.0
150.0


116


Figure 9.19: All the four liposome preparations with 10 mM sodium nitrate and sucrose equivalent to 90
mM sodium nitrate as mobile phase. Only one parallel of each sample shown



Figure 9.20: All the liposome samples prepared in 10 mM sodium nitrate and diluted 1:10 with 10 mM
sodium nitrate. Only one parallel of each sample shown

E-80 liposomes produced with 10 mMsodiumnitrate, diluted 1:10 with 10 mMsodiumnitrate
gfedcb
E-80 liposomes produced with 10 mMsodiumnitrate, diluted 1:10 with 10 mMsodiumnitrate and sucrose equivalent of 90 mMsodiumnitrate
gfedcb
E-80 liposomes produced with 10 mMsodiumnitrate and sucrose equivalent of 90 mMsodiumnitrate, diluted 1:10 with 10 mMsodiumnitrate
gfedcb
E-80 liposomes produced with 10 mMsodiumnitrate and sucrose equivalent of 90 mMsodiumnitrate, diluted 1:10 with 10 mMsodiumnitrate and sucrose equivalent of 90 mMsodiumnitrate
gfedcb
time (min)
10.0 15.0 20.0 25.0 30.0 35.0 40.0
g
e
o
m
e
t
r
i
c

r
a
d
i
u
s

(
n
m
)
0.0
50.0
100.0
150.0
10 mMsodiumnitrate and sucrose equivalent of 90 mMsodiumnitrate as mobile phase
gfedcb
10 mMsodiumnitrate as mobile phase
gfedcb
100 mMsodiumnitrate as mobile phase
gfedcb
time (min)
10.0 15.0 20.0 25.0 30.0 35.0 40.0
g
e
o
m
e
t
r
i
c

r
a
d
i
u
s

(
n
m
)
0.0
50.0
100.0
150.0


117



Figure 9.21: All the liposome samples prepared in 10 mM sodium nitrate and diluted 1:10 with 10 mM
sodium nitrate and sucrose equivalent to 90 mM sodium nitrate. Only one parallel of each sample shown


Figure 9.22: All the liposome samples prepared in 10 mM sodium nitrate and sucrose equivalent to 90 mM
sodium nitrate, diluted 1:10 with 10 mM sodium nitrate. Only one parallel of each sample shown
10 mMsodiumnitrate and sucrose equivalent of 90 mMsodiumnitrate as mobile phase
gfedcb
10 mMsodiumnitrate as mobile phase
gfedcb
100 mMsodiumnitrate as mobile phase
gfedcb
time (min)
10.0 15.0 20.0 25.0 30.0 35.0 40.0
g
e
o
m
e
t
r
i
c

r
a
d
i
u
s

(
n
m
)
0.0
50.0
100.0
150.0
10 mMsodiumnitrate and sucrose equivalent of 90 mMsodiumnitrate as mobile phase
gfedcb
10 mMsodiumnitrate as mobile phase
gfedcb
100 mMsodiumnitrate as mobile phase
gfedcb
time (min)
10.0 15.0 20.0 25.0 30.0 35.0 40.0
g
e
o
m
e
t
r
i
c

r
a
d
i
u
s

(
n
m
)
0.0
50.0
100.0
150.0


118



Figure 9.23: All the liposome samples prepared in 10 mM sodium nitrate and sucrose equivalent to 90 mM
sodium nitrate, diluted 1:10 with 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate.
Only one parallel of each sample shown
10 mMsodiumnitrate and sucrose equivalent of 90 mMsodiumnitrate as mobile phase
gfedcb
10 mMsodiumnitrate as mobile phase
gfedcb
100 mMsodiumnitrate as mobile phase
gfedcb
time (min)
10.0 15.0 20.0 25.0 30.0 35.0 40.0
g
e
o
m
e
t
r
i
c

r
a
d
i
u
s

(
n
m
)
0.0
50.0
100.0
150.0

2

Acknowledgements
This master thesis was carried out at the Department of Pharmaceutics and Biopharmaceutics, Institute of Pharmacy at the University of Tromsø from September 2007 to May 2008.

First of all I would like to thank my two supervisors, Professor Martin Brandl and PhD student Stefan Hupfeld for giving me the opportunity to work with this project, but also for inspiration during my work. Stefan , thank you for your valuable advice and useful input on the writing of this thesis. Thank you for all the help in the lab, for always being available and for all encouragement. Martin, thank you for your constructive feedback and good advice in writing of this thesis. Thanks to everyone in the department for their day-to-day support and conversation. Merete Skar deserves my thanks for always being helpful and giving me lots of technical advice in the lab. Rahul Haware, thank you so much for all the help with the viscosity measurements. I wish also to express my gratitude to dr.scient Roy Lysaa for illustrating two of the figures in my thesis. Thanks to all my friends for their support throughout these years and for motivating me to achieve my academic goals. A special thank to all the girls in the master student office, your optimistic spirit and support have been incredible. Kent and Linn, thank you for everything you have done for me these last months. Last but not least, I would like to thank my parents, Kirsten and Nils-Arnt, and my two sisters, Vibeke and Marie; I could not have done this without you. Thank you for always believing in me and encouraging me throughout these years. Vibeke deserves an extra appreciation for reading through the thesis and giving me helpful advice about the writing.

A lot of people deserve my sincere gratitude and thankfulness for giving me the motivation and feedback that made me able to do this. To all of you that are not mentioned by name; you are not forgotten!

Tromsø, May 2008 Helene Moen

3

4

CONTENTS
1 2 3 ABSTACT ....................................................................................................................................... 7 ABBREVIATIONS ......................................................................................................................... 9 INTRODUCTION ......................................................................................................................... 11 3.1 3.1.1 3.1.2 3.1.3 3.1.4 3.1.5 3.1.6 3.2 3.3 4 5 Liposomes .............................................................................................................................. 11 Definitions and background ........................................................................................... 11 Classification of liposomes ............................................................................................ 15 Stability of liposomes .................................................................................................... 16 Pharmaceutical use of liposomes ................................................................................... 16 Challenges with liposome formulations......................................................................... 17 Size analysis of liposomes ............................................................................................. 17 Influence of osmotic stress on liposome size......................................................................... 18 Previous studies ..................................................................................................................... 19

AIM................................................................................................................................................ 21 MATERIAL AND METHODS ..................................................................................................... 23 5.1 5.2 5.3 5.4 5.4.1 5.4.2 5.4.3 5.5 5.5.1 5.5.2 5.5.3 5.5.4 5.5.5 Chemicals .............................................................................................................................. 23 Equipment .............................................................................................................................. 24 Media and solutions ............................................................................................................... 27 Preparative methods............................................................................................................... 29 Preparation of multilamellar vesicles (MLVs) .............................................................. 29 Reduction of lamellarity ................................................................................................ 29 Reduction of liposome size ............................................................................................ 31 Analytical methods ................................................................................................................ 32 Characterization of particle size by Photon Correlation Spectroscopy.......................... 32 Determination of osmolality in solutions....................................................................... 38 Determination of viscosity in solutions ......................................................................... 39 Determination of refractive index in solutions .............................................................. 40 Characterization of liposomes, Flow Field-Flow Fractionation .................................... 42

5

6

RESULTS AND DISCUSSION .................................................................................................... 51 6.1 6.1.1 6.1.2 6.1.3 6.1.4 6.1.5 6.2 6.2.1 6.2.2 6.2.3 6.3 6.3.1 6.3.2 Preliminary experiments ........................................................................................................ 51 Osmolality measurements .............................................................................................. 51 Viscosity measurements ................................................................................................ 52 Refractive index measurements ..................................................................................... 54 Influence of viscosity and refractive index on the accuracy of PCS size measurements of latex bead standards................................................................................................... 54 Freeze-thaw experiments ............................................................................................... 56 Influence of osmotic stress on liposome size, measured by PCS .......................................... 59 Hypertonic osmotic stress experiment with 10 mM NaNO3 and glucose equivalent to 90 mM NaNO3 as dilution medium ............................................................................... 59 Hypertonic osmotic stress experiment with 10 mM NaNO3 and sucrose equivalent to 90 mM NaNO3 as dilution medium ............................................................................... 62 Hypotonic osmotic stress experiment with 10 mM NaNO3 as dilution medium ........... 64 Influence of osmotic stress on liposome size, measured by AF4 .......................................... 65 Hyperosmotic osmotic stress experiment with 10 mM NaNO3 and sucrose equivalent to 90 mM NaNO3 as dilution medium ........................................................................... 67 Hypotonic osmotic stress experiment with 10 mM NaNO3 as dilution medium ........... 73

7 8 9

CONCLUSION.............................................................................................................................. 81 REFERENCES .............................................................................................................................. 83 APPENDICES ............................................................................................................................... 87 Appendix 1......................................................................................................................................... 87 Appendix 2......................................................................................................................................... 88 Appendix 3......................................................................................................................................... 89 Appendix 4....................................................................................................................................... 110

6

7 . liposomes that were smaller than the pore size of the filter used for extrusion were found to shrink in hyperosmotic medium but stay quite constant in size in hypo-osmotic medium. A hypothesis is presented to explain this behavior. the intention was to find out if the retention behavior and calculated geometric radius of liposomes obtained by flow field-flow fractionation in combination with multi-angle light scattering is affected by the osmotic pressure of the medium used for diluting the liposomes and/or running the AF4. liposomes that were larger than the pore size of the filter were found to shrink in hyperosmotic medium and swell in hypo-osmotic medium. this project has demonstrated that a change in osmotic pressure. But. osmotic stress was found to affect liposomes of different sizes in a different manner. It has earlier been discovered that it is feasible to determine the size distribution of vesicle dispersions in a reliable manner and it appears especially useful to employ the combination of SEC fractionation. PCS and the enzymatic PC quantitation. This indicated a demand for a different fractionation method which does not have the limitations that the SEC method described above has. Neutral liposomes were found very dependent of the ionic strength when it comes to elution time. To this end flow field-flow fractionation was chosen. A detailed and reliable knowledge of vesicle size is therefore necessary in order to interpret results of physical and biological investigations in a correct manner. In this study. with constant ionic strength. In a previous study the influence of some key factors such as ionic strength of the eluent as well as pore size of the semipermeable membrane on liposome fractionation behavior has been evaluated. In conclusion.1 ABSTACT Liposomes are currently being investigated as potential parenterally used drug carriers. A drawback discovered was for certain liposome dispersions that in some of the dispersions SEC fractionation showed incomplete recovery of the vesicles. In contrast. affects both elution time and calculated size of liposomes that were prepared by high pressure filter extrusion. The main factor that influences the in vivo behavior of such liposomes is their vesicle size.and disaccharides. In order to exclude ionic-strength effects the salt concentration was kept constant while the osmotic pressure was varied by using mono.

8 .

P.2 ABBREVIATIONS AF4 cP E-80 g HPLC kD kHz L LAF LN2 LS LUVs MALS Min MLVs mM mPa·s Mw NaNO3 nm No. PC PCS QELS RI rms Rn Rw Rz Asymmetrical flow field-flow fractionation Centipoise Unsaturated egg phosphatidyl choline Gram High performance liquid chromatography KiloDalton Kilohertz Liter Laminar air flow Liquid nitrogen Light scattering Large unilamellar vesicles Multi-angle light scattering Minutes Multi lamellar vesicles Millimolar Milli pascal-second Molecular weight Sodium nitrate Nanometer Number Polydispersity index Phosphatidylcholine Photon correlation spectroscopy Quasi-elastic light scattering Refractive index Root mean square Number-average mean square radius Weight-average mean square radius z-average mean square radius 9 .I.

w/w Second Standard deviation Size exclusion chromatography Small unilamellar vesicles Half-life Retention time Micron/micrometer Microseconds Ultraviolet Ultraviolet-visible Versus Weight ratio 10 .s SD SEC SUVs t1/2 tr m Sec UV UV/VIS Vs.

Phosphatidyl choline (PC) molecules are not truly soluble in water.1 INTRODUCTION Liposomes 3. Therefore. Phospholipids are amphipathic.3 3. they self-assemble to liquid crystalline aggregates upon contact with aqueous media.1 Definitions and background Liposomes are spherical vesicles which can be thought of as a hollow sphere whose size ranges from approximately 20 nanometers (nm) to some microns ( m). the hydrophilic part of the phospholipid interacts with the water and the lipophilic part of the molecule avoids the water. for review see (Liposomes: a practical approach Torchilin and Weissig 2003). Liposomes do form spontaneously when phospholipids are mixed with aqueous medium. Liposome membranes can be composed of naturally-derived phospholipids with mixed lipid chains and a variation of head groups or of pure synthetic lipids with defined acyl chains and head groups. They are composed of a bilayer membrane which entraps an aqueous core.1.An Introduction to Lipid Nanoparticles Sciences 2008). when added to water.e. i.1. The membrane is composed of phospholipid molecules. This structure is known as a phospholipid bilayer of lamellar structure as shown to the right in figure 3.1. In order to accomplish this. that is. the phospholipids align themselves side-by-side with their lipophilic heads orienting themselves towards each other as shown in the middle figure below (Technical Summary . part of their structure is hydrophilic and the other part is hydrophobic. 11 . the same type of molecules cell membranes are comprised of.

reviewed in (Liposomes: from physics to applications Lasic 1993). 12 .Figure 3.com/company. (Figure taken with permission from: http://www.encapsula. The thickness of a bilayer is about 4 nm. from phospholipid molecules to a unilamellar vesicle.1: The formation of liposomes. Small liposomes usually consist of only one bilayer but bigger liposomes can consist of multiple bilayers or several smaller liposomes can be formed inside the bigger liposome.1.html) The vesicles formed may consist of one or more lamellae. Lipophilic of amphiphilic drug are incorporated into the membrane and hydrophilic drugs are entrapped in the aqueous core for review see (Liposomal formulations of anticancer drugs: selectivity and effectiveness Massing and Fuxius 2000). Materials can either be entrapped in the aqueous core or incorporated within the membrane for review see (Liposomes as drug carriers: a technological approach Brandl 2001).

1.A multi lamellar liposome is shown in figure 3. PC is amphiphilic and is composed of a hydrophilic head group consisting of the quaternary ammonium moiety choline linked to the glycerol-backbone via a phosphor-ester and two lipophilic acyl chains.2. PC can be derived from natural sources as egg yolk and soyabeans or be made synthetically (Liposomes: a practical approach Torchilin and Weissig 2003). PC is zwitterionic and liposomes made of it have no net charge. The most common phospholipid used in liposomal drug carriers is phosphatidyl choline.1. The green rods are lipid-soluble drugs which are incorporated in the lipid membrane. The pink dots are water-soluble drugs which are entrapped in the core or in the aqueous space between the bilayers. phosphodiglycerides and sphingolipids.2: Drug encapsulation in liposomes. As the phosphate is negatively charged at physiological pH.1. PC belongs to the group of phosphodiglycerides.3 13 . There exist two sorts of phospholipids.html) The choice of lipids for liposomal drug carriers depends on the desired stability of the liposome formulation.encapsula.com/company. A schematic presentation of PC is shown in figure 3. Figure 3. and the drug which should be incorporated into the liposomes. We can see that there are many phospholipid bilayers with water in between the layers. (Figure taken with permission from: http://www. the water-soluble drugs (shown in pink) are entrapped in the aqueous compartments and the lipid-soluble drugs (shown in green) are entrapped within the membrane.

ca/gallant/biology/biology.4. When drugs are incorporated into the liposome one usually wants to prevent leaking and loss of drug through the membrane.nbed. Blends of PC with other lipids are used primarily to improve both in-vitro and in-vivo stability of the liposomes (Liposomes as drug carriers: a technological approach Brandl 2001).Figure 3.1.1. cholesterol will induce a tighter packing of the membrane and reduce the fluidity of the membrane.3: A schematic representation of PC (Figure taken with permission from: http://kvhs.html) PC is hardly ever used alone in liposomal lipid formulations. as shown in figure 3. 14 .nb. A normal way to prevent leaking is adding cholesterol to the membrane.

Small unilamellar vesicles (SUVs) are defined as the smallest phospholipid vesicles possible (approximately 20 nm) and up to 50 nm. 15 . The size depends on the ionic strength of the aqueous medium and the lipid composition in the membrane. (Figure taken with permission from: http://www. Lipids with a net charge reduce both size and number of lamellae of the liposome. They usually consist of one lamella. Multi lamellar vesicles (MLVs) are vesicles covering a size range from 100-1000 nm and consist of five or more lamellae. composition of the medium and the mechanical stress exerted during swelling.1. for review see (Liposomes: a practical approach Torchilin and Weissig 2003).uic. but they only have one lamella.edu/classes/bios/bios100/lecturesf04am/lect08.htm 3.4: Phospholipid bilayer with cholesterol incorporated in the membrane. Size and lamellarity of liposomes formed by spontaneous swelling depend on the type of lipid. from 1001000 nm.1.2 Classification of liposomes Liposomes are often classified according to their size. Large unilamellar vesicles (LUVs) are vesicles in the same size range as MLVs.Figure 3.

The objective is to achieve selective localization of active drug in disease sites as tumors and inflammation sites. 3. The liposomal preparations may have the potential to change this with time.1. It recognizes only the liposome and the intrinsic pharmacokinetics of the drug is masked by the pharmacokinetic behavior of the liposomal vesicle. Aggregation is the process where liposomes form aggregates. Hence. The process most often happens to very small liposomes with a diameter of approximately 20 nm. 16 . the cells in tumors are often not as closely packed as in healthy tissue. Blood vessels in tumors are leakier than normal blood vessels because of their fast growth.1.3 Stability of liposomes For phospholipids there are two major degradation reactions known which affect their chemical stability in aqueous dispersion. Examples of this kind of instability are aggregation and fusion. such as size and size distribution. EPR-effect. This is a reversible process which can be resolved by stirring. excretion and degradation in the blood stream. In addition. Fusion is the phenomenon where vesicles fuse together and make bigger liposomes. preparations based on submicron particles are emerging as an important tool for achieving either controlled or targeted delivery of the active compound. hydrolysis and oxidation (Liposomes as drug carriers: a technological approach Brandl 2001). Physical instability might affect the particle size of liposomes. The physical properties of the liposomes. hence it protects the drug from premature recognition.3. Most of the cancer drugs on the marked now have dose-limiting toxicity problems and thus relatively low efficacy. The systemic environment does not recognize the drug when it is incorporated into a liposome. reviewed in (Liposomal formulations of anticancer drugs: selectivity and effectiveness Massing and Fuxius 2000). there is a great demand on detailed and reliable information about this subject. Examples of such drug carriers are polymeric and solid lipid nanoparticles as well as liposomes. play an important role in the work of developing successful drug formulations. The liposomes are also able to accumulate in tumors because of the enhanced permeability and retention effect.4 Pharmaceutical use of liposomes In the field of drug delivery. A potential field that is very interesting is cancer therapy. This is not a reversible process and therefore a much bigger problem.

Upon entering the blood pool. Some of the methods mentioned above such as SEC and ultracentrifugation are rather time consuming. there is clearly a need to develop methods that are not only able to measure the size and the size distribution of liposome dispersions. use of time should be evaluated.5 Challenges with liposome formulations Intravenous injection is regarded as the most promising route of administration for liposomal drug delivery.1. The role of a liposomal drug carrier is to circulate in the blood pool and reach the desired organ or tissue. The lipid composition and lamellarity is also important since it together with the physiochemical properties of the drug determines the retention of the active ingredient within the liposome reviewed in (Liposomal formulations of anticancer drugs: selectivity and effectiveness Massing and Fuxius 2000). 3. Big liposomes (diameter >200 nm) are quite rapidly taken up and disappear from the circulation. Liposomes with a diameter between 70 and 200 nm tend to circulate long enough in the blood stream to reach the desired organ. and in a reproducible manner.6 Size analysis of liposomes The main factor that influences the in vivo behavior is the size of the liposomes. ultracentrifugation and flow field-flow fractionation (AF4). Some of the techniques which have been used are various electron microscopic methods. but in a next step generates liposomes of defined size. liposomes should avoid to be taken up by macrophages.3. Due to these facts. and when developing a method for routine size analysis of liposomes. 17 . photon correlation spectroscopy and methods based on fractionation of liposomes according to size such as size exclusion chromatography (SEC). which unfortunately is difficult to achieve. Because of that fact it is important to have methods for determining the size and size distributions in a reliable manner. The pharmacokinetics and biodistribution of the carrier primarily depend on the size and surface characteristics of the liposome. Smaller liposomes with a diameter under 70 nm show shorter circulation time due to extravasation through the capillary walls of the liver reviewed in (Liposomes as drug carriers: a technological approach Brandl 2001). Preferable are methods which are able to give a qualitative and quantitative overview over the full size range.1.

causing it to swell. which exhibit similar permeability properties to biological permeability membranes represents a convenient model system to study osmotic stress (Osmotic properties and water permeability of phospholipid liquid crystals Bangham 1967).org/wiki/Tonicity#I http://en. movement of water in to the cell.wikipedia.The method should be able to quantify the amount of large particles and eventually aggregates in comparison to the amount of SUVs (Size analysis of submicron particles and liposomes by size exclusion chromatography and photon correlation spectroscopy Ingebrigtsen 2001) photon 2001). (Figure taken with permission from: http://en. Liposomes. 3. Figure 3.2. causing it to shrink (Tonicity Wikipedia 2008) In the opposite case. seen as a cell with a semi-permeable membrane and an aqueous core.2. Both of these principles are shown in principles figure 3.wikipedia. A liposome can be 1967). The difference in osmotic pressure inside meable and outside of the membrane causes a net movement of water out of the cell.org/wiki/Tonicity#Isotonicity) 18 .1.1: Effect of hypertonic and hypotonic solutions on blood cells. a hypotonic environment causes a net 2008).2 Influence of osmotic stress on liposome size It is likely that liposomes diluted in a hypertonic medium will shrink and become smaller than they originally were. When a cell comes in permeable membrane contact with a hypertonic environment it is surrounded by a higher concentration of impermeable solute than exists inside of the cell.

In the second part of his study he employed SEC. Previous studies of the size distribution of liposomes gained qualitative results of various qualities. for certain ratios and for certain sizes. It can be explained in terms of aggregation of these rather small vesicles which subsequently got stuck on the SEC column.e. He saw a shift of the liposome peak to later retention times when an eluent with a higher 19 . A drawback discovered was for certain liposome dispersions that in some of the dispersions SEC fractionation showed incomplete recovery of the vesicles. He tried to evaluate the influence of some key factors such as ionic strength of the eluent as well as pore size of the semi-permeable membrane on liposome fractionation behavior. if asymmetrical flow field-flow fractionation was convenient for fractionation of liposomes. Ingebrigtsen checked how reliable results photon correlation spectroscopy (PCS) can give when it is used for routine particle analysis of latex beads of submicron sizes.or multimodal size distributions.3 Previous studies Lars Ingebrigtsen and Christer Bakke Frantzen investigated in their master theses accomplished in December 2001 and May 2003 respectively. But it was obvious that PCS is inappropriate to resolve bimodal size distributions with a broader overlap or tri. the described method is very time consuming.3. PCS and the enzymatic PC quantitation. He found out that PCS was able to resolve bimodal size distributions of the heterogeneous samples within certain limits. In addition. PCS and a quantitative enzymatic PC assay. This clearly indicated a demand for different fractionation method which does not have the limitations that the SEC method described above has. Dominik Albert Ausbacher investigated in his diploma thesis accomplished in October 2007. but none of them provided quantitative information. He found that it was feasible to determine the size distribution of vesicle dispersions in a reliable manner and it appears especially useful to employ the combination of SEC fractionation. i. Neutral liposomes were found very dependent of the ionic strength when it comes to elution time. whether a combination of size exclusion chromatography with subsequent size analysis performed by photon correlation spectroscopy combined with a quantitative assay achieved a total qualitative as well as quantitative insight into the size distribution of liposome dispersions and thus was suited as a routine analysis method.

but he was not able to say whether the change in retention time of liposomes in higher ionic strength media was due to osmotic stress or zeta-potential. his master project can only be seen as a first step in paving on the way towards routine AF4-multi-angle light scattering (MALS) liposome analysis. These previously executed master projects form the basis of this master project. Nevertheless.ionic strength was used. 20 .

21 . with constant ion strength. In more detail. our aim was to find out if the retention behavior and calculated geometric radius of liposomes obtained by flow field-flow fractionation in combination with multi-angle light scattering is affected by the osmotic pressure of the medium used for diluting the liposomes and/or running the AF4. affects both elution time and calculated size of liposomes.4 AIM The purpose if this study was to investigate how a change in osmotic pressure. In order to execute ionic-strength effects the salt concentration was kept constant while the osmotic pressure was varied by using mono.and disaccharides.

22 .

5 5. Oslo.3: Chemicals Chemical Ethanol Quality 96 % Batch number N/A Manufacturer Arcus Kjemi AS. Norway Purified Water N/A N/A Prepared in-house by Millipore water purification system 23 . 1031492-11/904 Manufacturer Lipoid GmbH. Germany Table 5.1: Lipid Name of lipid Unsaturated egg phosphatidyl choline Lipoid E-80 Batch numbers 1031492-9/904. 1031492-11/902. Ludwigshafen.1.1 MATERIAL AND METHODS Chemicals Table 5.1. CA. Vestby. USA Table 5. Freemont. Norway Liquid nitrogen N/A N/A AGA AS. Trondheim.1. Norway Glucose For parenteral use 1A102/4 Norsk Medisinaldepot.2: Latex bead Name of latex bead Nanosphere TM size standards Mean diameter 102 nm ± 3 nm Batch number 28570 Manufacturer Duke Scientific Corporation.

Dernbach.Sodium chloride solution. 142 mm diameter Freezer (-80 °C) Forma Laboratory Freezer. Göttingen. Santa Rosa. Ing. OH. 1100 series LAF (laminar air flow) bench Holten maxisafe 2000 Heto Holten A/S.2. Germany 24 . Corp. Poole. Darmstadt. Berlin. CA. Denmark MALS-detector Dawn EOS Wyatt Tech.2 Equipment Table 5. USA Glass tubes for PCS analysis Borosilicate glass disposable culture tubes. Herbert Knauer GmbH. Karlskoga. Allerød. USA HPLC variable-wavelengthUV/VIS-detector G1314A.a. 400 mOsm/kg Sodium nitrate N/A 5484C41 Dr. Darmstadt. England 5. Germany K33825286 524 VWR International Ltd. Sweden Agilent Technologies Europe. Europe. Marietta.1: Equipment Type Equipment Filtration device. Germany Sucrose Ph Eur K341881187 544 Merck KGaA. Germany p. Germany ThermoQuest/ Forma Scientific Division. A571737 519 Merck KGaA. 6 x 50 mm VWR International AB. model 738 SM 16275 Manufacturer Sartorius AG.

CA. Germany Water bath Büchi Waterbath B-480 Büchi Labortechnik AG. Dernbach. Molsheim. 25 cm3 RI-detector Optilab rEX Stainless steel filtration vessel. Corp. Germany Branson Ultrasonic Cleaner 1510E-MT Branson Ultrasonic Corporation. Ing. Flawil. Wertheim. Germany Millipore S. Germany Nicomp Particle Sizing Systems. capillary type 0c Ubbelohde viscometers. Germany Wyatt. France Brand GMBH + CO KG. No. Germany Pycnometer Specific gravity bottle. Molsheim. CT. Berlin. Danbury. Schott-Geräte.A. Type ML. Herbert Knauer GmbH. Hofheim. France 25 .PCS Submicron Particle Sizer Model 380 Prototype filter extruder Continuous consisting of Lewa diaphragh pump type LDB 1 and Millipore 47 mm high pressure filter holder Custom made. Santa Barbara.. A0299 Wissenschaftliche Gerätebau Dr. Europe. Tech. USA Viscometer Capillary viscometer. USA Photon Correlation Spectrometer. Göttingen. Switzerland Water purification system Millipore water purification system Millipore S.A.Osmometer Knauer Semi-Micro Osmometer. 5 liter Ultrasonic bath SM 1753 Sartorius AG. Lewa GmbH. Leonberg.

A. Ann Arbor. France Millipore S.4 m filter. 0. batch no: R8MM92556 0. Millipak 20 Express 0.V. 142 mm Nitrocellulose VCWP 0. MI. Molsheim.2 m filter.Filters for: Filtration device.22 m filter. batch no: MPGP02001 Milli-Q Synthesis Quantum EX Ultrapure organic Cartridge. MA. batch no: R8NM25306 Millipore Ireland B..2 m filter. batch no: H5JN02152 Millipore Corporation. Cork. Molsheim. USA Liposome filter extruder Millipore Isopore Membrane filters: 0.1 m filter. Ireland Milli-Q water system. Billerica. batch no: F5HN65923 Millipore S. batch no: R5SN28296 0.1 m filter.A. France 26 . USA Syringe filter Acrodisc syringe filter. batch no: 21182 Pall Corporation...

5.0 ml 0.4 mM sucrose solution Sodium nitrate Sucrose Purified water ad Osmolality measurements 27 .6940 g + 1000.2358 g + 1000.8499 g + 3.0 ml Osmolality measurements 50 mM sodium nitrate solution Sodium nitrate Purified water ad Osmolality measurements 10 mM sodium nitrate and 19.8499 g + 5.8499 g + 14.0 ml 4.6 mM sucrose solution Sodium nitrate Sucrose Purified water ad Osmolality measurements 10 mM sodium nitrate and 79 mM glucose solution Sodium nitrate Glucose Purified water ad Osmolality measurements 10 mM sodium nitrate and 66.0 ml 0.2495 g + 1000.1: Media and solutions Name of medium 10 mM sodium nitrate solution Content Sodium nitrate Purified water ad 0.0 ml 0.3 Media and solutions For all the solutions the composition is given per 1 liter: Table 5.6998 g + 1000.8499 g + 1000.8 mM glucose solution Sodium nitrate Glucose Purified water ad Osmolality measurements 10 mM sodium nitrate and 16.0 ml Application Hydration medium in MLV production and as diluting agent before PCS measurements Also used for diluting liposome dispersion 1:10 prior to investigation of size changes as a result of osmotic stress and as mobile phase in the AF4 experiments 20 mM sodium nitrate solution Sodium nitrate Purified water ad 1.8499 g + 22.7780 g + 1000.5590 g + 1000.3.0 ml 0.

0 ml liposome dispersion 1:10 prior to investigation of size changes as a result of osmotic stress and as diluting agent before PCS measurements 10 mM sodium nitrate and 149.0 ml Hydration medium in MLV production and as diluting agent before PCS measurements Also used for diluting liposome dispersion 1:10 prior to investigation of size changes as a result of osmotic stress and as mobile phase in the AF4 experiments 100 mM sodium nitrate solution Sodium nitrate Purified water ad 8. The concentrated solution was then diluted by adding water up to the desired volume (1 liter) in a volumetric flask.2490 g + 1000.0306 g + Also used for diluting 1000.0 ml Mobile phase in the AF4 experiments All the different solutions were prepared according to the same procedure.499 g + 1000.6 mM glucose solution Sodium nitrate Glucose Purified water ad 0. 28 .10 mM sodium nitrate and 177.1 m nitrocellulose filter.8499 g + 51. the solid components were weighted in and transferred to a volumetric flask where they were dissolved with some water. 32.3 mM sucrose solution Sodium nitrate Sucrose Purified water ad 0.8499 g + Viscosity measurements. All the solutions were filtered through a 0.

Aqueous medium 5g 45 g E-80 and the aqueous medium were weighed in directly in a round bottom flask. Experiment: MLVs were prepared according to the hand-shaken method: Hand-shaken method: 10 % w/w E-80 (unsaturated egg phosphatidyl choline) in different aqueous media 1. The freezing and thawing cycles cause the MLVs to rupture and re-assemble such as the aqueous layers between concentric lamellae increase in thickness. which takes approximately 45 minutes. 5. Standish 1965). The components were stirred using a magnetic stirrer until E-80 was finely dispersed. E-80 2.4 Preparative methods 5.4.2 Reduction of lamellarity Theory: To increase the proportion of unilamellar vesicles in preparations it is a common practice to subject MLVs to freeze-thaw cycles prior to extrusion.1 Preparation of multilamellar vesicles (MLVs) Theory: MLVs form spontaneously when phospholipids are blended with excess aqueous medium (Diffusion of univalent ions across the lamellae of swollen phospholipids Bangham.4. (Liposome technology Mui and Hope 2006) 29 .5. this probably reduces the number of closely associated bilayers. (Osmotic properties of large unilamellar vesicles prepared by extrusion Mui. Cullis 1993).

Freeze-thawing was performed before extrusion.Experiment: In order to find out which freeze-thawing method would give the best result. 3. The liposome dispersion was frozen in liquid nitrogen (LN2) and then thawed on a 50°C water bath. The liposome dispersion was frozen in a -80°C freezer for 1 hour and then thawed on a 50°C water bath. Freeze-thawing was performed after extrusion. 30 . The liposome dispersion was frozen for one hour in a -80°C freezer and thawed on a 50°C water bath. Freeze-thawing was performed before extrusion. 4. Freeze-thawing was performed between extrusion through 400 nm filer and 200 nm filter. 2. The freeze-thaw cycle was repeated three times. The liposome dispersion was frozen in a -80°C freezer for one hour and thawed on a 50°C water bath. 1. The freeze-thaw cycle was repeated three times. four preliminary experiments were executed. The freeze-thaw cycle was repeated three times. The freeze-thaw cycle was repeated three times.

as shown in figure 5. 200 nm and 100 nm.4.4. 31 . Due to this fact. The pores have been formed by chemical etching along ion tracks. When the MLV preparation is squeezed through the filter pore under pressure a process of membrane rupture and resealing occurs.3 Reduction of liposome size Theory: MLVs have a broad particle size distribution and have multiple internal compartments. The extrusion was performed on a custom made extruder. Filter extrusion involves the process of forcing the liposome preparations through pores of membrane filters with defined pore sizes. The pump of the extruder was running at the same speed every time.5. (Liposome technology Mui and Hope 2006) Experiment: The MLV dispersion obtained by the hand-shaken method was extruded using filters with decreasing pore sizes of 400 nm. In order to achieve liposomes with homogeneous size. unprocessed MLVs have limited use in medical research. The transfer of liposomes through the filter was repeated 10 times.1. After about 10 cycles through filters with 100-nm pores a homogeneous population of vesicles with a mean diameter of approximately 100 to 120 nm is obtained. this process generates large to small unilamellar vesicles with a mean vesicle diameter usually slightly larger than the pore size of the polycarbonate membranes. The preferred filter type for reducing the size of liposomes is made of polycarbonate with straight-through. cylindrical pores. filter extrusion was accomplished. as recommended by (Liposome technology Mui and Hope 2006).

A laser light beam (typically 5 mW Helium and Neon laser) is focused on a glass tube containing a diluted suspension of particles. The technique has proven to be especially powerful in measuring particles with a diameter of approximately 20-200 nm. Each of the particles scatters light in all directions and the intensity of scattered light varies with the particles molecular weight. The difference in refractive indices of the particle and the surrounding medium also play an important role (Windows based software.1 Characterization of particle size by Photon Correlation Spectroscopy Theory: PCS is an analytical tool to determine the size distribution of submicron particles suspended in an aqueous medium. Dynamic light scattering theory User Manual 1997).1: The custom made extruder 5. 32 .4. size and shape.5.Figure 5.5 Analytical methods 5.

This is the physical phenomenon known as interference. the phase of each of the scattered light waves that is arriving at the detector will fluctuate randomly in time due to the random positions of the particles (Size analysis of submicron particles and liposomes by size exclusion chromatography and photon correlation spectroscopy Ingebrigtsen 2001). of particles increases with increasing temperature. The next step is to determine the diffusion coefficient. Equation 1: È k = Boltzmann´s constant (1. η. which measures the net scattering intensity at a 90 degrees scattering angle.PCS measures the fluctuations in the scattered light intensity. This is primarily due to the temperature dependent viscosity of the solvent. All the different waves interfere at a distant slit on the face of a photomultiplier detector. T.38 X 10-16 erg K-1) T = temperature (°K. From D it is possible to calculate the particle diameter using the Stokes-Einstein equation. As a consequence of these motions. Scattered light intensity fluctuates with time because many individual waves add coherently. = °C + 273) η = shear viscosity of the solvent ds = Stokes particle diameter From equation 1 we can see that the diffusion coefficient. of the particles from the raw data. 33 . D. Small particles will move around faster and give rapid fluctuation of the light intensity. The suspended particles move around randomly in the medium by Brownian motion. The fluctuation of light intensity is dependent on the size of the particle. D.

t-t´.Σ Is (t) 2 B = Σ Is(t) 2 Variable τ is the characteristic decay time constant of the exponential function.Autocorrelation is the mathematical process of extracting quantitative information as the size of the particles and their size distribution in a sample from the fluctuation of the intensity of the scattered laser light (Size analysis of submicron particles and liposomes by size exclusion chromatography and photon correlation spectroscopy Ingebrigtsen 2001). The value of τ describes the duration of a major fluctuation in the scattered intensity Is. the larger the particles. Such comparisons are carried out for many values of (t) in order to get a statistical meaningful average value for C (t´). the slower fluctuations in Is and the longer the decay constant τ. The correlation function can be expressed: Equation 2: { { HJ { { 0 HJ { . The autocorrelation function is used to study the similarity between the value of Is (light intensity) at a given time and the value of Is at an earlier time. È { - A = Σ Is2 (t) . As expressed below in equation 3. { One can describe the autocorrelation function as an exponential function that gradually decreases as the value of (t´) increases. Hence. Equation 3: { { J {. We are able to predict the diffusion coefficient of the particles from the decay constant τ. 34 .

If the value of Chi squared is over three. but any value under three is regarded well enough.Equation 4: { È H {{ È { K = scattering wavevector (A constant which depends on the laser wavelength in the solvent and the angle between the laser beam and where the detector is placed. the PCS software suggests that the Gaussian model is inappropriate. NICOMP is used for bi. and NICOMP distribution analysis will consequently not be further explained. A higher baseline adjust value is indicative for large particles or aggregates in the sample.or multimodal size distribution and Gaussian is used for a unimodal size distribution. Gaussian distribution: The PCS software will indicate how well the measured results fit with the normal distribution or the Gaussian model. and the NICOMP model should be used instead. using either the NICOMP model or the monomodal Gaussian model. collected by the detector. 35 . The ideal value is zero. The Gaussian analysis is restricted to simple. Any value close to or below one indicates an exceptionally good fit. The value for baselines adjust is indicating an adjustment needed to obtain a low value of Chi squared. unimodal particle size distributions which are the case in this thesis. The quality of this fit is stated by the statistical value Chi squared. The Gaussian model states how good a fit is approaching a normal distribution.) Fitting and interpretation of the results: The PCS software fits the raw data.

I. Dynamic light scattering theory User Manual 1997). value thus indicates a homogenous size distribution. A low P. It is very important that the intensity level is correct because the correlator´s input counter must not receive more photons than it can count in a single sample time otherwise the correlation function will be distorted (Particle size analysis in pharmaceutics and other industries: theory and practice Washington 1992). To avoid particle contamination the dilution medium was filtrated through a sterile filter with 0. In brief. value close to zero is therefore most desirable. the test tubes used for PCS measurements were sonicated for 10 minutes and then rinsed with the dilution medium.I. They were diluted with the same medium as used for producing the liposomes. and PCS analysis and enzymatic assay of lipid content Ingebrigtsen and Brandl 2002).I. Before any measurement was carried out the instrument parameters were set according to the values listed in table 5. approaches one (Windows based software.I.Polydispersity index (P.5. a cycle of 5 minutes was run for each sample in order to calculate how long a sample need to be run to ensure count rates above 1000 K (1 million) in channel no. A P. All the preparative work was done in a laminar airflow bench to avoid particle contamination.1. For highly polydisperse samples the P.) is stating how broad the distribution is around the mean particle size.2 m pore size. Samples were diluted using particle free medium. 36 . until the intensity was between 250 and 350 kilohertz (kHz). For statistical accuracy. 1. Experiment: Measurements were carried out as described by (Determination of the size distribution of liposomes by SEC fractionation.

Values for the solutions that contained sucrose and glucose were measured with the Optilab rEX refractive index detector Intensity setpoint Laser wavelength Scattering angle 300 ± 50 kHz 632.5.5.) The viscosity values for the solutions that contained glucose or sucrose were measured with a capillary viscometer. (E. The values were obtained from a table in the PCS manual. values of viscosity of water was used.333 is the literature value of water.g.9325 cP.8 nm 90 ° Figure 5. if the temperature was 23 °C then the viscosity would be 0. usually 23-25 °C If the liquid only contained sodium nitrate. Liquid index of refraction 1.1: PCS parameters Parameter Channel width Temperature Liquid viscosity Value Auto set Room temperature. and the same value was used when the solution only contained water and sodium nitrate.1: Block diagram of the PCS (NICOMP Model 380 submicron particle sizer) 37 .Table 5.

1 means that 100 % dissociates.. The equation to determine the osmolality of a solution is shown in equation 5: Equation 5: J JI 0 J 0 JI Ф = osmotic coefficient. when one mole of non-ionic solute is added to one kilogram of water.86 °C. NaNO3 is dissolved in a kilogram of water it will yield almost twice as many particles since NaNO3 dissociates almost completely into one mole Na+ and one mole NO3.g. 1 for sucrose.86 °C.5. in ideal solutions.. The osmotic strength of a solution can be measured by an osmometer. 38 . the freezing point goes down 1.2 Determination of osmolality in solutions Theory: Osmolality is a measure of the osmoles of solute.ions (Refractive index 2008). 2 for NaNO3) The unit of osmolality is Osm/kg (osmole per kilogram).g.5. n = number of particles into which the molecule can dissociate (e. Ф is between 0 and 1. E. that number of particles that would reduce the freezing point of the solvent by 1.. which accounts for the degree of non-ideality of the solution.g. per kilogram of solvent. An osmole is the amount of substance that yields. When one mole ionic solute e. Currently available osmometers use the colligative properties of freezing point depression or vapour pressure depression.

Measurements were carried out by Knauer semi-micro osmometer to ensure that the calculations were correct.2: Knauer semi-micron osmometer 5. The measurement was executed on an osmometer which measured the freezing point depression of the solutions. it would prove that the calculated amount of glucose was correct. E. If the measured values were equal to each other.3 Determination of viscosity in solutions Theory: Viscosity is a measure of the fluids resistance to flow. Figure 5.5. One of the most common and most accurate instruments for measuring kinematic viscosity of Newtonian fluid´s is the glass capillary viscometer. one solution with 20 mM NaNO3 was compared to a solution with 10 mM NaNO3 and an amount of glucose equivalent to 10 mM NaNO3. Three parallels were measured for every solution.Experiment: Calculations of the osmolalities were performed according to the calculation method described in appendix 1.52 shows the osmometer used for these experiments.. Dynamic viscosity coefficient is calculated from kinematic viscosity by multiplying the dynamic viscosity by the density of the Newtonian fluid (Viscosity Wikipedia 2008).5. Figure 5. 39 .g. Viscosity can be measured by various types of viscometers.

The viscosity of both the solutions that contained sucrose and NaNO3. To calculate the dynamic viscosity the density of the solution is needed.5. Experiment: Measurements of viscosity were carried out because the knowledge of the accurate viscosity is crucial when PCS analysis is performed. Four parallels for every solution were measured. and the average value was used in the PCS software. The refractive index of vacuum is by definition 1. 5. The kinematic viscosity was measured using a glass capillary viscometer. besides viscosity. or glucose and NaNO3 were measured. The density of the solutions was measured with a pycnometer. except those who only contained NaNO3 because it was assumed that the viscosity would not change noticeably.4 Determination of refractive index in solutions Theory: Refractive index is the other parameter that needs to be determined to get reliable results from the PCS measurements. 40 . The refractive index of a solution is a measure of how much the speed of light is reduced inside the medium or the bending of a ray of light when passing from one medium into another.333.The equation to calculate dynamic viscosity from kinematic viscosity is shown in equation 6: Equation 6: 0 η = dynamic viscosity ν = kinematic viscosity ρ = density The unit of dynamic viscosity is mPa·s (pascal-second). and the refractive index of water is 1. Every solution that was used as dilution medium in PCS was measured.

333 = 0. which means that both the glass cells in the instrument are flushed with medium and the absolute refractive index is measured. To prevent dilution of the medium that should be measured. For the measurements the respective solution was injected into the instrument with a syringe. All solutions used during this project were measured.333 means that light travels at 1 / 1.A refractive index of 1.75 times the speed in vacuum (Refractive index Wikipedia 2008). It was necessary to set the Optilab rEX into purge mode. the instrument was flushed until the value of refractive index did not change anymore. 41 . The refractive index can be defined by equation 7: Equation 7: J n = the refractive index c = phase velocity of a wave vp = phase velocity of the medium itself Experiment: The refractive indices were measures using the Optilab rEX on-line refractive index detector.

cfm) 5. AF4 is fractionating particles according to their size and determining size distribution of polydisperse particle samples from an observed retention profile (Size characterization of liposomes by flow field-flow fractionation and photon correlation spectroscopy Effect of ionic strength and pH of carrier solutions Moon.5.5 Characterization of liposomes. Flow Field-Flow Fractionation Theory: Asymmetrical flow field-flow fractionation is a one-phase chromatography technique which allows separation of heterogeneous samples and is able to perform fractionation ranging from the 1 nm up to 10 microns (Changes in Liposome Morphology Induced by Actin Polymerization in Submicrometer Liposomes Nickels 2003).com/solutions/hardware/Refractive_Index_Detector-OptilabrEx.3: Picture of the Optilab rEX refractive index detector (Figure used with permission from: http://www. This is done by the application of field force generated by the transverse movement of carrier liquid (cross flow) across the channel.Figure 5.5. Park 1998). However. the fractionation of samples takes place in a separation channel instead of a separation column. 42 . The instrumental Set-Up of an AF4 system is comparable to a HPLC (high performance liquid chromatography) system.wyatt. Particles are separated by flow in aqueous media.

due to the particles diffusive transport. large particles have a small diffusion coefficient and are therefore driven closer to the accumulation wall.5. different flows and forces during A4F (Figure used with permission from Dominik Ausbacher (A4F/MALS-Analysis of Liposomes Influence of Key Factors on Fractionation Behavior and Evaluation of MALS Fit Routines Ausbacher 2007)) Upon injection into the AF4 channel particles are driven toward the bottom of the channel wall by the cross flow. the permeable frit. The upper channel plate is impermeable.4: Channel setup. covers the bottom plate to prevent the sample from penetrating the channel (How Asymmetric Field Flow Fractionation (AFFF) Theory Works Technology 2008). The Brownian motion of the particles or vesicles leads them to be differentially distributed over the accumulation wall according to their size. The small particles will move around faster and float further from the accumulation wall hence they are displaced by the fast flow stream and are eluted earlier than the larger ones. The upper block contains the channel inlet. on the other hand. An ultra filtration membrane with a typical size barrier of 10 kD. 43 . as can be seen in figure 5. After injection the sample is focused on a small band near the injection point by applying an inverse flow through the channel outlet. Equilibrium positions are established away from the accumulation wall. the membrane and the spacer.5 (A4F/MALS-Analysis of Liposomes Influence of Key Factors on Fractionation Behavior and Evaluation of MALS Fit Routines Ausbacher 2007).5. channel in injection port field = generated by cross flow upper block channel height provided by spacer eluent back-diffusion (focusing) sample membrane + frit Cross flow out force by flow lower block eluent channel out Figure 5.The channel consists of a lower block which contains the cross flow outlet. but the bottom channel plate. The spacer foil has a typical thickness of 100 to 500 m. the injection port and the channel outlet as shown in figure 5. is permeable. The thickness and the form of the spacer foil are defining the dimensions of the actual channel.4.5.

the channel flow is split and introduced both at the inlet and at the outlet of the channel (Asymmetric Flow Field-Flow Fractionation Analytics 2008). focusing and elution.5. relaxation and focusing are quite simultaneous and are followed by the elution. the next step is the experiment is the so-called elution mode. In AF4 is retention time (tr) of a particle given by equation 8. Equation 8: 0 V = channel flow Vx = cross flow w = channel thickness D = diffusion coefficient 44 . After focusing. The first three steps injection.Figure 5. These are injection. is divided into four steps. In elution mode both the cross flow and channel flow active and fractionation can take place. relaxation. In the first step.5: Side view of particle migration according to the size in the AF4 channel The separation based on AF4 technology.

45 . the particles size is measured by MALS. A4F can give a direct measure of liposome size since separation is based on the difference in hydrodynamic radius of the particles (Size characterization of liposomes by flow field-flow fractionation and photon correlation spectroscopy Effect of ionic strength and pH of carrier solutions Moon. In a MALS detector several photo diodes are arranged in a circle around a glass cell with a bore where the sample runs through. LS (light scattering) will occur in all directions as demonstrated in figure 5.6. If we link equation 1 and equation 8 we get equation 9. When light from a polarized laser light beam hits a sample molecule. The mathematical basis for this is provided by the Stokes-Einstein equation shown in equation 1. The diffusion coefficient can be used for calculating the molecular dimensions in the form of the Stokes diameter (On-line coupling of flow field-flow fractionation and multi-angle laser light scattering Roessner and Kulicke 1994). the retention time is directly proportional to the square of the channel thickness. The resulting scattered light will then be detected by the photo diodes at the different angles from 10° to 160°. Park 1998). Equation 9:  $ 0 0 Vesicle diameter can readily be calculated from experimental retention time (tr) when the experimental parameters are known.As we can see in equation 8. which gives the dependence of the retention time on the material and experimental parameters. and inversely proportional to the diffusion coefficient. The wavelength of the laser light used to illuminate the solution containing the sample is 690 nm (Wyatt Technology Corporation User Manual 2007). Theory: Instead of determining the hydrodynamic radius from the retention time of particles in an AF4 run. However a direct determination of hydrodynamic radius was not performed in this work because the applied method requires more complex mathematics for calculating the hydrodynamic radius which is not available at the time.5.

(Figure taken with permission from: Wyatt Technology (Introduction to Light Scattering. The excess Rayleigh ratio is a ratio of the scattered light and incident light intensities that take into account different factors which are shown in equation 10. called the excess Rayleigh ratio. for the solute or particle alone (Wyatt Technology Corporation User Manual 2007).6: Laser light scattering. Equation 10: {  { Iθ = scattered intensity Iθ. It is called the excess ratio because it is for scattered light in excess of scattered light from the solvent.5.Figure 5. The symbol used to describe angle-dependent light scattering is Rθ. Light Scattering University Technology 2006)) One big advantage with light scattering experiments is that the solute can be measured in solution in a non-invasive manner. Rθ is defined in equation 10. solvent = scattered intensity of the solvent I0 = intensity of the beam V = volume of the scattering medium r = distance between the scattering volume and the detector 46 .

Equation 11:  H { {. The intensity carries information about the molar mass. 47 . This makes Rθ the most important measured quantity in light scattering (Wyatt Technology Corporation User Manual 2007). where n0 is the refractive index of the solution. $ IH $ $ { { Rθ = excess Rayleigh ratio (cm-1) K = optical constant (=4π2n02(dn/dc)2λ0-4NA-1).If we know Rθ at a number of different angles it leads directly to the weight average molar mass and mean square size of the solute molecules. λ0 is the radiation wavelength in vacuum expressed in nanometers. It can hence give information about both the particle size and the molar mass of the particle. z-average mean square radius (equation 13) and weight-average mean square radius (equation 14). Astra calculates an rms (root mean square) radius moments for each peak. NA is Avogadro´s number and dn/dc is the differential refractive index of the solvent-solute concentration c = concentration M = molar mass (g/mol) P(θ) = theoretically-derived form factor A2 = second virial coefficient (mol mL/g2) Astra is the software which processes the MALS data. The calculations given from the MALS detector software are based on equation 11. All summaries are taken over one peak (Wyatt Technology Corporation User Manual 2007). The different rms radius´s measured are numberaverage mean square radius (equation 12). while the angular dependency carries information about the size of the macromolecule.

All the samples were diluted 1:10 with the mobile phase prior to the measurements. The flow field-flow fractionation is coupled on-line with a Dawn EOS 18 angle light scattering.0 to 0. 48 .m spacer was applied and a main flow of 1. A cross flow gradient was applied. The liposomes are assumed to be hollow spheres in which each of the lipid molecules acts as an isotropic scattering element. the cross flow was reduced from 1.Equation 12: J$ 2 F Equation 13: J$ 2 { { { { Equation 14: J$ 2 { { ci = mass concentration Mi = molar mass < r2 > = mean square radius of the ith slice Experiment: AF4 experiments were performed using the Eclipse 2 instrument system from Wyatt Technology Europe. A 250. The angular dependence of the scattering is expressed by the so-called form factor or shape-factor. a single wavelength UV detector and an Optilab rEX differential refractive index detector (RI-detector). 10 l. P(θ).15 ml/min. The injection volume was the same in every experiment.0 ml/min.

For processing the received data the ASTRA (version) 5. as has been measured using X-ray diffraction (Characterization of vesicles by classical light scattering Van Zanten and Monbouquette 1991). It is also called the particle scattering function (Introduction to Light Scattering. shape and structure. 49 . Light Scattering University Technology 2006).7 nm for phosphatidylcholine vesicles.1.The form factor is assuming a shell thickness of 3.5 and Eclipse software from Wyatt Technology were used. The form factor is the mathematical relationship describing the angular variation of the scattered intensity as a function of particle size.

50 .

6
6.1

RESULTS AND DISCUSSION
Preliminary experiments

6.1.1 Osmolality measurements In order to expose the liposomes to osmotic stress it was necessary to prepare solutions of distinct osmolalities. In addition to calculating the amount of solute that is needed for a solution with a given osmolality it was decided to measure the osmolality of different solutions to check the calculated values and to check that sodium nitrate dissociates completely. We calculated how much glucose or sucrose is needed to make a solution with the same osmolality as a solution with a known sodium nitrate concentration, as describes in appendix 1. The calculated amounts are given in table 6.1.1.

Table 6.1.1: Amount of sucrose, glucose and sodium nitrate needed to make solutions of various given osmolalities

Amount solid (given in gram per liter) 3.5592 g glucose + 0.8499 g NaNO3 14.2363 g glucose + 0.8499 g NaNO3 0.5694 g sucrose + 0.8499 g NaNO3 2.2778 g sucrose + 0.8499 g NaNO3 0.8499 g NaNO3 4.2495 g NaNO3

Concentration of the solution Equivalent to 20 mM sodium nitrate Equivalent to 50 mM sodium nitrate Equivalent to 20 mM sodium nitrate Equivalent to 50 mM sodium nitrate 20 mM sodium nitrate 50 mM sodium nitrate

We prepared two different solutions, one with 20 mM sodium nitrate and one with 50 mM sodium nitrate. Corresponding solutions that contained 10 mM sodium nitrate and an amount of glucose or sucrose (equivalent to either 10 mM sodium nitrate or 40 mM sodium nitrate) that made the osmolality equal to the corresponding sodium nitrate solution were also prepared. Both corresponding solutions contained the same amount of salt because we wanted to have the same ionic strength in all the solutions.

51

The freezing points of all the solutions were measured using an osmometer, against a sodium chloride calibration solution of known osmolality. The results, expressed in mOsm/kg, are summarized in table 6.1.2.

Table 6.1.2: Results from the osmolality measurements

Solution 20 mM sodium nitrate 10 mM sodium nitrate and glucose equivalent to 10 mM sodium nitrate 10 mM sodium nitrate and sucrose equivalent of 10 mM sodium nitrate 50 mM sodium nitrate 10 mM sodium nitrate and glucose equivalent to 40 mM sodium nitrate 10 mM sodium nitrate and sucrose equivalent to 40 mM sodium nitrate

Measured value (average ± SD of three parallels) 40.0 mOsm/kg ± 0 mOsm/kg 42.0 mOsm/kg ± 0 mOsm/kg

39.0 mOsm/kg ± 1 mOsm/kg

95.3 mOsm/kg ± 0.58 mOsm/kg 101.0 mOsm/kg ± 1 mOsm/kg

98.0 mOsm/kg ± 0 mOsm/kg

The results show that the measured osmolalities of the salt solutions and corresponding combined salt and sugar solutions were within 5 % variability. The calculated values of sugar needed to prepare a solution with a distinct osmolality were thus confirmed. It was decided that when other solutions with different osmolality were to be made, it would be adequate only to calculate the values and not measure every solution with the osmometer.

6.1.2 Viscosity measurements It is also important to determine the exact viscosity of the various dispersion media used for PCS measurements. The reason why viscosity is so important can be described with the Stokes-Einstein equation expressed in equation 15:

52

Equation 15:

È

From D (the diffusion coefficient) in the Stokes-Einstein equation it is easy to calculate the particle radius as described in section 5.5.1. η in equation 15 is liquid viscosity and it is affecting the size calculation. If an incorrect viscosity value is entered in the PCS software the calculation will be wrong, resulting in an incorrect mean Stokes diameter.

Both media that contained sugar (glucose or sucrose) were measured. For the solution just containing sodium nitrate the viscosity was assumed to be the same as for water. In table 5.3.1 we can see that the solution with 10 mM sodium nitrate contained 0.8499 g/L or 0.8499 % sodium nitrate. The literature value for viscosity of an aqueous solution with 0.5 % sodium nitrate is 1.0016 cP (20°C), and for a solution with 1.0 % the viscosity is 1.0050 cP (20°C). If we compare those values to the viscosity of water 1.0020 cP (20°C) we can see that the amount of sodium nitrate in 10 mM sodium nitrate solution would not affect the viscosity markedly (CRC Handbook of Chemistry and Physics Lide 2008). The measurements were executed as described in section 5.5.3, with a glass capillary viscometer. The results are given in table 6.1.3. A calculation example is given in appendix 2.

Table 6.1.3: Results from the viscosity measurements

Solution 10 mM sodium nitrate and glucose equivalent to 90 mM sodium nitrate 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate

Measured viscosity 1.001 cP (mPa·s)

0.9898 cP (mPa·s)

53

but also on the difference in refractive indices of the particle and the surrounding solvent. in 10 mM sodium nitrate and glucose medium and in 10 mM sodium nitrate and sucrose medium.4. They were diluted in 10 mM sodium nitrate medium.6.4 Influence of viscosity and refractive index on the accuracy of PCS size measurements of latex bead standards Latex particles with a specified size of 102 ± 3 nm were used.1.4. Therefore it is of great significance to know the exact refractive index of the dilution medium when PCS measurements are executed.339 6. 54 .1.5. Latex bead standards were diluted in all the different media used to dilute liposomes for PCS measurements.1. Table 6. The refractive indices of media were measured with a RI (refractive index)-detector in the batch mode as described in section 5.3 Refractive index measurements The intensity of light scattered by a single.1.336 1.4: Results from refractive index measurements Solution 10 mM sodium nitrate 10 mM sodium nitrate and glucose equivalent to 90 mM sodium nitrate 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate Measured refractive index 1. The PCS results are given in table 6. Firstly the viscosity and refractive index values of water were used in the PCS software.5. isolated particle depends on its molecular weight and overall size and shape.333 1. The results are summarized in table 6.1.

5 nm ± 0. The PCS results are given in table 6.6 nm ± 4.Table 6.88 nm 107. The number of valid parallels used for calculations of mean vesicle sizes and standard deviations are given in table 9.7 nm ± 1.52 nm Number weighting 101. 55 .6.3 nm ± 1. In the next step the PCS results were recalculated size using the measured values for both refractive index and viscosity of the sugar solutions.15 in appendix 3 Sample Mean vesicle size ± SD (from PCS measurements) Intensity weighting Volume weighting 103.97 nm From the table above it is possible to see that the viscosity and refractive index values play a significant role in the size measurements executed by PCS.6 nm ± 0.41 nm 110.20 nm 117.1 nm ± 1. As we can see the mean diameter is around 15 nm larger than the defined diameter when sucrose is used as medium with the viscosity and refractive index values of water.6 nm ± 0.5: PCS mean diameters of latex beads calculated on the basis of viscosity and refractive index values of water.21 nm 113.86 nm 115.6 nm ± 1.1.9 nm ± 0.39 nm 116.97 nm Latex bead standards diluted with 10 mM sodium nitrate Latex bead standards diluted with 10 mM sodium nitrate and glucose equivalent to 90 mM sodium nitrate Latex bead standards diluted with 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate 105.13-9.1.

6: PCS mean diameters of latex beads calculated on the basis of measured viscosity and refractive index values. continuous filter extrusion. The number of valid parallels used for calculations of mean vesicle sizes and standard deviations are given in table 9.4.7 nm ± 3. 6.64 nm 102. It is a common practice to increase the proportion of unilamellar vesicles in preparations by subjecting MLVs to freeze-thaw cycles prior to extrusion (Liposome technology Mui and Hope 2006).6 nm ± 1.3.8 nm ± 1. The mean diameter of the latex particles is smaller as compared to the data based on the viscosity and refractive index value of water.Table 6.4.52 nm As we can see in table 6.1. Extrusions were carried out following the method described in section 5.6 the measured viscosity and refractive indices yield diameters closer to the value specified by the manufacturer.8 nm ± 0.6 nm ± 0. with subsequent extrusion of the raw MLV dispersion through polycarbonate filters.16-9.74 nm Number weighting 100. and is close to 102±3 nm as specified by the producer.1.2 nm ± 0.86 nm Latex bead standards diluted with 10 mM sodium nitrate and glucose equivalent to 90 mM sodium nitrate Latex bead standards diluted with 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate 106. 56 . Experiments were executed to investigate how different freezing methods affected the size distributions of liposomes.34 nm 103.1.17 in appendix 3 Sample Mean vesicle size ± SD (from PCS measurements) Intensity weighting Volume weighting 103.40 nm 104.5 Freeze-thaw experiments Liposomes were prepared by the hand-shaken method according to section 5.1.

the liposomes were broken and formed much bigger aggregates.4 nm ± 0.4. four preliminary freeze-thaw experiments were executed as described in section 5.67 nm 57 . Liposome dispersion frozen in -80°C freezer before extrusion 2.6 nm ± 0.1.5 nm ± 0.1 nm ± 1.9 nm ± 0. Liposome dispersion frozen in -80°C freezer after extrusion Experiment description 116.3 nm ± 1.5 nm ± 1.81 nm 1.17 nm 23.5 nm ± 0. Liposome dispersion frozen in -80°C freezer during extrusion 32. It was not possible to measure the liposomes by PCS.7 nm ± 0.62 nm 23. Liposome dispersion frozen in LN2 before extrusion 3. The number of valid parallels used for calculations of mean vesicle sizes.1 nm ± 1.28 nm 91.70 nm Not possible.23 in appendix 3 Experiment description Mean vesicle size ± standard deviation Intensity weighting Volume weighting 116.9 nm ± 2. Liposome dispersion frozen in LN2 before extrusion 3.5 nm ± 0. In total.17 nm 114. All the different liposome preparations were thawed on a 50°C water bath for approximately 20 minutes.62 nm 1.4 nm ± 1. In addition we investigated whether it would make a difference if the freeze-thawing cycles were done before.81 nm 31.3 nm ± 2.45 nm 86. The results from the PCS measurements are given in table 6.-80°C freezer and liquid nitrogen were used for freezing the liposome dispersions.35 nm 112.1.38 nm 29. Table 6.7 nm ± 0.1 nm ± 2.7: PCS mean diameter ± standard deviation and distribution width ± standard deviation from freeze-thawing experiments.17 nm 31.7.38 nm 111.2.25 nm 114. Distribution width ± standard deviation Intensity weighting Volume weighting 32. Liposome dispersion frozen in -80°C freezer before extrusion 2.18-9.40 nm Number weighting 89. Liposome dispersion frozen in -80°C freezer during extrusion 4.1 nm ± 1. until the liposome dispersion was completely thawed.83 nm Number weighting 24.1 nm ± 0. after or during the extrusion process. distribution widths and standard deviations are given in table 9.62 nm 29.

This doubled the loss of dispersion and the extruder had to be cleaned twice.1. there is no major difference in the measured mean sizes for the first three preparation methods. It was decided to perform a two sample t-test to see if there was a significant difference between the different freeze-thawing methods. 58 . or they can alter their shape and hence pass through the filter even though the diameter is bigger than 100 nm.7 show. It seems that it does not make a big difference whether the freeze-thawing cycles are done before extrusion or during extrusion. It was discovered that there was a significant difference between the liposome dispersion frozen in a -80° freezer before extrusion and the liposome dispersion frozen in a -80° freezer during extrusion. This can be explained by the flexibility of the liposomal membranes. Again only small differences could be seen when looking at the standard deviations of the different methods. There is not a large size difference from the liposomes frozen in liquid nitrogen to the liposomes frozen in -80°C freezer. There is a minor difference. All liposomes show a slightly larger mean diameter than the pore size of the filter used for the extrusions. Liposomes that are substantially bigger then 100 nm will either break and form smaller liposomes that are able to pass through the polycarbonate filter. All the t-tests were executed with a significance level of 0.As the results in table 6. The third setup/experiment was rather impractical because the extrusion process had to be stopped.05. When the other liposomes dispersions were compared it was not discovered a statistical significant difference. For being the most time saving method the first procedure was chosen for further preparations. but it is too small to say whether one method should be preferred above another. because ideally the liposomes should be more homogenous in size after freeze-thawing cycles and then the standard deviation would decrease. Lipoid E-80 forms very flexible liposomes that are assumed to alter their shapes while squeezing through the filter pores (Liposome technology Gregoriadis 2006). It might help to look at the distribution width (expressed as standard deviation).

2.1. Every liposome dispersion was made as a 10 % lipid dispersion. We could have used more sodium nitrate to adjust the osmolality.6. produced by the hand-shaken method.4. The liposome dispersion was freeze-thawed as described in section 6.5 before extrusion.3.4.1 Hypertonic osmotic stress experiment with 10 mM NaNO3 and glucose equivalent to 90 mM NaNO3 as dilution medium The first experiment was executed with liposomes prepared in 10 mM sodium nitrate medium.2 Influence of osmotic stress on liposome size. sodium nitrate and glucose solution or sodium nitrate and sucrose solution. 24 hours and 48 hours after dilution.4.2. but it would also influence the ionic strength and thus potentially the retention behavior in the flow field-flow fractionation experiments (A4F/MALS-Analysis of Liposomes Influence of Key Factors on Fractionation Behavior and Evaluation of MALS Fit Routines Ausbacher 2007). The liposome size was measured by PCS 1 hour. and the mean diameter was measured by PCS right after preparation. described in section 5. to see if the osmotic stress affected their size. 59 . The results from the PCS measurements are given in table 6.1. 6. The liposome dispersion was diluted 1:10 with 10 mM sodium nitrate and glucose equivalent to 90 mM sodium nitrate (a hyperosmolal medium). The dispersion media varied between sodium nitrate solution.2. measured by PCS All the liposome dispersions made throughout this project were prepared as the liposomes in experiment 1 in section 5. as described in section 5.1 with subsequent filter extrusion.

36 nm 78.44 nm 96.8 nm ± 1.11 nm 113.3 nm ± 2.73 nm 120. Volume weighting reflects the relative particle diameter vs. 60 .1: The liposomes mean diameters during osmotic stress experiments (hyperosmolal dilution) with glucose.1-9. and they are also calculated assuming that the particles are spheres. the value of the volume-weighted particle size distribution assumes that the particles are spheres of uniform density. Volume weighting and mass weighting are equal terms.7 nm ± 0.60 nm Obviously the mean sizes after dilution with hyperosmotic medium changed. almost 27 nm with volume weighting and over 10 nm with number weighting.77 nm 110. Number weighting displays the relative number of particles in a sample run vs. Intensity weighting reflects the relative intensity of scattered light vs.8 nm ± 0.8 nm ± 4.7 nm ± 1.93 nm 93.4 in appendix 3 Experiment description Mean vesicle size ± SD (from PCS measurements) Intensity weighting Volume weighting Number weighting Liposome dispersion prepared with 10 mM sodium nitrate Liposome dispersion 1 hour after dilution with 10 mM sodium nitrate and glucose equivalent to 90 mM sodium nitrate Liposome dispersion 24 hours after dilution with 10 mM sodium nitrate and glucose equivalent to 90 mM sodium nitrate Liposome dispersion 48 hours after dilution with 10 mM sodium nitrate and glucose equivalent to 90 mM sodium nitrate 119.5 nm ± 2.4 nm ± 0. diameter for a sample run.4 nm ± 3.96 nm 77.2.9 nm ± 0. diameter. diameter.91 nm 86.7 nm ± 0.Table 6. 8 nm ± 0.32 nm 113.02 nm 76. The mean diameter has reduced by almost 6 nm with intensity weighting.35 nm 95. Valid parallels used for calculations of mean vesicle sizes and standard deviations are given in table 9.

As we could expect it does look like the liposomes have shrunk. half-life (t1/2) = 1 hour. there is a small difference and it could well be that the glucose starts to penetrate the membrane and hence reverse the shrinking to some extent. Cullis 1993) glucose will penetrate the liposome membrane. their membrane contained cholesterol which will make the membrane stiffer and less permeable. Since Lipoid E-80 forms a less stiff membrane one could assume that glucose will penetrate the membrane even faster. Since we were not sure how fast glucose was able to penetrate through our liposome membrane it was decided to also perform the next experiment with sucrose. The mean size does not change much from 1 hour to 24 hours. If glucose to some extent penetrates the membrane the osmotic stress would not be as large as it was supposed to be. Since we did not know if that could occur in our experiment it was decided to perform a similar experiment with sucrose instead of glucose.The value of the mean diameter can vary significantly with the choice of weighting. water will diffuse out of the liposomes. irrespective of weighting a clear change of size can be seen when the liposomes were diluted with the hyperosmolal solution. When the osmotic pressure inside the liposomes is smaller than the osmotic pressure outside.16 g/mol while the sucrose has an Mw of 342. Dynamic light scattering theory User Manual 1997). This may explain why the measured size difference is so deviating when looking at the different weightings. .30 g/mol. The biggest mean size difference can be seen 1 hour after dilution. at 45°C. 61 . According to (Osmotic properties of large unilamellar vesicles prepared by extrusion Mui. One big particle will hence affect the mean size distribution in volume weighting more than number weighting. But. (Osmotic properties of large unilamellar vesicles prepared by extrusion Mui. in order to equal the concentration inside and outside the liposomes. depending on the width of the gaussian-like distribution (Windows based software. It was reported that glucose is slowly membrane permeable. Sucrose is a bigger molecule (almost twice as big as glucose) and should thus penetrate the liposome membrane to an even lower extent than glucose. The molecular weight (Mw) of glucose is 180. Cullis 1993) used another liposome membrane.

6. It is the same experiment as the one described in section 6.2. The size was measured by PCS right after preparation.2. 24 hours and 48 hours after dilution.2. and the liposome dispersion was diluted 1:10 with 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate (a hyperosmolal medium). Results from the PCS measurements are given in table 6. This was done to see if a size difference from the original undiluted sample could be detected.1. with 10 mM sodium nitrate as dispersion medium. the only difference is that glucose is replaced by sucrose. and how fast it happened. The liposome size was measured by PCS 1 hour.2.2 Hypertonic osmotic stress experiment with 10 mM NaNO3 and sucrose equivalent to 90 mM NaNO3 as dilution medium Liposomes again were prepared according to the hand-shaken method as described before. 62 .

71 nm 73.5 nm ± 0.3 nm ± 0.1 nm ± 2.5 nm ± 3. Valid parallels used for calculations of mean vesicle sizes ± SD are given in table 9.8 nm ± 2.97 nm 116.10 nm 74.6 nm ± 0.46 nm 97.54 nm Again a difference in the sizes before and after dilution with the sucrose medium is seen.Table 6.29 nm 93.4 nm ± 3. a somewhat bigger size difference can be seen here. The size difference in this experiment is almost 17 nm (number weighting) and almost 25 nm (volume weighting).73 nm 116.3 nm ± 0.2.34 nm Liposome dispersion prepared with 10 mM sodium nitrate Liposome dispersion 1 hour after dilution with 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate Liposome dispersion 24 hours after dilution with 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate Liposome dispersion 48 hours after dilution with 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate 118.7 nm ± 1.67 nm Number weighting 90.8 in appendix 3 Experiment description Mean vesicle size ± SD (from PCS measurements) Intensity weighting Volume weighting 118.8 nm ± 4.2: Mean diameters during osmotic stress experiment (hyperosmolal dilution) with sucrose.29 nm 95.04 nm 77.5-9.61 nm 114. If we compare these results with the results when glucose was used.0 nm ± 3. 63 .6 nm ± 0.

The liposomes were then diluted with 10 mM sodium nitrate (hypoosmolal medium). The exact viscosity and refractive index of the sucrose medium was already determined.25 nm 94.4 nm ± 0.3 Hypotonic osmotic stress experiment with 10 mM NaNO3 as dilution medium After it could be seen that is was possible to discover a size difference when liposomes were diluted with a hyperosmolal solution.49 nm 118.28 nm From the table above we can see that the mean size of the liposomes increases after they are diluted in a hypoosmolal medium at least when looking at volume weighting and number weighting.86 nm Liposome dispersion prepared with 10 mM NaNO3 and sucrose equivalent to 90 mM NaNO3 Liposome dispersion 1 hour after dilution with 10 mM NaNO3 Liposome dispersion 24 hours after dilution with 10 mM NaNO3 Liposome dispersion 48 hours after dilution with 10 mM NaNO3 121. 64 . Table 6.26 nm 118. 24 hours and 48 hours.2.36 nm 118.3: Liposome sizes during osmotic stress experiment (hypoosmolal dilution) with sucrose.8 nm ± 2. The results from the PCS measurements are given in table 6. The intensity weighting shows only little change and it looks like the liposomes have shrunk because the mean diameter is smaller after the dilution.40 nm 96. Valid parallels used for calculations of mean vesicle sizes ± SD are given in table 9.1 nm ± 4.12 in appendix 3 Experiment description Mean vesicle size ± SD (from PCS measurements) Intensity weighting Volume weighting 103.34 nm 118.5 nm ± 0.3.1 nm ± 2.2.6.4 nm ± 0.36 nm 118.4 nm ± 0.20 nm Number weighting 85. And the sizes were measured by PCS after one hour.6 nm ± 0.37 nm 95.1 nm ± 2. so these values were used when the size was measured by PCS.5 nm ± 0.2.35 nm 118.3 nm ± 0.4 nm ± 3. the liposome dispersion was prepared with 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate.9-9.

But the change in diameter is only 3.3 Influence of osmotic stress on liposome size.2. We had already learned. The mobile phase in the AF4 experiments was always the same medium as the one used as dilution medium. When we look at the size changes with volume weighting we can see a much bigger effect. As one could predict. from the PCS measurements. 65 . measured by AF4 The liposomes used for these following experiments were prepared in the same manner as those described in section 6. than outside of the liposome and as a consequence water will diffuse from the surrounding liquid into the liposome. the mean diameter increases with almost 15 nm. that the change of particle size by osmotic pressure occurs within the first hour. Every sample was analyzed by AF4 one hour after dilution.3.1. Two different liposome batches were prepared. Again was chosen to adjust different osmotic pressures by adding sucrose. After the previous experiments with both 10 mM sodium nitrate and glucose solution and 10 mM sodium nitrate and sucrose solution as dilution agent it was decided to only use the sucrose solution in the following experiments.2 nm and it is not considered as a change due to the osmotic stress the liposomes have been exposed to. as shown in table 6. Both of the two batches were then diluted 1:10 with either the same medium as the one used for preparing the liposome or the other medium giving four different experiment.1. one with 10 mM sodium nitrate and one with 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate as dispersion medium. because glucose might penetrate the membrane as discussed earlier is section 6. 6. Increasing the sodium nitrate concentration is at the same time influencing the ionic strength which however could influence retention time to an unknown degree (A4F/MALS-Analysis of Liposomes Influence of Key Factors on Fractionation Behavior and Evaluation of MALS Fit Routines Ausbacher 2007). The concentration is bigger inside.2. it seems that the liposomes have swollen when they were diluted with the hypoosmolal medium.

1: The four different liposome preparations used for analysis by the flow field-flow fractionation Liposome sample 100 nm liposomes prepared in 10 mM sodium nitrate 100 nm liposomes prepared in 10 mm sodium nitrate Dilution medium and mobile phase 10 mM sodium nitrate 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate 100 nm liposomes prepared in 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate 100 nm liposomes prepared in 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate 10 mM sodium nitrate 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate It was decided to compare the liposome samples prepared in the same medium. a volume weighted mean diameter of 103. If we look at table 6.3. we can see the mean size distribution of the liposomes prepared in 10 mM sodium nitrate and we can compare them to table 6.2.6 nm and a number weighted mean diameter of 90. 66 .3 where we can see that the mean size distribution of the liposomes prepared in 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate.2. The liposomes prepared in 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate have an intensity weighted mean diameter of 121.8 nm and a number weighted mean diameter of 85.6 nm.5 nm. We can see that the mean diameter of the liposomes is not the equal when the liposomes are prepared in the two different media. because they were from the same batch and hence we knew that they had the same size before dilution and that they generally had the same physical properties.Table 6. The liposomes prepared in 10 mM sodium nitrate have an intensity weighted mean diameter of 118. We could not know if the liposomes made in 10 mM sodium nitrate and the liposomes made in 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate had the same size after preparation.4 nm. but it is unknown how parameters like different viscosity of the dispersion medium were influencing the size distribution and the properties of the liposomes.2.3 nm. The extrusion of the two different batches was performed in the same manner. a volume weighted mean diameter of 118.

8 relative scale UV/VIS absorbance 0.0 15.3.0 25.0 40.6 0. shown in figure 6.2 0. 6. the UV/VIS-absorbance and the Rayleigh ratio were recorded over time. 1.0 20.0 time (min) 30.) Each of the samples was run in five parallels and both.1: UV/VIS absorbance vs.3. UV/VIS-detection is common in flow field-flow fractionation.3. (The first two experiments listed in table 6.3.1 presents the absorbance vs. time for the two liposomes samples prepared in 10 mM sodium nitrate. diluted in either 10 mM sodium nitrate or 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate (five parallels each) 67 .1. time.0 Figure 6.And it is unknown if the liposomes prepared in different media would react differently when they are diluted in a hypo-/hyperosmolal solution.0 35.1 Hyperosmotic osmotic stress experiment with 10 mM NaNO3 and sucrose equivalent to 90 mM NaNO3 as dilution medium First we looked at the two samples prepared in 10 mM sodium nitrate.0 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate as dilution medium and mobile phase 10 mM sodium nitrate as dilution medium and mobile phase 0.4 0. diluted in either 10 mM sodium nitrate or 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate. The first plot.

0 25.5. Figure 6.2 0.5. time.The Rayleigh ratio is a quantity used to characterize the scattered intensity as a function of scattering angle and is defined in section 5.4 0.0 Figure 6. 1. smaller particles will elute earlier from the channel as described in section 5.0 40.3.6 0.3. This indicates that the liposomes prepared in 10 mM sodium nitrate solution and diluted in 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate (hypertonic) solution elutes before the liposomes prepared and diluted in 10 mM sodium nitrate solution. diluted in either 10 mM sodium nitrate or 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate (five parallels each) As we can see are the two plots very similar. whereas the two samples behave markedly different: The curves of the liposome dispersion diluted in 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate appear shifted left in both plots.0 20.5. time for the two liposome samples prepared in 10 mM sodium nitrate.0 35.8 relative scale 0.0 15.0 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate as dilution medium and mobile phase 10 mM sodium nitrate as dilution medium and mobile phase 0.2 show a plot of Rayleigh ratio vs. According to the AF4 theory. The parallels for the same sample are very close to each other.0 time (min) 30.5.2: Plot of Rayleigh ratio vs. 68 .

3. the liposome sample diluted in 10 mM sodium nitrate and the liposome samples diluted in 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate. We can assume thus that the liposomes have become smaller when they were blended with the hypertonic medium since they eluted earlier.3. The lower part of figure 6.2. 69 . but here the area between 20 and 25 min is enlarged to show the difference between the two samples clearer. Next.3 shows the same plot as the one above. We can see in figure 6.3 shows the same samples as figure 6.3. Since we used a sphere model for the sample is the radius presented as geometric radius (Wyatt Technology Corporation User Manual 2007). elution type.3 that both the samples show very similar sizes over time.When we are comparing these two samples we can see that the sample diluted in the hypertonic medium elutes before the sample diluted in the isotonic medium. geometric radius vs.3.3 shows the resulting size. Figure 6.3. Here it is possible to see that there is a slight shift to smaller sizes for a given elution time for the liposomes in hypertonic medium. The geometric radii reach from approximately 25 nm to 70 nm for both. Figure 6.1 and 6.3. from the angular dependence of the light scattering signal the geometric radii were calculated for all slices over the whole elution.

3.3: Geometric radius of the two liposome samples prepared in 10 mM sodium nitrate.0 g eom etric radiu s (nm ) 40.0 25.0 time (min) 23.0 10 mM sodium nitrate as dilution medium and mobile phase 42.0 20.0 22.0 25.0 44.80.0 38.0 time (min) 30.0 60.0 15.0 20.0 24.0 Figure 6. diluted in either 10 mM sodium nitrate or 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate (five parallels of each).0 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate as dilution medium and mobile phase 36.0 20.0 0.0 21.0 35.0 geo m etric rad ius (nm ) 40. The lower picture shows an enlarged section of the upper plot 70 .

2 is due to a difference in the liposome size after the sample has been diluted in a hypertonic medium. geometric radius. diluted in either 10 mM sodium nitrate or 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate Finally a plot was constructed where the measured UV absorbance was plotted over the calculated geometric radii.6.01 mM sodium nitrate as of 90 dilution medium and mobile phase 0.003 0.001 0 20 30 40 50 60 10 mM sodium nitrate as dilution medium and mobile phase 70 80 Geometric radius (nm) Figure 6.006 0. we can make a plot of absorbance vs. In figure 6.007 Absorbance (AU) 0. For example.10 mM sodium nitrate and sucrose equivalent0.4.008 0. as shown in figure 6.3.3.1 and figure 6. The size distribution of the liposomes in the hypertonic medium appears shifted towards smaller sizes as compared to liposomes in isotonic medium.004 0.002 0. figure 9.4 only one parallel of each sample is shown. 71 . This confirms that the shift in elution time seen before in figure 6. a parametric plot.5 and figure 9.005 0. This is done because it gives a more perspicuous plot.3. A parametric plot is a plot that generates a new data set for two different types of x-y data that share the same x-axis (Wyatt Technology Corporation User Manual 2007). The five parallels of each sample are shown in appendix 3. The size distributions obtained this way are clearly different from each other.009 0.3.3.4: Parametric plot of the two liposome samples prepared in 10 mM sodium nitrate.

72 .1 nm ± 0.26 in appendix 3 Sample Liposomes prepared in 10 mM sodium nitrate.08 nm 59.2: Mean radii and standard deviation of the liposome samples prepared in 10 mM sodium nitrate.2. diluted in 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate Liposomes prepared in 10 mM sodium nitrate. measured by PCS. The parallel shift of the whole curve to the left in figure 6.In the parametric plot we can see that all the liposomes from the lower end of the distribution (approximately 30 nm radius) to the upper end of the distribution (approximately 75 nm radius) have been equally affected by the osmotic stress they have been exposed to. from table 9.0 nm ± 0.figure 6. This corresponds with the result as we have seen from the plots in figure 6.26 nm Rz ± SD 55. diluted in either 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate or 10 mM sodium nitrate.8 nm ± 0.3.3.8 nm ± 0.3. Here we can see that the number-average mean square radius decrease after hypertonic dilution by 6. of the same two liposome samples as the liposome samples in table 6.5 nm ± 0. The liposomes diluted in the hypertonic medium are in mean smaller than the liposomes diluted in the isotonic medium.3.3.2 shows the mean diameter.8 nm ± 0.49 nm Rw ± SD 51.3 nm.4 indicates that all the different sized liposomes are the sample is almost equally affected by the osmotic stress.2 shows the mean radius of two liposome samples prepared in 10 mM sodium nitrate as calculated from AF4 analysis. Table 6.3 nm. Table 6.32 nm Table 6.3.2 nm and the z-average mean square radius decrease by 4.23 nm 53. diluted in 10 mM sodium nitrate Rn ± SD 46.4.2. the weightaverage mean square radius decrease by 5.35 nm 57.1.

the last two experiments in table 6.The same hyperosmolal experiment are performed in both cases.2 Hypotonic osmotic stress experiment with 10 mM NaNO3 as dilution medium Secondly we looked at the liposome samples that were prepared in 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate. 6. In figure 6.2 0.0 35.2 we saw that the liposomes shrunk when they were diluted in the hyperosmolal medium.0 20. the only difference is that the liposome sizes are measured with two different techniques.0 15.1 and 6.0 Figure 6. the liposome shrink and the size decrease when it is diluted in a hypertonic medium.0 time (min) 30. This is the exact opposite experiment where the liposomes have a greater concentration inside than outside.1.4 0.0 40.0 25. diluted in either 10 mM sodium nitrate or 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate (five parallels each) 73 . diluted in either the same medium or in 10 mM sodium nitrate (hypotonic solution).3. As we can see from the results.8 UV/VIS absorbance relative scale 0.3. 1. If they became larger as we predicted they would elute last.5: UV/VIS plot of absorbance vs.6 0.3.3.0 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate as dilution medium and mobile phase 10 mM sodium nitrate as dilution medium and mobile phase 0. Here we would expect that the liposomes diluted in the hypoosmolal medium would expand and become larger. the same principles are constituted.3. time for the two liposomes samples prepared in 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate.

time for the two liposome samples prepared in 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate. As in figure 6.4 0.8 relative scale 0.5 the absorbance is plotted against elution time.6 Rayleigh ratio is plotted against elution time.0 15.0 Figure 6. 74 .3.3.0 time (min) 30.0 35.3.3.0 25.Both the plots in figure 6. In figure 6.3.2 0.3.0 40.0 20.6 shows the same liposome samples which were prepared in 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate.5 both of the liposome samples elute at almost the same time. It looks like both the liposome samples elute roughly at the same time.6 0. 1.6: Plot of Rayleigh ratio vs.5 and figure 6. diluted in either 10 mM sodium nitrate or 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate (five parallels each) In figure 6. We can see it is no major difference in the two graphs.0 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate as dilution medium and mobile phase 10 mM sodium nitrate as dilution medium and mobile phase 0.

0 36.3.0 38. diluted in either 10 mM sodium nitrate or 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate (five parallels of each).0 22.0 20.0 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate as dilution medium and mobile phase 21.0 geo m etric radius (nm ) 40.0 20.0 25.0 15.0 60.0 24.7: Geometric radius of the two liposome samples prepared in 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate.0 0.0 geo m etric radius (nm ) 40.0 44.0 25.0 10 mM sodium nitrate as dilution medium and mobile phase 42.0 time (min) 30. The lower picture shows an enlarged section of the upper plot 75 .80.0 35.0 Figure 6.0 time (min) 23.0 20.

Furthermore the size-over-time lines are not fully parallel.008 mobile phase 0. Absorbance (AU) 10 mM 0. When the concentration is higher inside of the liposomes than outside of the liposome.3.003 0.3. but here the area between 20 and 25 min is enlarged to show the difference between the two samples clearer.007 0.7 shows a plot of sizes over time of the samples prepared in 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate.7 shows the same plot as the upper plot.009 of 90 mM sodium nitrate as dilution medium and 0.002 0.01 nitrate sodium and sucrose equivalent 0. The geometric radius of the liposomes diluted in the hypotonic medium ranges from approximately 20 nm to 65 nm. The liposomes diluted in the isotonic medium ranges from approximately 20 nm to a little less than 60 nm.5 and 6.001 0 20 30 40 50 60 10 mM sodium nitrate as dilution medium and mobile phase 70 80 Geometric radius (nm) Figure 6. It shows the same two samples as figure 6.3.6.Figure 6.3.3. The liposomes that were diluted in a hypotonic medium appear to have become larger.006 0. As a consequence the liposome will swell and become bigger.005 0. water will diffuse through the membrane into the core of the liposomes to equal the osmotic pressure. When we compare these two samples it is possible to see a clear difference in sizes. The lower part of figure 6. diluted in either 10 mM sodium nitrate or 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate 76 .004 0.8: Parametric plot of the two liposome samples prepared in 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate.

As we have seen before there is a difference in the liposome size after the sample has been diluted in a hypotonic medium. inner curve is the liposomes diluted in the isotonic medium.3.8.3. In figure 6. these liposomes is shown on the left side of the curve.10 the phenomenon is illustrated which we think might explain the size difference we saw in the parametric plot. but all the five parallels are shown in appendix 3. there we could clearly see that the liposomes diluted in the hypotonic medium were bigger than the liposomes diluted in the isotonic medium. The liposomes that are smaller then 100 nm will pass through the filter without problem. There will always be some liposomes that are smaller then the rest when they are prepared by extrusion. This is the same information we received from the geometric radius plots.8 shows the liposomes that have been diluted in the hypoosmolal medium.9 and figure 6. Only one parallel of each sample is shown in figure 6. And it is agreeing with the theory. A conceivable explanation can be that the smaller liposomes have a different shape than the bigger liposomes and hence can change less in size. This information is also shown in figure 6. The difference between the two samples was in the maximum size. where the liposomes diluted in the hypotonic solution has almost the same minimum diameter as the liposomes diluted in the isotonic solution. On that side it is not possible to see a size difference between the two liposome samples.3. But the liposomes that are larger than 100 nm need to either break and form smaller spheres or change their conformation when they are squeezing through the pores on the filter. and they will maintain their original conformation.3.3.8 we can see that not all the liposomes have been affected to the same extend when they are diluted in the hypotonic medium.3. We could see from the plot that both of the liposome samples had geometric radius reaching from approximately 20 nm. figure 9.7 and figure 9. In the parametric plot in figure 6. The outer curve in figure 6. The two curves are crossing when the geometric radius is approximately 25 nm.8. 77 . On the right side of the curve we can see that the liposomes that have been diluted in the hypoosmolal solution have become bigger.8.

then it is diluted in either hypo.    Figure 6.or hypertonic medium 78    .9: A liposome bigger than 100 nm squeezing itself through a 100 nm filter by altering its conformation.3.

10: A liposome smaller than 100 nm passing through a 100 nm filter. then it is diluted in either hypo.or hypertonic medium 79    .    Figure 6.3.

Table 6. The number-average mean square radius increase with 1.Table 6. The liposomes diluted in 10 mM sodium nitrate (hypotonic medium) are slightly bigger than the liposomes diluted in the isotonic medium. the liposome swell and the size increase after the liposome has been diluted in the hypoosmolal medium.1 nm ± 0. 80 .3. First was the liposomes measured by PCS and then by AF4.3. diluted in either 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate or 10 mM sodium nitrate.3. from table 9.47 nm Rz 50.1 nm ± 0. the only difference is that the sizes are measured with different techniques. Both methods show the same principle.2.8 nm ± 0.1 nm ± 0.15 nm Rw 48.18 nm Table 6.3 shows the mean radius of the liposomes prepared in 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate.3 shows the mean diameter of the same liposomes as the liposomes shown in table 6.1 nm ± 0.09 nm 50.7 nm. diluted in 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate Liposomes prepared in 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate.9 nm ± 0.2 nm.3: Mean radius of the liposome samples prepared in 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate.10 nm 53.3.70 nm 45. diluted in 10 mM sodium nitrate Rn 44. the weight-average mean square radius increase with 2 nm and the z-average mean square diameter increase with 2.26 in appendix 3 Sample Liposomes prepared in 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate.

osmotic stress was found to affect liposomes of different sizes in a different manner.7 CONCLUSION In conclusion. liposomes that were smaller than the pore size of the filter used for extrusion were found to shrink in hyperosmotic medium but stay quite constant in size in hypo-osmotic medium. liposomes that were larger than the pore size of the filter were found to shrink in hyperosmotic medium and swell in hypo-osmotic medium. with constant ionic strength. thus aqueous media with the same osmotic pressure as the liposome dispersion should be used as dilution medium/mobile phase when liposomes are fractionated by AF4. Sugar should be added to adjust the osmotic pressure of the mobile phase to avoid osmotic stress artifacts and to avoid changes in ionic strength. But. A hypothesis is presented to explain this behavior. affects both elution time and calculated size of liposomes. 81 . Ideally. this project has demonstrated that a change in osmotic pressure. It was found that the retention behavior and calculated geometric radius of liposomes obtained by flow field-flow fractionation in combination with multi-angle light scattering is affected by the osmotic pressure of the medium used for diluting the liposomes and/or running the AF4. In contrast. which could alter the elution behavior. This gives some limitations with the AF4 method.

82 .

Institute of Pharmacy. Standish MM. Size analysis of submicron particles and liposomes by size exclusion chromatography and photon correlation spectroscopy. 2001. Ingebrigtsen L. Liposomes: from physics to applications.wyatt. Ausbacher DA. 2006. 2001. 83 . 1967. 2007. 1993.13(1):238-52. Biotechnol Annu Rev. 1965.8 REFERENCES Asymmetric Flow Field-Flow Fractionation. Available from: http://www. Amsterdam: Elsevier. 3rd ed. Diffusion of univalent ions across the lamellae of swollen phospholipids. and PCS analysis and enzymatic assay of lipid content. How Asymmetric Field Flow Fractionation (AFFF) Theory Works. Liposomes as drug carriers: a technological approach.com/content/english/theory/theory_fff. Bangham AD.com/theory/fieldflowfractionation/. New York: Informa Healthcare. Ingebrigtsen L. Liposome technology. 2008 [updated 2008]. Gregoriadis G.3(2). Brandl M. 2002. Watkins JC. Tromsø: University of Tromsø. Determination of the size distribution of liposomes by SEC fractionation.1:225.7:59-85. Available from: http://www.postnova. Lasic D. 2008 [updated 2008]. Chemistry and physics of lipids. Osmotic properties and water permeability of phospholipid liquid crystals. A4F/MALS-Analysis of Liposomes Influence of Key Factors on Fractionation Behavior and Evaluation of MALS Fit Routines. Brandl M. Bangham AD. AAPS PharmSciTech.htm. Journal of molecular biology.

Massing U.html.687(2):249. 2000. 2008. Park I. Taylor and Francis Group.wikipedia.813(1):91. Tonicity. Available from: http://en. Journal of chromatography. Journal of chromatography. Liposomes: a practical approach. Moon MH. In: Gregoriadis G. 2003.hbcpnetbase. Evans EA. Technical Summary .org/wiki/Refractive_index. Nickels JCH. 84 . 2008 [updated 2008]. 2nd ed. Roessner D. editor. Available from: http://www. Changes in Liposome Morphology Induced by Actin Polymerization in Submicrometer Liposomes. Liposome technology. 1993. 2003. Oxford: Oxford University Press. Fuxius S. 2006. Liposomal formulations of anticancer drugs: selectivity and effectiveness. Weissig V. LLC. Available from: http://www.wikipedia.19(25):10581. CRC Handbook of Chemistry and Physics.3(3):171-7. Torchilin VP. 3rd ed. Cullis PR. Available from: http://en. Refractive index. editors. Langmuir. Kulicke W-M. Hope MJ.com/company. Osmotic properties of large unilamellar vesicles prepared by extrusion. 1994. Drug resistance updates. Kim Y. 2008 [updated 2008]. 2008 [updated 2008].64(2):443-53. Madden TD. Biophysical Journal. New York: Informa Healthcare. On-line coupling of flow field-flow fractionation and multi-angle laser light scattering. p.org/wiki/Tonicity#Isotonicity. Mui BL.com/. Mui BL. 324.Lide DR. 1998.An Introduction to Lipid Nanoparticles.encapsula. Size characterization of liposomes by flow field-flow fractionation and photon correlation spectroscopy Effect of ionic strength and pH of carrier solutions.

146(2):330. User Manual Nicomp Model 380. 1991. Light Scattering University. Available from: http://en. Particle size analysis in pharmaceutics and other industries: theory and practice. New York: Ellis Horwood. Characterization of vesicles by classical light scattering. Dynamic light scattering theory.User Manual ASTRA V User´s Guide. 1992. Viscosity. 2008 [updated 2008]. Washington C. Wyatt Technology Introduction to Light Scattering.wikipedia. 2006.org/wiki/Viscosity. Wyatt Technology Corporation. 2007. Van Zanten JH. Monbouquette HG. 1997. 300 p. Journal of colloid and interface science. Windows based software. 85 .

86 .

01 mol/l = 0.569 g The amount needed from glucose: 5.694 g – 0.499 g/l Amount after correction with the E-value (NaNO3): 8.125 g Amount glucose needed (when corrected with the E-value of glucose): 0.694 g Amount NaNO3 in 10 mM solution: 84.99 g/mol × 0.16 × X = 5.67 = 0.67 E-value glucose = 0.569 g = 5.67 = 5.499 g × 0.8499 g/l Amount after correction with the E-value (NaNO3): 0.16 If we should make a solution with 10 mM NaNO3 and glucose equivalent to 90 mM NaNO3 Amount NaNO3 in a 100 mM solution: 84.125 g 32.8499 g × 0.99 g/mol × 0.9 APPENDICES Appendix 1 Calculation of osmolality in solutions Example: E-value NaNO3 = 0.031 g 87 .1 mol/l = 8.

9923 mm2/s Dynamic viscosity (η): 0 ρ = density Example: η = 0.003 mm2/s2 × (331.9923 mm2/s × 1.61 s) u = 0.Appendix 2 Calculation of dynamic viscosity of solutions Kinematic viscosity (u): H 0{ .0014 mPa × s 88 . { ϑ = Hagenbach correction for average flow time (s) (from manual) K = constant (mm2/s2) (from manual) t = time (seconds) Example: u = 0.39 s – 0.0092 g/cm3 η = 1.

00 % 0.35 0.0 Sec 13. Table 9.47 0.2 nm 119.1: PCS measurements of 100 nm liposomes.2 nm 86.2 nm 119.00 % 0.9 nm Volume weighting 119.0 Sec 13.8 nm 121.00 % 89 .6 nm Channel width 13.0 nm 119.08 % 0. and baseline adjust should be ≤ 0.6 nm 119.4 nm 86.6 nm 90.0 Sec 13.6 nm 122. All results are from Gaussian distributions.0 Sec 13.0 Sec 13.0 nm 88.92 Baseline adjust 0.05 %.6 nm 119.5 nm 120.1 nm 119.00 % 0.33 0.6 nm 121.32 1.0 Sec 13.0 Sec 13.2 nm 86.0 Sec 13.54 0.7 nm 120.33 0.4 nm Number weighting 84.0 nm 119.3 nm 119.0 nm 120.7 nm 87.03 % 0.Appendix 3 PCS results All the results marked red in appendix 3 are excluded from the summary because they did not fulfill the requirements that chi squared should be < 3.8 nm 119.0 nm 119.00 % 0.0 Sec 13.00 % 0. prepared with 10 mM NaNO3 Intensity weighting 119.8 nm 85.1 nm 120.99 0.22 0.00 % 0.8 nm 85.0 Sec Chi squared 0.50 0.5 nm 90.2 nm 120.00 % 0.

0 nm 109.23 0.01 % 0.0 Sec Chi squared Baseline adjust 2.6 nm 98.8 nm 83.00 % Table 9.8 nm 114.7 nm 112.0 Sec 13.51 0.0 Sec 13.05 % 0.00 % 0.4 nm Volume weighting.0 nm 94.6 nm 91.7 nm 110.0 Sec 15.82 0.38 1. prepared with 10 mM NaNO3.5 nm 93.5 nm Channel width 15.10 0.8 nm 92.0 Sec 15.4 nm 94.Table 9.8 nm 111.0 Sec 15.0 Sec 13.5 nm 113.5 nm 93.00 % 0.8 nm 91.0 Sec 13.2: PCS measurements of 100 nm liposomes prepared with 10 mM NaNO3.0 Sec 13.44 1.52 0.0 nm 111.1 nm 97.0 Sec 15.0 Sec 13.1 nm 76.0 Sec 15.44 1.9 nm 114.00 % 0.8 nm 73.0 nm 97.6 nm Channel width 13.7 nm 75.00 % 0.0 nm 75.6 nm 111.0 nm 111.3 nm 113.0 Sec 13.53 0.6 nm 113.2 nm Number weighting 74.0 nm 98.3 nm 114.03 2.80 2.3 nm 78.3 nm 80.4 nm 113.7 nm 110.3 nm 78.0 Sec 13.00 % 0. 91.71 0.4 nm 110.00 % 0.1 nm 76.00 % 0.9 nm 113.3 nm 75.6 nm 83.80 6.15 0.8 nm 99.00 % 0.5 nm 94.00 % 0.7 nm 94.94 1. 1 hour after dilution with 10 mM NaNO3 and glucose equivalent of 90 mM NaNO3 Intensity weighting 109.00 % 0.0 nm 81.59 1.0 Sec 15.7 nm 95.4 nm 94.00 % 0. 24 hours after dilution with 10 mM NaNO3 and glucose equivalent of 90 mM NaNO3 Intensity weighting 113.00 % 0.02 % 0.0 Sec 15.3 nm 72.4 nm 83.00 % 90 .3: PCS measurements of 100 nm liposomes.4 nm 92.4 nm Volume weighting 100.23 2.7 nm 75.0 nm 75.1 nm 74.00 % 0.68 0.0 Sec 15.00 % 0.0 Sec 15.7 nm Number weighting 87.7 nm 94.0 Sec 13.62 0.00 % 0.0 Sec Chi squared Baseline adjust 1.4 nm 76.32 1.5 nm 114.

0 Sec 14.4 nm 118.00% 0.0 Sec 14.1 nm 118.3 nm 114.8 nm 113.8 nm Number weighting 76. 48 hours after dilution with 10 mM NaNO3 and glucose equivalent of 90 mM NaNO3 Intensity weighting 113.08 0.0 Sec Chi squared Baseline adjust 0.00 % 0.0 Sec 14.00 % 0.4 nm 118.00 % 0. prepared with 10 mM NaNO3.7 nm 119.03 % 0.9 nm 114.87 0.00 % 0.96 0.0 Sec 14.4: PCS measurements of 100 nm liposomes.28 0.35 0.5: PCS measurements of 100 nm liposomes.4 nm 77.Table 9.0 Sec 14.0 Sec 14.1 nm 117.0 Sec 14.0 nm 81.0 Sec 14.8 nm 95.0 Sec 14.8 nm 96.1 nm 96.00 % 0.6 nm 113.3 nm Volume weighting 94.7 nm 117.20 0.6 nm 98.37 1.4 nm 92.0 nm 95.00 % 0.06 0.2 nm 90.00 % 91 .76 0.00 % 0.00 % 0.2 nm 118.6 nm 86.9 nm 113.8 nm 117.0 Sec 14.00 % 0.9 nm 78.2 nm 98.3 nm 118.9 nm 118.0 Sec 14.9 nm 76.6 nm 117.36 1. prepared with 10 mM NaNO3 Intensity weighting 118.00 % 0.6 nm 113.0 Sec 14.8 nm 78.7 nm 75.03 % 0.00 % Table 9.98 1.8 nm Volume weighting 119.8 nm 96.9 nm 91.69 2.4 nm Channel width 14.0 Sec 14.92 3.2 nm 94.2 nm 118.4 nm 93.8 nm 89.0 Sec 14.7 nm 119.4 nm 118.7 nm 79.00 % 0.4 nm 94.7 nm 113.90 1.37 1.3 nm Channel width 14.32 1.00 % 0.2 nm 113.06 0.9 nm 119.9 nm 96.91 0.7 nm 88.2 nm 95.2 nm 79.08 % 0.0 Sec 14.5 nm 118.15 3.3 nm 118.0 Sec Chi squared Baseline adjust 0.8 nm 76.0 Sec 14.32 0.00 % 0.3 nm 118.0 Sec 14.00 % 0.0 Sec 14.3 nm 114.9 nm Number weighting 86.

63 0.3 nm 114.00 % 0.13 1.5 nm 94.00 % 0.1 nm 116.0 Sec 14.4 nm 117.0 Sec Chi squared Baseline adjust 1.1 nm Volume weighting 95.6 nm 113.0 Sec 15.0 nm 114.2 nm Volume weighting 93.9 nm 73.32 1.04 1.0 Sec 15.2 nm 69.4 nm 96.48 0.0 Sec 15.6: PCS measurements of 100 nm liposomes.00 % 0.0 Sec 14.0 Sec 15.3 nm 116.7 nm Channel width 14.0 Sec 14.5 nm Number weighting 72.2 nm 75. 1 hour after dilution with 10 mM NaNO3 and sucrose equivalent of 90 mM NaNO3 Intensity weighting 114. 24 hours after dilution with 10 mM NaNO3 and sucrose equivalent of 90 mM NaNO3 Intensity weighting 116.00 % 0.00 % 0.59 1.7 nm 91.63 0.38 0.1 nm 81.00 % 0.7 nm 116.4 nm 114.0 Sec Chi squared Baseline adjust 0.00 % 0.9 nm 76.00 % 0.00 % 0.34 0.6 nm 101.6 nm 74.35 2.0 Sec 14.0 Sec 15.3 nm 74.27 1.0 Sec 14.69 0.0 nm 116.0 Sec 14.0 Sec 14.9 nm 74.6 nm 114.00 % 0.05 1.7 nm 84.Table 9.9 nm 96.00 % 0.00 % 0.3 nm 77.00 % Table 9.9 nm 95.00 % 0.1 nm 96.2 nm 114.00 % 0.0 Sec 15.3 nm 91.0 nm 70.0 Sec 15.2 nm Channel width 15.0 Sec 15.6 nm 115.79 1. prepared with 10 mM NaNO3.0 nm 114.7 nm 116.30 2.0 Sec 15.8 nm 99.32 0.9 nm 100.6 nm 94.00 % 0.2 nm 97.0 Sec 14.5 nm 90.00 % 0.5 nm 80.6 nm 76.7: PCS measurements of 100 nm liposomes.9 nm 77.0 Sec 14.4 nm 115.4 nm 85.2 nm 95.8 nm Number weighting 76.01 % 0.00 % 92 .2 nm 116.2 nm 94.0 nm 74.00 % 0.7 nm 97.5 nm 93.6 nm 93.3 nm 78.59 1.69 0.7 nm 114.6 nm 117.54 2. prepared with 10 mM NaNO3.26 4.6 nm 68.

00 % 0.03 % 0.0 Sec 14.8 nm 117.8: PCS measurements of 100 nm liposomes.0 Sec 14.33 0.91 3.3 nm 121.65 0.0 Sec 15.2 nm 116.00 % 0.1 nm Volume weighting 104.9 nm 99.5 nm 87.5 nm 120.0 Sec 15.1 nm 75.9 nm 93.9 nm 122.6 nm 101.39 0.0 Sec 14.05 % 0.23 2. prepared with 10 mM NaNO3.00 % 0.0 Sec 15.2 nm 104.9: PCS measurements of 100 nm liposomes.46 1.0 nm 121.1 nm 83.0 nm 71.1 nm 81.1 nm 78.2 nm 92.00 % 0.1 nm 97.7 nm 94.51 0.00 % 93 .0 Sec 15.5 nm 116.90 0.00 % 0.7 nm Number weighting 79.00 % 0.00 % 0.00 % 0.2 nm 73.4 nm 116.65 0.4 nm 82.00 % 0.0 Sec Chi squared Baseline adjust 1.0 nm 92.9 nm Channel width 14.00 % 0.28 0.6 nm 116.00 % 0.0 Sec 15.0 Sec 14.0 Sec Chi squared Baseline adjust 0.93 1. 7 nm 101.2 nm 93.0 Sec 15.2 nm 102.8 nm 82.9 nm 122.46 0.7 nm 121.1 nm 102.0 nm 116.00 % 0.44 1.34 0.0 Sec 15.95 4.00 % 0.30 1.0 nm 82.7 nm 117.41 4.6 nm 121. 48 hours after dilution with 10 mM NaNO3 and sucrose equivalent of 90 mM NaNO3 Intensity weighting 117.3 nm 121.0 Sec 15. prepared with 10 mM NaNO3 and sucrose equivalent of 90 mM NaNO3 Intensity weighting 121.0 Sec 15.9 nm 106.14 1.1 nm 99.5 nm 90.3 nm Volume weighting 98.1 nm 80.57 0.4 nm 72.5 nm 121.0 Sec 14.30 0.0 Sec 14.7 nm 82.9 nm 103.6 nm 102.00 % 0.0 Sec 14.00 % Table 9.8 nm 98.1 nm 117.2 nm 116.0 Sec 14.7 nm 96.6 nm 107.01 % 0.0 Sec 14.00 % 0.8 nm Number weighting 87.0 nm 82.Table 9.1 nm 70.5 nm Channel width 15.7 nm 84.

00 % 0.44 0.07 % 0.00 % 0.00 % 0.0 Sec 12.6 nm Number weighting 92.6 nm 98. 24 hours after dilution with 10 mM NaNO3 Intensity weighting 118.6 nm 118.4 nm 117.2 nm Channel width 12.00 % 94 .3 nm 97.8 nm 118.0 Sec 12.3 nm 118. prepared with 10 mM NaNO3 and sucrose equivalent of 90 mM NaNO3.5 nm 118.97 0.3 nm 118.3 nm 118. 1 hour after dilution with 10 mM NaNO3 Intensity weighting 118.1 nm 94.30 0.0 nm 118.7 nm 118.0 Sec 12.0 Sec 13.Table 9.54 0.0 nm 118.3 nm 118.7 nm 118.2 nm 97.34 0.35 0.0 Sec 12.51 0.0 Sec 13.14 2.2 nm 118.9 nm 96.8 nm 118.7 nm 119.74 0.00 % 0.8 nm 95.11 PCS measurements of 100 nm liposomes.0 Sec 13.5 nm 92.68 0.9 nm 118.8 nm Channel width 13.0 nm 92.5 nm Volume weighting 118.00 % 0.8 nm 118.9 nm 96.72 0.7 nm 118.4 nm 118.7 nm 90.9 nm 93.1 nm 95.9 nm 97.17 0.0 Sec 13.05 % 0.60 1.1 nm 91.0 Sec 13.0 Sec 12.00 % 0.2 nm 118.7 nm 90.9 nm 118.1 nm 118.0 Sec 13.0 Sec 12.3 nm 118.00 % 0.00 % Table 9.0 Sec 12.00 % 0.0 Sec 12.5 nm 118.00 % 0.6 nm 118.5 nm 117.1 nm 118.1 nm 92.0 Sec Chi squared Baseline adjust 3.0 Sec 12.0 Sec 13.0 Sec 13.00 % 0.00 % 0.00 % 0.0 Sec 13.37 0.00 % 0.2 nm 118.1 nm 118.34 0.1 nm 118.00 % 0.8 nm 95.7 nm 118.5 nm 118.0 Sec Chi squared Baseline adjust 0.3 nm 98.5 nm Volume weighting 118.1 nm 118.10: PCS measurements of 100 nm liposomes.57 1.2 nm 118.0 nm 118.00 % 0.9 nm 119.7 nm Number weighting 95.75 2.20 0.00 % 0.54 0. prepared with 10 mM NaNO3 and sucrose equivalent of 90 mM NaNO3.36 0.

2 nm 102.0 nm 92.45 2.49 0.07 % 0.9 nm 118.01 % 0.7 nm 118.00 % 0.3 nm 105.06 0. prepared with 10 mM NaNO3 and sucrose equivalent of 90 mM NaNO3.00 % 0.1 nm 118.2 nm 118.00 % 0.0 nm 118.0 Sec 13.3 nm Volume weighting 100. 48 hours after dilution with 10 mM NaNO3 Intensity weighting 117.6 nm 105.13: Latex bead standards.6 nm 105.7 nm Number weighting 94.0 nm 118.0 nm 119.00 % 0.00 % 95 .0 Sec 13.0 Sec 12.6 nm Number weighting 94.6 nm 118.58 0.00 % Table 9.12: PCS measurements of 100 nm liposomes.26 0.9 nm 118.01 % 0.9 nm 103.9 nm 104.41 1.60 0.Table 9.00 % 0.0 nm Channel width 13.00 % 0.4 nm 118.3 nm 102.44 0.3 nm 105.00 % 0.6 nm 104.9 nm 118.5 nm 104.4 nm 105.39 0.0 Sec 13.4 nm 104.36 0.4 nm 104.38 0.0 Sec Chi squared Baseline adjust 1.0 Sec 13.77 0.3 nm 97.0 Sec 13.7 nm 105.0 Sec 13.0 Sec 12.43 0.5 nm 118. diluted with 10 mM NaNO3. 6 nm 105.4 nm 104.0 Sec 12.02 % 0.6 nm Volume weighting 118.0 Sec 12.00 % 0.9 nm 105.0 Sec 12.7 nm 94.25 2.4 nm 117.4 nm 98.08 1.4 nm 117.5 nm 118.0 Sec 13.2 nm 118.26 0.7 nm 105.3 nm 100.0 Sec 12.3 nm 103.0 Sec 12.73 0.3 nm 105.0 Sec 13.3 nm 104.8 nm 102.1 nm 98.00 % 0.2 nm 95. PCS mean sizes calculated on the basis of viscosity and refractive index values of water Intensity weighting 105.05 % 0.9 nm 94.8 nm 103.0 nm Channel width 12.00 % 0.3 nm 96.05 % 0.4 nm 95.32 0.0 Sec Chi squared Baseline adjust 0.0 Sec 12.07 % 0.6 nm 105.00 % 0.7 nm 118. 100 nm.0 nm 118.81 0.5 nm 90.3 nm 103.26 1.0 Sec 13.0 Sec 12.

5 nm 109.5 nm 97.9 nm Volume weighting 116.3 nm 117.5 nm 112.1 nm Number weighting 115.8 nm 117.4 nm 105.0 Sec Chi squared Baseline adjust 0.05 % 0.00 % 0.40 0.0 Sec 13.30 0.7 nm 113.03 % 0.8 nm Volume weighting 111.0 Sec 13.9 nm 116.8 nm 113.5 nm 117.8 nm 111.9 nm 113.6 nm 117.0 Sec 13.8 nm 117.1 nm 106.14: Latex bead standards.0 nm 114.34 0.0 Sec 13. 100 nm.9 nm 114.6 nm 116.39 0.7 nm 117.00 % 0.6 nm 117.00 % 0.4 nm 113.40 0.0 nm 110.7 nm Number weighting 109.02 % 0.9 nm 117.04 % 0.33 0.1 nm 113.57 0.0 Sec 13.0 Sec 13.02 % 0.1 nm 106.00 % 0.02 % Table 9.0 nm 113.38 0.0 Sec 13.0 Sec 13.0 Sec 13.74 0.Table 9. PCS mean sizes calculated on the basis of the viscosity and refractive index values of water Intensity weighting 117.15: Latex bead standards.4 nm 109.7 nm 117.00 % 0. 100 nm.0 Sec 13. PCS mean sizes calculated on the basis of viscosity and refractive index values of water Intensity weighting 112.5 nm 117.42 2.26 0.5 nm 115.5 nm 117.0 Sec 13. diluted with 10 mM NaNO3 and sucrose equivalent to 90 mM NaNO3.0 Sec 13.5 nm 108.0 Sec 13.00 % 0.1 nm Channel width 13.7 nm 117.00 % 0.80 0.2 nm 111.8 nm 110.0 Sec 13. diluted with 10 mM NaNO3 and glucose equivalent of 90 mM NaNO3.6 nm 111.0 Sec 13.31 0.0 Sec 13.7 nm Channel width 13.00 % 0.87 0.0 nm 111.0 nm 108.6 nm 116.42 0.56 0.9 nm 115.1 nm 111.04 % 0.50 0.67 0.0 Sec 13.1 nm 113.31 0.13 % 0.01 0.4 nm 117.3 nm 110.3 nm 113.2 nm 117.10 % 96 .0 Sec Chi squared Baseline adjust 0.5 nm 112.1 nm 107.0 Sec 13.9 nm 109.07 % 0.8 nm 113.9 nm 115.00 % 0.4 nm 116.3 nm 114.6 nm 118.00 % 0.

1 nm 106. diluted with 10 mM NaNO3 and sucrose equivalent of 90 mM NaNO3.5 nm 106.0 Sec 13.7 nm 100.6 nm 104.7 nm 101.31 0.6 nm 104.0 Sec 13.0 nm 106.4 nm 105.1 nm 106.Table 9.0 Sec 13.7 nm 102.1 nm 102.00 % 0.6 nm 102.2 nm 103.31 0. 100 nm.1 nm 99.40 0.02 % 0.8 nm 100.6 nm 104.00 % 0.0 Sec 13.68 0.0 Sec 13.80 0.55 0.73 0.28 0.0 Sec 13.0 Sec 13.00 % 0.1 nm 103.0 Sec Chi squared Baseline adjust 0.2 nm 103.3 nm 104.07 % 0.6 nm Volume weighting 104.1 nm Number weighting 103.37 0.41 0.0 Sec 13.5 nm 106.05 % 0.8 nm 106.41 2.0 nm 100.0 Sec 13.9 nm 91.02 % Table 9.0 Sec 13.5 nm Channel width 13.03 % 0. PCS mean sizes calculated on the basis of measured viscosity and refractive index values Intensity weighting 105.00 % 0.00 % 0.26 0.4 nm 103. 100 nm. diluted with 10 mM NaNO3 and glucose equivalent of 90 mM NaNO3.4 nm 104.10 % 97 .4 nm 104.50 0.0 nm 102.0 Sec 13.4 nm 99.0 Sec 13.5 nm 104.0 Sec 13.13 % 0.01 0.2 nm Channel width 13.0 Sec 13.00 % 0.56 0.0 Sec 13.7 nm 105.0 Sec 13.6 nm 103.40 0.1 nm 102.2 nm 102.4 nm 104.3 nm 104.43 0.8 nm 101.00 % 0.04 % 0. PCS mean sizes calculated on the basis of measured viscosity and refractive index values Intensity weighting 104.8 nm 104.0 Sec Chi squared Baseline adjust 0.7 nm 104.78 0.34 0.3 nm 103.00 % 0.1 nm 104.6 nm 99.7 nm Volume weighting 104.16: Latex bead standards.04 % 0.9 nm 106.8 nm 100.8 nm 104.5 nm 104.5 nm 102.2 nm Number weighting 102.03 % 0.5 nm 103.2 nm 98.2 nm 105.6 nm 104.00 % 0.33 0.7 nm 103.4 nm 104.4 nm 105.2 nm 102.00 % 0.0 Sec 13.17: Latex bead standards.0 Sec 13.

4 nm Channel width 12.5 nm 116.6 nm 85.0 Sec 12.0 Sec 12.00 % 0. before 200 nm filter.8 nm 113.9 nm 86.4 nm 116.4 nm 114.0 Sec 12. Frozen in -80 °C freezer.05 % 0.5 nm 89.00 % 0.0 nm 114.0 Sec 12.0 nm 113.4 nm 92.9 nm 89.19: PCS measurements of 100 nm liposomes.00 % 0.3 nm 88.03 % 98 .32 0.0 Sec 14.0 Sec 12.07 % 0.0 Sec 14.9 nm 88.62 0.06 2.00 % 0.2 nm 116.9 nm Number weighting 90.00 % 0.42 0.35 0.22 0.7 nm 91. after 400 nm filter.1 nm 115.7 nm 92.00 % Table 9.0 Sec 14.2 nm 114. thawed on a 50°C water bath Intensity weighting 116.0 nm 116.0 Sec 14.0 nm 114.81 1.0 Sec 12.4 nm 114.Table 9.30 1.00 % 0.0 Sec 14.0 nm 114. freeze-thawed (3 cycles) during extrusion.0 Sec 12.8 nm 117.1 nm 116.61 0.1 nm 94.3 nm 116.6 nm 116.0 Sec 12.36 0.6 nm 114.4 nm 114.00 % 0.3 nm 114.2 nm 116.7 nm 90.1 nm Number weighting 92.31 4.9 nm 116.8 nm 114.27 2.05 % 0.9 nm Volume weighting 116.0 nm 116.00 % 0.9 nm 116. freeze-thawed (3 cycles) before extrusion.4 nm Volume weighting 114.00 % 0.00 % 0.94 0.18: Liposomes 100 nm.31 0. Frozen in -80 °C freezer.9 nm 114.03 % 0.55 0.6 nm 87.55 0.0 Sec 14.5 nm 90.8 nm 114.1 nm Channel width 14.4 nm 114.4 nm 92.0 Sec 14. thawed on a 50°C water bath Intensity weighting 114.8 nm 116.5 nm 113.49 0.9 nm 95.0 Sec 14.00 % 0.0 Sec 14.0 Sec Chi squared Baseline adjust 0.00 % 0.0 Sec Chi squared Baseline adjust 0.0 nm 92.0 Sec 12.6 nm 94.8 nm 116.5 nm 114.67 0.7 nm 117.8 nm 116.8 nm 114.5 nm 116.00 % 0.6 nm 89.53 0.90 1.00 % 0.

0 Sec 13.04 % 0.1 nm 89.4 nm 85.61 0.0 Sec 13.8 nm 111.6 nm 111.3 nm 86.2 nm 111.3 nm 112.00 % 0.0 Sec 13.9 nm 112.2 nm 112.0 Sec 13.8 nm 25.4 nm 30.5 nm 34.9 nm 24.7 nm Channel width 13.4 nm 111.0 Sec 13.5 nm 24. Frozen in -80 °C freezer.5 nm 34.6 nm 31.2 nm 31.3 nm 31.0 nm 84.72 0.1 nm 31.1 nm Volume weighting 111.36 2.6 nm 112.2 nm 112.00 % 0.8 nm 87.3 nm Number weighting 24.6 nm Number weighting 89.8 nm 110.Table 9.7 nm 113.1 nm 83.4 nm 99 .0 Sec 13.2 nm 32.73 0.3 nm 112.26 2.4 nm 30.00 % 0.01 % 0.8 nm 35.00 % 0.6 nm 24.38 0.6 nm 30.00 % 0.0 Sec 13.3 nm Volume weighting 31.8 nm 112. freeze-thawed (3 cycles) before extrusion. thawed on a 50°C water bath Intensity weighting 31.01 % 0. Frozen in liquid nitrogen (LN2).20: PCS measurements of 100 nm liposomes.9 nm 87.1 nm 85.2 nm 112.8 nm 32.3 nm 31.7 nm 24.0 Sec 13.9 nm 29.4 nm 23.6 nm 24.0 Sec Chi squared Baseline adjust 1.2 nm 32.5 nm 111.8 nm 35.0 nm 29.6 nm 24.4 nm 25.22 0.72 0.5 nm 111.5 nm 112.30 4. freeze-thawed (3 cycles) before extrusion. thawed on a 50°C water bath Intensity weighting 112.0 Sec 13.00 % Table 9.00 % 0.0 nm 83.5 nm 111.8 nm 32.58 2.21: Distribution width from PCS measurements of 100 nm liposomes.

1 nm 23.0 nm 24.0 nm 30.9 nm 29.6 nm Number weighting 23.4 nm 27.1 nm 24.6 nm 28.9 nm 24. Frozen in liquid nitrogen (LN2).7 nm 30.7 nm 28.0 nm 22.7 nm 31.5 nm 23. freeze-thawed (3 cycles) during extrusion.6 nm 32.9 nm 30.2 nm 28.5 nm 30.4 nm 30.6 nm 30.8 nm 30. after 400 nm filter. thawed on a 50°C water bath Intensity weighting 28.7 nm 23.22: Distribution width from PCS measurements of 100 nm liposomes. freeze-thawed (3 cycles) before extrusion.1 nm 28.4 nm 32.3 nm 32.9 nm 22. Frozen in -80 °C freezer.9 nm 31.4 nm 23.4 nm 32.8 nm 32.5 nm 29.3 nm 23.9 nm Number weighting 23.9 nm 31.1 nm 31.6 nm 30.1 nm 30.8 nm Volume weighting 29.2 nm 31.7 nm 28.0 nm 24.4 nm 24.8 nm 30.0 nm 27.9 nm 24.8 nm 30.3 nm 23.5 nm Table 9.6 nm 29.8 nm 100 .9 nm 29.2 nm 24.2 nm 27. thawed on a 50°C water bath Intensity weighting 29.0 nm Volume weighting 28.0 nm 22.3 nm 23.23: Distribution width from PCS measurement of 100 nm liposomes. before 200 nm filter.2 nm 31.1 nm 24.6 nm 32.Table 9.3 nm 27.

73 116. liposomes prepared with 10 mM NaNO3 From table 9.67 nm 90.4 nm ± 0.60 nm 118.35 nm 95.3 nm ± 0.02 nm 76.96 nm 77.5 nm ± 2.24: A summary of all the results presented in the tables above. liposomes 24 hours after dilution with 10 mM NaNO3 and glucose From table 9.7 nm ± 0.4.46 nm 97. 8 nm ± 0. liposomes 24 hours after dilution with 10 mM NaNO3 and sucrose Intensity weighting 119.3 nm ± 2.11 nm 113.0 nm ± 3. liposomes 1 hour after dilution with 10 mM NaNO3 and sucrose From table 9.8 nm ± 4. Mean diameters and standard deviations for all the valid results.91 nm Number weighting 86.04 nm 77.34 nm 114.3 nm ± 0.7 nm ± 1.29 93.4 nm ± 3.6 nm ± 0. Sample From table 9.7.93 nm 93.44 nm 96. liposomes prepared with 10 mM NaNO3 From table 9.7 nm ± 0.5 nm ± 0.6.5 nm ±3.73 nm Volume weighting 120.32 nm 113.8 nm ± 0.61 nm 118.5.2. liposomes 1 hour after dilution with 10 mM NaNO3 and glucose From table 9.8 nm ± 1.3.71 73.36 nm 78.9 nm ± 0. liposomes 48 hours after dilution with 10 mM NaNO3 and glucose From table 9.7 nm ± 1.8 nm ± 4. The results that had a too high value for chi squared or baseline adjust are not included.1.Table 9.8 nm ± 2.97 nm 101 .77 nm 110.

3 nm ± 0.13.35 nm 118.52 nm 101.5 nm ± 0.34 nm 118.1 nm ± 2. liposomes 48 hours after dilution with 10 mM NaNO3 and sucrose From table 9. liposomes prepared with 10 mM NaNO3 and sucrose equivalent to 90 mM NaNO3 From table 9.97 nm 113.36 nm 103.15.11.26 nm 118.3 nm ± 1.6 nm ± 0.6 nm ± 0.7 nm ± 1.39 nm 116.1 nm ± 2.41 nm 110.54 nm 121.1 nm ± 2.6 nm ± 4.86 nm 115.20 nm 85.1 nm ± 4.86 nm 118.29 nm 95.4 nm ± 0.8 nm ± 2.14.25 nm 94.From table 9. liposomes 48 hours after dilution with 10 mM NaNO3 From table 9.20 nm 102 .49 nm 118.8. Duke size standards diluted with 10 mM NaNO3 From table 9.4 nm ± 3.12.10 nm 74.9.6 nm ± 1.6 nm ± 0.6 nm ± 0.97 nm 117.21 nm 103.36 nm 118.4 nm ± 0.9 nm ± 0.37 nm 95. liposomes 24 hours after dilution with 10 mM NaNO3 From table 9.5 nm ± 0.4 nm ± 3.4 nm ± 0.1 nm ± 1. liposomes 1 hour after dilution 10 mM NaNO3 From table 9. Duke size standards diluted with 10 mM NaNO3 and glucose 116.88 nm 107.5 nm ± 0.28 nm 105.10.40 nm 96. Duke size standards diluted with 10 mM NaNO3 and sucrose From table 9.

liposomes frozen in -80°C freezer and thawed during extrusion From table 10.34 nm 103.5 nm ± 0.8 nm ± 0.38 nm 111.8 nm ± 1.40 nm 89.3 nm ± 2.6 nm ± 1.19.6 nm ± 0.18. Duke size standards diluted with 10 mM NaNO3 and glucose From table 9.1 nm ± 0.4 nm ± 0.52 nm 106.From table 9.7 nm ± 3.40 nm 103.2 nm ± 0. liposomes frozen in -80°C freezer and thawed before extrusion From table 9.20.17.74 nm 100.86 nm 116.17 nm 103 .7 nm ± 0.28 nm 91.35 nm 116.16. liposomes frozen in liquid nitrogen and thawed before extrusion 104.81 nm 114.1 nm ± 2.9 nm ± 2.25 nm 114. Duke size standards diluted with 10 mM NaNO3 and sucrose From table 9.64 nm 102.70 nm 112.45 nm 86.6 nm ± 0.5 nm ± 0.

62 nm 29. liposomes frozen in liquid nitrogen and thawed before extrusion Intensity weighting 32.5 nm ± 0.1 nm ± 1.9 nm ± 0.7 nm ± 0.62 nm 23.17 nm 31.1 nm ± 1.22. The results that had a too high value for chi squared or baseline adjust are not included.17 nm 23.4 nm ± 1. liposomes frozen in -80°C freezer and thawed during extrusion From table 9.25: Mean distribution width and standard deviations for all the valid results.1 nm ± 1.67 nm 31.Table 9.62 nm 29.83 nm Number weighting 24.38 nm 104 . liposomes frozen in -80 °C freezer and thawed before extrusion From table 9.81 nm Volume weighting 32. Sample From table 9.21.23.3 nm ± 1.5 nm ± 1.

10 nm Rz (nm) 55.3 (1 %) 48.Table 9.5 %) 59.8 %) 55.8 nm ± 0.26 Rn (nm) 46.26 3.8 %) 45.7 (0.3 (2 %) 50.5 (0.6 %) 55. diluted in 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate 4.7 %) 50.6 (0.1 nm ± 0.6 %) 55.0 (0.5 %) 52.32 nm 50.5 %) 59.4 (3 %) 2.6 %) 45.1 nm ± 0.6 %) 55.6 %) 51.6 (1 %) 46.1 (1 %) 44.7 (0. Liposomes prepared in 10 mM sodium nitrate and diluted in 10 mM sodium nitrate 53.2 (0. Liposomes prepared in 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate.7 %) 52.3 (2 %) 44.8 nm ± 0. from table 9.5 %) 48.6 %) 53.26 nm 57. Liposomes prepared in 10 mM sodium nitrate and diluted in 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate Rn (nm) 47.1 (0.7 (0.5 %) 53.2 (0.1 (0.6 %) 45.08 nm 48.5 %) 55.6 %) 60.8 nm ± 0.1 nm ± 0.8 (0.26: The different liposome sizes (radius) gained from the AF4 Sample 1.5 %) 52.from table 9.6 %) 48.7 %) 57.26 4.09 nm Rw (nm) 51.6 (0.1 nm ± 0.5 %) 50.3 (0.0 (0.6 %) 3.7 (2%) 50.6 %) Table 9.7 (0.6 (3 %) 47.7 %) 46. diluted in 10 mM sodium nitrate 44.7 (0. from table 9.9 (0.0 (0.8 nm ± 0.6 %) 57.1 (0.2 (2 %) 43.26 Sample 1.6 %) 50.3 (0.5 %) 51.0 (0.0 (0.5 (0.0 nm ± 0.49 nm 53.8 (3 %) 51.5 %) 57.3 (0.9 (0.23 nm 59.2 (0.0 (2 %) 47.5 (0.7 %) 45.35 nm 44.5 %) 50.2 (0.7 (0.7 %) 60.7 (0.6 %) 53.47 nm 50.1 (4 %) 45.6 %) 52.8 %) 51.5 %) 57.7 (0.6 % ) 46.7 %) 53.8 %) Rw (nm) Rz (nm) 52.7 %) 50.3 (0.7%) 52.0 (0.8 (0.0 (0.27: Mean radii and standard deviations for all samples in table 9. Liposomes prepared in 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate.9 (3%) 44.5 nm ± 0.8 (0.0 (0.2 (0.15 nm 45.1 nm ± 0.5 %) 59.70 nm 53.6 %) 53.2 (1 %) 50.9 nm ± 0. from table 9.5%) 56.7 %) 46.7 (0.9 (0.1 (0.26 2.9 (0.3 (0.18 nm 105 .9 (0.9 (0.9 (0.5 %) 51.

0 20.4 g b c d e f 4.0 time (min) 30.b c d e f g 150.5 100.0 0.0 40.0 40.0 Figure 9.0 2.0 g eom etric radiu s (n m ) 50.0 25.5 100.1 g b c d e f 2.0 35.0 time (min) 30.2 g b c d e f 2. 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate as mobile phase b c d e f g 150.0 35. diluted 1:10 in 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate. diluted 1:10 in 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate.0 4.1: The five parallels of liposomes produced in 10 mM sodium nitrate.0 0.3 g b c d e f 4.0 10.2: The five parallels of liposomes produced in 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate.0 15.4 g b c d e f 2. 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate as mobile phase 106 .0 20.0 25.0 15.3 g b c d e f 2.0 geom etric radius (nm ) 50.0 Figure 9.0 10.1 g b c d e f 4.2 g b c d e f 4.

0 0.0 40.0 25.0 35.5 100.1 g b c d e f 5.b c d e f g 150.0 time (min) 30.0 10.3: The five parallels of liposomes produced in 10 mM sodium nitrate. diluted 1:10 in 10 mM sodium nitrate. 10 mM sodium nitrate as mobile phase b c d e f g 150.0 20.0 7.0 time (min) 30.3 g b c d e f 5.3 g b c d e f 7.0 40.0 25. 10 mM sodium nitrate as mobile phase 107 .4 g b c d e f 5.2 g b c d e f 7.4 g b c d e f 7.4: The five parallels of liposomes produced in 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate. diluted 1:10 in 10 mM sodium nitrate.5 100.0 15.0 0.0 20.1 g b c d e f 7.0 35.0 Figure 9.0 geo m etric rad ius (nm ) 50.0 5.0 g eom etric radiu s (n m ) 50.0 15.2 g b c d e f 5.0 10.0 Figure 9.

012 0.003 0.001 0 20 30 40 50 Geometric radius (nm) 60 70 3 5 Figure 9.004 0.002 0 20 30 40 50 60 Geometric radius (nm) 70 80 Figure 9.006 Absorbance (AU) 0.002 0.007 0. 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate solution used as dilution medium and as mobile phase (five parallels) 108 .0.006 5 0.5: Parametric plot of the liposome sample prepared in 10 mM sodium nitrate solution. 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate solution used as dilution medium and as mobile phase (five parallels) 0.008 1 3 2 4 0.005 1 2 4 0.004 0.008 0.01 Absorbance (AU) 0.6: Parametric plot of the liposome sample prepared in 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate solution.

005 0. 10 mM sodium nitrate solution used as dilution medium and as mobile phase (five parallels) 0.002 0.003 0.004 0.8: The five parallels of the liposome sample prepared in 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate solution.01 0.009 0.001 0 20 30 40 50 60 Geometric radius (nm) 70 80 1 3 5 2 4 Figure 9.0.006 0.008 Absorbance (AU) 0.005 0.007 0.003 0.002 0.001 0 20 30 40 Geometric50 radius (nm) 60 70 80 1 3 5 2 4 Figure 9.7: Parametric plot of the liposome sample prepared in 10 mM sodium nitrate solution.006 0.007 0.008 Absorbance (AU) 0.01 0.009 0.004 0. 10 mM sodium nitrate 109 .

Appendix 4 The liposome samples which also were analyzed by AF4. but not included in the thesis Liposome dispersion medium 10 mM sodium nitrate Dilution medium 10 mM sodium nitrate Mobile phase 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate 10 mM sodium nitrate 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate 10 mM sodium nitrate 10 mM sodium nitrate 10 mM sodium nitrate 10 mM sodium nitrate 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate 100 mM sodium nitrate 100 mM sodium nitrate 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate 10 mM sodium nitrate 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate 10 mM sodium nitrate 10 mM sodium nitrate 10 mM sodium nitrate 100 mM sodium nitrate 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate 100 mM sodium nitrate 110 .28: Summary of the liposome samples which were analyzed by AF4. but not were included in the thesis. Table 9.

0 3.0 10.0 g eom etric radiu s (n m ) 50.3 g b c d e f 3.4 g b c d e f 3. diluted 1:10 in 10 mM sodium nitrate.2 g b c d e f 3.b c d e f g 150.0 0.0 15.0 35.5 100.1 g b c d e f 3. 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate as mobile phase 111 .0 20.1 g b c d e f 1.0 20.0 15.0 Figure 9.4 g b c d e f 1.0 geo m etric rad ius (nm ) 50.0 25.0 25.2 g b c d e f 1. diluted 1:10 in 10 mM sodium nitrate.10: The five parallels of liposomes produced in 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate.0 time (min) 30.0 time (min) 30.0 1.0 40. 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate as mobile phase b c d e f g 150.0 35.0 10.9: The five parallels of liposomes produced in 10 mM sodium nitrate.3 g b c d e f 1.0 40.0 0.5 100.0 Figure 9.

0 10.b c d e f g 150.0 15.11: The five parallels of liposomes produced in 10 mM sodium nitrate.4 g b c d e f 6. diluted 1:10 in 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate.0 15.4 g b c d e f 8.3 g b c d e f 8.5 100.5 100.0 20.0 25.1 g b c d e f 8.0 10.0 20.0 time (min) 30.0 geometric radius (nm) 50.3 g b c d e f 6.0 25. 10 mM sodium nitrate as mobile phase b c d e f g 150.0 0.1 g b c d e f 6. 10 mM sodium nitrate as mobile phase 112 .0 40.0 Figure 9. diluted 1:10 in 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate.0 geometric radius (nm) 50.0 8.0 time (min) 30.0 35.2 g b c d e f 8.0 Figure 9.0 40.12: The five parallels of liposomes produced in 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate.0 6.2 g b c d e f 6.0 35.0 0.

100 mM sodium nitrate as mobile phase b c d e f g 150.4 g b c d e f 9.5 100.5 100.0 10.0 40.0 0.1 g b c d e f 9.0 35.0 35.3 g b c d e f 9.0 10.0 geom etric rad ius (n m ) 50.0 40.3 g b c d e f 10.0 time (min) 30.4 g b c d e f 10.2 g b c d e f 10.b c d e f g 150.2 g b c d e f 9.0 20.0 15.0 20.1 g b c d e f 10.0 25.13: The five parallels of liposomes produced in 10 mM sodium nitrate.0 0.0 Figure 9.0 time (min) 30.0 15.0 25.0 Figure 9.0 g eom etric radiu s (n m ) 50. diluted 1:10 in 10 mM sodium nitrate. diluted 1:10 in 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate.14: The five parallels of liposomes produced in 10 mM sodium nitrate.0 10. 100 mM sodium nitrate as mobile phase 113 .0 9.

b c d e f g 150.0 25.0 10.0 10.0 25. 10 mM sodium nitrate as mobile phase 114 .0 20.0 12.16: The five parallels of liposomes produced in 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate. diluted 1:10 in 10 mM sodium nitrate.0 time (min) 30.0 40.0 20.3 g b c d e f 11.1 g b c d e f 12.0 35.0 geo m etric rad ius (nm ) 50.0 15.0 15.15: The five parallels of liposomes produced in 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate. 100 mM sodium nitrate as mobile phase b c d e f g 150.5 100.4 g b c d e f 11.0 Figure 9.0 40.0 0.2 g b c d e f 12.3 g b c d e f 12.0 0.0 Figure 9.0 time (min) 30. diluted 1:10 in 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate.1 g b c d e f 11.0 35.0 geo m etric rad ius (nm ) 50.5 100.0 11.4 g b c d e f 12.2 g b c d e f 11.

0 Figure 9.0 Figure 9.0 time (min) 30.0 50. diluted 1:10 w ith 10 mM sodium nitrate E-80 liposomes produced w ith 10 mM sodium nitrate. diluted 1:10 w ith 10 mM sodium nitrate E-80 liposomes produced w ith 10 mM sodium nitrate.0 0.0 0.0 20.0 15.0 25.0 35.0 40.0 35. diluted 1:10 w ith 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate geom etric radius (nm ) 100. Only one parallel of each sample shown 115 .0 15.0 10.0 E-80 liposomes produced w ith 10 mM sodium nitrate.0 10.b c d e f g b c d e f g b c d e f g b c d e f g 150.0 40.0 20. diluted 1:10 w ith 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate E-80 liposomes produced w ith 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate. diluted 1:10 w ith 10 mM sodium nitrate E-80 liposomes produced w ith 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate.0 25.0 50. diluted 1:10 w ith 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate geo m etric rad iu s (nm ) 100.17: All the four liposome preparations with 100 mM sodium nitrate as mobile phase. diluted 1:10 w ith 10 mM sodium nitrate E-80 liposomes produced w ith 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate.0 time (min) 30.0 E-80 liposomes produced w ith 10 mM sodium nitrate. diluted 1:10 w ith 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate E-80 liposomes produced w ith 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate.18: All the four liposome preparations with 10 mM sodium nitrate as mobile phase. Only one parallel of each sample shown b c d e f g b c d e f g b c d e f g b c d e f g 150.

0 20.0 40.0 20.0 15.b c d e f g b c d e f g b c d e f g b c d e f g 150.0 40.0 25.0 Figure 9. diluted 1:10 w ith 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate geometric radius (nm) 100.0 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate as mobile phase g b c d e f 100 mM sodium nitrate as mobile phase 10 mM sodium nitrate as mobile phase 100. Only one parallel of each sample shown 116 .0 50.0 geom etric radius (nm ) 50.0 35.0 Figure 9.0 E-80 liposomes produced w ith 10 mM sodium nitrate.0 10.0 15.20: All the liposome samples prepared in 10 mM sodium nitrate and diluted 1:10 with 10 mM sodium nitrate.0 35.0 25. diluted 1:10 w ith 10 mM sodium nitrate E-80 liposomes produced w ith 10 mM sodium nitrate.0 0.0 time (min) 30. Only one parallel of each sample shown b c d e f g b c d e f g 150.19: All the four liposome preparations with 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate as mobile phase.0 10. diluted 1:10 w ith 10 mM sodium nitrate E-80 liposomes produced w ith 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate. diluted 1:10 w ith 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate E-80 liposomes produced w ith 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate.0 time (min) 30.0 0.

0 geom etric radius (nm ) 50.b c d e f g b c d e f g 150. Only one parallel of each sample shown 117 .0 35.0 Figure 9.0 20. Only one parallel of each sample shown b c d e f g b c d e f g 150.0 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate as mobile phase g b c d e f 100 mM sodium nitrate as mobile phase 10 mM sodium nitrate as mobile phase 100. diluted 1:10 with 10 mM sodium nitrate.0 25.0 Figure 9.22: All the liposome samples prepared in 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate.0 40.0 10.0 25.0 time (min) 30.0 10.0 15.0 15.21: All the liposome samples prepared in 10 mM sodium nitrate and diluted 1:10 with 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate.0 geom etric radius (nm ) 50.0 time (min) 30.0 20.0 40.0 0.0 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate as mobile phase g b c d e f 100 mM sodium nitrate as mobile phase 10 mM sodium nitrate as mobile phase 100.0 35.0 0.

0 0.0 Figure 9.0 35.0 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate as mobile phase g b c d e f 100 mM sodium nitrate as mobile phase 10 mM sodium nitrate as mobile phase 100.23: All the liposome samples prepared in 10 mM sodium nitrate and sucrose equivalent to 90 mM sodium nitrate. diluted 1:10 with 10 mM sodium nitrate and sucrose equivalent of 90 mM sodium nitrate.0 15.0 time (min) 30.0 10.b c d e f g b c d e f g 150.0 40.0 25. Only one parallel of each sample shown 118 .0 20.0 geometric radius (nm) 50.

Sign up to vote on this title
UsefulNot useful