Controlling
the Formation of Vesicle Structures
and
Their Fixation by Polymerization
vorgelegt von
Master of Science - Chemistry
Hacer Yalcinkaya
geboren in Of, Türkei
von der Fakultät II - Mathematik und Naturwissenschaften
der Technischen Universität Berlin
zur Erlangung des akademischen Grades
Doktor der Naturwissenschaften
-Dr. rer. nat.-
genehmigte Dissertation
Promotionsausschuss
Vorsitzender: Prof. Dr. Reinhard Schomäcker (Technische Universität Berlin)
Gutachter: Prof. Dr. Michael Gradzielski (Technische Universität Berlin)
Gutachter: Prof. Dr. Helmut Schlaad (Universität Potsdam)
Tag der wissenschaftlichen Aussprache: 04.03.2019
Berlin 2019
i
ii
To Yusuf Yalçınkaya
(31.10.2018)
iii
iv
Acknowledgements
First of all, I would like to acknowledge my supervisor Prof. Dr. Michael Gradzielski for his
endless support, valuable guidance, and advices during my PhD work. I am very grateful for
being a member of his research team in Berlin and greatly appreciate his scientific discussions
as well as his friendly contributions on my professional and personal times during my PhD.
I would like to thank Prof. Dr. Helmut Schlaad for kindly being a reviewer of the present thesis.
I am also thankful to Prof. Dr. Reinhard Schomäcker for being the chairman of the examination
of this work. I am very grateful to Dr. Olga Koshkina for her friendly help on reading and
correcting the thesis and her fruitful inputs and discussions.
I am thankful to Prof. Thomas Zemb (Marcoule, France) and Dr. Christoph Herfurth
(Fraunhofer IAP, Potsdam) for their valuable scientific discussions and advices on the present
work. I am especially grateful to Prof. Yeshayahu Talmon (Technion, Haifa, Israel) and his
student Maor Ram-on for cryo-TEM measurements together with his very helpful discussions
and advices. I am additionally very thankful to Dr. Ingo Hoffmann for NSE measurements, later
his friendly help on analysing and interpreting of the data.
I am deeply thankful to Dr. Katharina Bressel, for all her scientific guidance, discussing many
times in patience, and encouraging me almost my entire PhD time.
I have appreciated a lot to work with many people during the neutron beamtimes. As local
contacts during SANS beamtimes I would like to thank Dr. Artem Feoktystov, Dr. Gaetano
Mangiapia and Dr. Marie-Sousai Appavou from MLZ, Garching; Dr. Peter Lindner and Dr.
Sylvain Prevost from ILL, Grenoble. I have had the pleasure to know them and work with them
and am grateful for all their support and scientific inputs.
I would like to thank Dr. Daniela Fliegner who has encouraged me a lot, willing to listen and
help me with her very friendly advices.
I am very much grateful to our secretaries Maria Bülth and Petra Erdmann, CTA’s Jana Lutzki,
Michaela Dzionara and Dr. Rene Straßnik for all their help to have a PhD time free of any
administrative, technical and experimental problems.
I am also thankful to Sven Thiel for rheology measurements and Michaela Dzionara for NMR
measurements.
v
Moreover, I would like to thank all the members of Stranski Laboratorium for their kindness
and friendship during my time in Berlin; especially my officemate Sven Riemar and Dr.
Leonardo Chiappisi for their friendly support, generosity and scientific inputs.
I would like to acknowledge the financial support by the Deutsche Forschungsgemeinschaft
(DFG) via the grant GR1030/17-1 that gave me the opportunity to work on this PhD project.
Special thanks to my 4F: Funda and Fulya Boztas, Fatma Ilgen and Fatma Durmus. Without
their support despite the distance, it would not be possible to have such success. I am very
grateful to my dear friend whom I know since my childhood, Dr. Nazmiye Dönmez for her
endless support. Additionally, I want to thank a lot to my refik-i hub Mojdeh Heidari and zuzu
Christoph Bartsch for our very nice friendship, their support and endless encouragement. My
dear friend Tuna from Bremen and Elif Gönül who was there all my PhD time listening and
advising me with all her kindness during our long Berlin walks, thank you so much.
Last but not least, I am deeply grateful to my entire family from 3 countries, my mum and dad,
Rahime and Hikmet Yalcinkaya, who trusted and supported me during my whole life in
patience; my aunt Ayten and her daughter Meltem for their endless support and being as a real
family to me in Germany, Aysegül and Mesut for being that nice siblings of me. I am very
thankful to Cenk Olgun for his continuous encouragement, valuable help in any case, and also
proofreading of the present thesis. I am very much grateful to all the above-mentioned people
for their love, trust and support.
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Publications
Some parts of this thesis have been or will be published as:
▪ H. Yalcinkaya, A. Feoktystov, M. Gradzielski, Formation of Well-Defined
Vesicles by Styrene Addition to a Nonionic Surfactant and their
Polymerization Leading to Viscous Hybrid System, Langmuir (2018), 34
(31), 9184-9194. DOI: 10.1021/acs.langmuir.8b01377.
▪ H. Yalcinkaya, K. Bressel, P. Lindner, M. Gradzielski, Controlled
Formation of Vesicles with Added Styrene and their Fixation by
Polymerization, Journal of Colloids and Interface Science, (2018), 531,
672-680. DOI: 10.1016/j.jcis.2018.07.097.
▪ H. Yalcinkaya, G. Mangiapia, M.-S. Appavou, I. Hoffmann, M. Ram-on,
Y. Talmon, M. Gradzielski, Formation of Vesicle Templated Polymer
Nanopcapsules and Calcein Encapsulation, (in prep.)
Other publications:
▪ L. Chiappisi, H. Yalcinkaya, V. K. Gopalakrishnan, M. Gradzielski, T.
Zemb, Catanionic surfactant systems-thermodynamic and structural
conditions revisited, Colloid Polymer Science, (2015), 293, 3131-3143.
DOI: 10.1007/s00396-015-3739-9.
vii
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Table of Contents
1 Introduction ..................................................................................................................................... 1
1.1 Theory ..................................................................................................................................... 1
1.1.1 Colloidal Forces .............................................................................................................. 2
1.1.2 Micelles ........................................................................................................................... 5
1.1.3 Vesicles ........................................................................................................................... 7
2 Motivation ..................................................................................................................................... 17
3 Materials and Methods .................................................................................................................. 21
3.1 Methods ................................................................................................................................. 21
3.1.1 Density ........................................................................................................................... 21
3.1.2 Refractivity Measurements ............................................................................................ 21
3.1.3 Refractive Index Increment Measurements ................................................................... 21
3.1.4 Viscosity ........................................................................................................................ 22
3.1.5 Light Scattering ............................................................................................................. 22
3.1.6 Small Angle Neutron Scattering .................................................................................... 25
3.1.7 Neutron Spin-Echo Spectroscopy (NSE) ...................................................................... 33
3.1.8 Cryogenic Transmission Electron Microscopy (cryo-TEM) ......................................... 34
3.1.9 Rheology ....................................................................................................................... 35
3.1.10 Nuclear Magnetic Resonance Spectroscopy (NMR) ..................................................... 36
3.1.11 UV-vis Spectrometry ..................................................................................................... 37
3.1.12 Fluorescence Spectroscopy ........................................................................................... 37
3.2 Materials and Experimental ................................................................................................... 39
3.2.1 Chemicals ...................................................................................................................... 39
3.2.2 Sample Preparation ........................................................................................................ 44
4 TDMAO/L35/Styrene System ....................................................................................................... 47
Introduction ....................................................................................................................................... 47
4.1 TDMAO/L35 /Styrene .......................................................................................................... 48
4.1.1 Phase Behaviour ............................................................................................................ 48
4.1.2 Light Scattering ............................................................................................................. 50
4.1.3 Small Angle Neutron Scattering (SANS) ...................................................................... 56
4.2 Polymerization of the TDMAO/L35/Styrene System ........................................................... 62
4.2.1 Nuclear Magnetic Resonance (NMR) ........................................................................... 63
4.2.2 Phase Behaviour ............................................................................................................ 63
4.2.3 Small Angle Neutron Scattering (SANS) ...................................................................... 64
4.2.4 Rheology ....................................................................................................................... 65
4.3 Summary ............................................................................................................................... 68
5 TDMAO/LiPFOS/L35/Styrene System ......................................................................................... 71
ix
Introduction ....................................................................................................................................... 71
5.1 TDMAO/LiPFOS/L35/Styrene ............................................................................................. 72
5.1.1 Phase Behaviour ............................................................................................................ 72
5.1.2 Light Scattering ............................................................................................................. 74
5.1.3 Small Angle Neutron Scattering (SANS) ...................................................................... 77
5.2 Polymerization of the TDMAO/LiPFOS/L35/Styrene System ............................................. 79
5.2.1 Phase Behaviour ............................................................................................................ 79
5.2.2 Light Scattering ............................................................................................................. 80
5.2.3 Small Angle Neutron Scattering (SANS) ...................................................................... 82
5.2.4 Nuclear Magnetic Resonance (NMR) ........................................................................... 83
5.3 Summary ............................................................................................................................... 85
6 The Effect of Acrylate Monomers ................................................................................................. 87
Introduction ....................................................................................................................................... 87
6.1 TDMAO/L35/Acrylate System ............................................................................................. 88
6.1.1 Phase Behaviour ............................................................................................................ 88
6.1.2 Light Scattering ............................................................................................................. 90
6.1.3 Small Angle Neutron Scattering (SANS) ...................................................................... 93
6.2 TDMAO/LiPFOS/L35/Acrylate Monomers.......................................................................... 99
6.2.1 Phase Behaviour ............................................................................................................ 99
6.2.2 Light Scattering ........................................................................................................... 101
6.2.3 Small Angle Neutron Scattering (SANS) .................................................................... 103
6.3 Polymerization of the TDMAO/LiPFOS/L35/Hexyl Acrylate System ............................... 111
6.3.1 Phase Behaviour .......................................................................................................... 111
6.3.2 Nuclear Magnetic Resonance (NMR) ......................................................................... 112
6.3.3 Light Scattering ........................................................................................................... 114
6.3.4 Small Angle Neutron Scattering (SANS) .................................................................... 117
6.3.5 Cryogenic Electron Microscopy (Cryo-TEM) ............................................................ 120
6.3.6 Neutron Spin Echo (NSE) ........................................................................................... 123
6.3.7 Encapsulation Efficiency ............................................................................................. 126
6.4 Summary ............................................................................................................................. 132
7 Summary and Outlook ................................................................................................................. 135
8 References ................................................................................................................................... 138
9 Appendix ..................................................................................................................................... 151
9.1 Appendix of Chapter 4 ........................................................................................................ 151
9.1.1 Refractive Index Increment ......................................................................................... 151
9.1.2 Determination of Aggregation Numbers ..................................................................... 152
9.1.3 Density of the aggregates ............................................................................................ 152
9.1.4 Kratky-Porod Plots ...................................................................................................... 153
x
9.1.5 Small Angle Neutron Scattering (SANS) of Polymerized Samples ............................ 155
9.2 Appendix of Chapter 5 ........................................................................................................ 156
9.2.1 Refractive Index Increment ......................................................................................... 156
9.2.2 Kratky-Porod Plots ..................................................................................................... 156
9.2.3 Small Angle Neutron Scattering (SANS) of Samples after 1.5 Years......................... 159
9.2.4 Small Angle Neutron Scattering (SANS) of samples before and after polymerization
160
9.2.5 Calculation of the Polymer Shell Thickness ................................................................ 162
9.3 Appendix of Chapter 6 ........................................................................................................ 163
9.3.1 Appendix to Studies with Dodecyl Acrylate Monomer .............................................. 163
9.3.2 Appendix to Studies with Isooctyl Acrylate Monomer ............................................... 164
9.3.3 Appendix to Studies with Hexyl Acrylate Monomer .................................................. 165
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xii
Abstract
In recent years, nanostructures have drawn much interest due to their potential applications in
biomedicine, energy conversion, detergency or electronics. Controlling their formation and
stability is one of the key issues to obtain such versatile structures with enhanced morphological
properties. Especially vesicles are interesting nanostructures with their hollow spherical
geometry, allowing encapsulation or transportation of hydrophobic or hydrophilic agents.
These structures can be enriched with the aid of polymers, either supporting the structures from
the outside or strengthening the inner side of their hydrophobic membrane through the insertion
of polymerizable monomers.
In that context, a spontaneously formed model vesicle system from nonionic
tetradecyldimethylamine oxide (TDMAO), anionic perfluoro surfactant (LiPFOS), and
Pluronic L35 copolymer was comprehensively studied in order to be stabilized by
polymerization and further used as nanocarriers. Earlier studies had shown that this system
generates small, monodisperse and well-defined vesicles. We have used this model system as a
template and by inserting various hydrophobic monomers into their bilayer, the effect of
monomer and its polymerization on the initial nanostructures were investigated systematically.
For that purpose, hydrophobic monomers (styrene, butyl-, hexyl-, dodecyl-, and isoosctyl
acrylate) were dissolved into the micellar TDMAO/L35 mixture prior to the vesicle formation.
Then monomer laden vesicles were prepared by mixing them with LiPFOS. Subsequently,
structures became permanently fixed by UV initiated polymerization process under mild
conditions, in water and at 18 °C. Then the systems were characterized elaborately using
different methods such as turbidity, UV-vis, Light Scattering (LS), Small Angle Neutron
Scattering (SANS), rheology, Nuclear Magnetic Resonance (NMR), Cryogenic Transmission
Electron Microscopy (cryo-TEM), Neutron Spin-Echo (NSE) and fluorescence spectroscopy.
In particular, scattering experiments revealed an interesting effect of styrene monomer in
micellar TDMAO/L35 mixtures which induces a structural evolution from rod-like micelles to
vesicles and then to the microemulsion droplets along with increasing the monomer
concentration. When polymerization was applied to the vesicle regime, it generated viscous
systems, which contain worm-like aggregates. On the other hand, it was shown by SANS
experiments that microemulsions with high styrene amounts lead to nanolattices with retaining
their size after polymerization. Secondly, it was shown that in the presence of LiPFOS, styrene
xiii
loaded vesicles were successfully stabilized via polymerization, yielding nanocapsules with
larger sizes and higher polydispersity values than the initial system.
As a third approach acrylate monomers in general showed an explicit transition from monomer
dissolved micelles to the vesicles, keeping the low polydispersity of the model vesicle system.
Among with the other acrylates, hexyl acrylate did not show any disruptive effect on the
formation of well-defined vesicles during its loading into the hydrophobic membrane.
Accordingly, small, and monodisperse monomer loaded vesicles were formed already with low
monomer content (10-30 mM). Therefore, hexyl acrylate was chosen for fixating the structures
via polymerization and structural fixation was confirmed by light scattering, SANS and cryo-
TEM measurements. In the last step, the encapsulation properties of the formed polymeric
nanocapsules were examined. Prepared nanocarriers were loaded with calcein, a water-soluble
fluorescence dye, which can be entrapped into the vesicle core and outside aqueous media.
Quenching of the unentrapped calcein in the presence of Cobalt (II) chloride, allowed us to
obtain the encapsulation efficiency of these polymeric nanocapsules. As a result, it was shown
that polymer stabilized vesicles have higher efficiency for being used as nanocarrier system.
xiv
Zusammenfassung
In den letzten Jahren haben nanostrukturierte Systeme aufgrund ihrer potenziellen
Anwendungen in Biomedizin, Energieumwandlung, Waschprozessen oder Elektronik viel
Interesse geweckt. Die Kontrolle ihrer Bildung und Stabilität ist eines der Hauptprobleme, um
solche vielseitigen Strukturen mit verbesserten morphologischen Eigenschaften zu
erhalten. Insbesondere Vesikel sind interessante Nanostrukturen mit ihrer hohlkugelförmigen
Geometrie, die die Einkapselung oder den Transport von hydrophoben oder
hydrophilen Agentien ermöglicht. Diese Strukturen können mit Hilfe von Polymeren
modifiziert werden, die entweder die Strukturen von außen unterstützen oder die Innenseite
ihrer hydrophoben Membran durch die Einführung von polymerisierbaren Monomeren
verstärken.
In diesem Zusammenhang wurde ein sich spontan bildendes Modellvesikelsystem aus
nichtionischem Tetradecyldimethylaminoxid (TDMAO), anionischem Perfluortensid
(LiPFOS) und Pluronic L35 Copolymer umfassend untersucht, um es durch Polymerisation zu
stabilisieren und als Nanoträger weiter zu verwenden. Frühere Studien hatten gezeigt, dass
dieses System kleine, monodisperse und wohldefinierte Vesikel erzeugt. In dieser Arbeit
wurde dieses Modellsystem als Templat verwendet und der Zusatz verschiedener hydrophober
Monomere in die Doppelschicht bezüglich des Einflusser von Monomer und des folgenden
Polymerisationsprozesses auf die anfänglich vorliegenden Nanostrukturen systematisch
untersucht. Zu diesem Zweck wurden unterschiedliche hydrophobe Monomere (Styrol, Butyl-,
Hexyl-, Dodecyl- und Isoosctylacrylat) vor der Vesikelbildung in der mizellaren TDMAO /
L35-Mischung gelöst. Monomer beladene Vesikel wurden dann hergestellt, indem sie mit
LiPFOS gemischt wurden. Anschließend wurden diese Strukturen unter milden Bedingungen
in Wasser durch UV-Licht bei 18 °C polymerisiert. Die Systeme wurden umfassend mit
verschiedenen Methoden wie Trübung, UV-Vis, Lichtstreuung (LS), Kleinwinkel-
Neutronenstreuung (SANS), Rheologie, Kernspinresonanz (NMR), Kryo-
Transmissionselektronenmikroskopie (Kryo-TEM), Neutronenspin-Echo (NSE) und
Fluoreszenzspektroskopie charakterisiert.
Insbesondere Streuexperimente zeigten eine interessante Wirkung von Styrol Monomer auf
mizellare TDMAO / L35-Gemische, nämlich mit wachsender Monomerkonzentration erfolgt
eine strukturelle Entwicklung von stäbchenförmigen Mizellen zu Vesikeln und dann zu den
xv
Mikroemulsionströpfchen. Polymerisation im Vesikelregime führte zu viskosen Systemen, die
wurmartige Aggregate enthalten. Dagegen zeigten SANS-Experimente, dass die
Polymerisation von Mikroemulsionen mit hohem Styrolanteil zu Nanolatices führt, wobei die
Größe erhalten bleibt. Zweitens wurde gezeigt, dass in Gegenwart von LiPFOS styrolbeladene
Vesikel erfolgreich durch Polymerisation stabilisiert wurden, was zu größeren Nanokapseln mit
höheren Polydispersitätswerten als das ursprüngliche System führte.
Als dritten Ansatz zeigten Acrylatmonomere im Allgemeinen einen expliziten Übergang von
gelösten Monomermizellen zu Vesikeln, wobei die geringe Polydispersität des Modellsystems
erhalten blieb. Von den Acrylaten war Hexylacrylat am geeignetsten und die Beladung der
hydrophoben Membran zeigte keine störende Wirkung auf die Bildung gut definierter
Vesikel. Dementsprechend wurden kleine und monodisperse monomerbeladene Vesikel bereits
mit geringem Monomergehalt (10-30 mM) gebildet. Daher wurde Hexylacrylat zum Fixieren
der Strukturen durch Polymerisation ausgewählt und die strukturelle Fixierung wurde durch
Lichtstreuung, SANS- und Kryo-TEM-Messungen bestätigt. Im letzten Schritt wurden die
Verkapselungseigenschaften der gebildeten polymeren Nanokapseln untersucht. Zubereitete
Nanoträger wurden mit Calcein beladen, einem wasserlöslichen Fluoreszenzfarbstoff, der im
Vesikelkern und außerhalb vorliegen kann. Das Quenchen des nicht eingeschlossenen Calceins
in Gegenwart von Cobalt (II) chlorid ermöglichte es, die Verkapselungseffizienz dieser
polymeren Nanokapseln zu bestimmen. Als Ergebnis wurde gezeigt, dass polymerstabilisierte
Vesikel eine höhere Effizienz aufweisen, um als Nanotransportsysteme verwendet zu werden.
xvi
Symbol Index
NA: Avogadro constant: NA = 6.022 x 1023 mol−1
κ: bending modulus
kB: Boltzmann constant: kB = 1.381 · 10−23 J/K
K: capillary constant
𝜇𝑔
0: chemical potential
C: coefficient in the interatomic pair potential
Γ : decay rate
τ or t: delay time
ρ: density at 25°C
ε: dielectric constant
D: diffusion coefficient
η: dynamic viscosity
𝐷𝑒𝑓𝑓: effective diffusion coefficient
J1: first order Bessel function of the first kind
Σ: sum formula
I(0): forward scattering
κ: Gaussian modulus
α: head group area
Rh: hydrodynamic radius
ν: kinematic viscosity
𝑙𝑐: length of the amphiphile
log: logarithm to the base 10
G': loss modulus
μ: mean location parameter
𝑣𝑚: molecular volume
ln: natural logarithm
ρ1 and ρ2: number of atoms per unit volume of two interacting particles
ω: oscillating frequency
U(r): pair interaction potential
r o R: particle radius
Vp: particle volume
ε0: permittivity of medium
Φ: phase or volume fraction of particles
xvii
n: refractive index
dn/dcg: refractive index increment
Δ𝜌 or ∆𝑆𝐿𝐷𝑠: scattering length density difference between shell and matrix
θ: scattering angle
q: modulus of the scattering vector
G0: shear modulus
𝛾: shear rate
𝛽: space average radius of gyration
G'. storage modulus
𝜎: surface charge density
t, t0, D, ΔR: thickness
τ: turbidity coefficient or relaxation time
σ: variance
S0: vesicle outer surface area
V: volume
ν: volume of the chain, and
𝑓𝑝: volume fraction of particles
λ: wavelength
cg: weight concentration of sample
wt%: weight fraction
η0: zero shear viscosity
xviii
Abbreviations
Agg: aggregate
Nagg: Aggregation number
Mwapp: apparent molecular weight
AIBN: azobisisobutyronitrile
Bkg: background
BPO: benzoyl peroxide
BA: Butyl acrylate
C0: bilayer’s spontaneous curvature
CTAB: cetyltrimethylammonium bromide
CTAT: cetyltrimethylammonium toluenesulfonate
CMC: critical micelle concentration
c: concentration
CEVS: controlled environment vitrification system
cryo-TEM: cryogenic transmission electron microscopy
D2O: deuterium oxide
DLVO: Derjaguin, Landau, Verwey, and Overbeek (theory)
AOT: Dioctyl sulfosuccinate sodium salt
DVB: divinyl benzene
DTAB: dodecyltrimethylammonium bromide
DLS: dynamic light scattering
Mweff: effective molecular weight
EE: encapsulation efficiency
P(q): form factor
FWHM: full width at half maximum
A: Hamaker constant or vesicle surface area or amplitude
HS: hard sphere or monomodal lognormal distributed spherical model
MLZ: Heinz Maier-Leibnitz Zentrum
HA: hexyl acrylate
ILL: Institut Laue-Langevin
I: intensity
S(q,t): intermediate scattering function
IUPAC: International Union of Pure and Applied Chemistry
Q: invariant
xix
K: inverse Debye length
IOA: isooctyl acrylate
JCNS: Jülicher Zentrum für Forschung mit Neutronen
LiPFOS: lithium perfluorooctylsulfonate
L1: micellar phase
Mw: molecular weight
NSE: neutron spin echo
NMR: nuclear magnetic resonance
1N: number density
N: number of segments in the tail
O/W: oil in water
p: packing parameter
L35: Pluronic L35
PDI: polydispersity index
PEO and EO: polyethylene oxide and ethylene oxide
PI- PCEMA: poly(isoprene)-block-poly(2-cinnamoyl methacrylate
PMOXA-PDMS-PMOXA: poly(2-methyl-oxazoline)-block-poly(dimethylsiloxane)-block-
poly(2-methyloxazoline)
PTFE: Polytetrafluoroethylene
c1 and c2: principal curvatures for bilayer
PO: propylene oxide
Rg: radius of gyration
Rtoluene: Rayleigh ratio for toluene
SD: sample to detector distance
SLD: scattering length density
L: segment length of surfactant tail
SANS: small angle neutron scattering
SDBS: sodium dodecylbenzenesulfonate
SDS: Sodium dodecyl sulfate
L3: sponge phase
SLS: static light scattering
S(q): structure factor
T: temperature or transmittance
TDMAO: tetradecyldimethylamine oxide
TEOS: tetraethyl orthosilicate
TMOS: tetramethyl orthosilicate
xx
t: time
UV: Ultraviolet
UV-vis: Ultraviolet–visible
Rves: vesicle outer radius
Lves: vesicle phase
DMPA: 2,2-Dimethoxy-2- phenylacetophenone
HDODA: 1,6-hexandiol diacrylate
xxi
1 Introduction
1.1 Theory
Amphiphilic molecules play an important role in colloidal science due to their dual function of
having hydrophobic and hydrophilic groups together within the same compound. In aqueous
media they arrange in such a way that the hydrophilic head group prefers to stay in water and
the hydrophobic tail reduces its contact with water to minimum. This more favourable
organisation of amphiphiles in water engenders at the air-water interface, a simple monolayer,
which lowers the surface tension and therefore surfactants are termed as surface active agents.
Surfactant molecules are rather dynamic species showing the tendency for self-assembly in
water. Along with the external environmental changes they rearrange and transfer themselves
rapidly to various aggregate types. As a result, different morphologies such as spherical, rod-
like micelles or vesicles can be formed depending on the molecular geometry which is described
by the packing parameter. Earlier Tanford developed the relation between the geometry of the
aggregates and amphiphile properties depending on the hydrophobic effect which in principle
leads to the aggregation 1. The term packing parameter was later proposed by Israelachvili in
1976, suggesting the relation between the molecular properties as head group area, volume of
hydrophobic tail, chain length and external variables such as temperature, ionic strength, pH or
salinity 2,3. The packing parameter is described by equation 1.1 and predicts the shape of the
spontaneously formed aggregates.
𝑝= ν
𝑎 .𝑙𝑐
(1.1)
Here ν,𝑎 and 𝑙𝑐 are the volume of the chain, head group area and length of the amphiphile
chain, respectively. Assembly into different geometries can be simply expected based upon the
shift of packing parameter for instance into spherical aggregates when p < 1/3, worm-like
micelles for 1/3 < p < 1/2, vesicles for 1/2 < p < 1 and planar bilayers for p ~ 1 (Figure 1.1).
The packing parameter is determined by the amphiphile structure or admixing cosurfactants
which are amphiphilic additives, not forming micelle themselves, but which can take a part in
micelle formation thereby typically lowering the interfacial curvature 4,5. These aggregates have
Chapter 1. Introduction
2
a wide application area from detergency to biomedicine and therefore their dynamic and static
behaviour has been studied thoroughly in the past years.
1.1.1 Colloidal Forces
During the self-assembly process, colloidal particles interact by virtue of various forces related
with all the components in the colloidal system. These forces influence the stability of the whole
dispersion and self-assembling efficiency, thus a detailed understanding is needed.
Brownian motion, suggested by Robert Brown in 1827, is the driving force for diffusion and
collision of colloidal particles. This random and continuous motion is driven by the thermal
energy, 𝟑
𝟐𝒌𝑻=𝟏
𝟐𝒎𝒗𝟐 where the translational velocity can be deduced. In this context, some
relations were developed for describing the particle motion. Einstein suggested the term
diffusion coefficient, relating the particle diffusion in a suspension with its size, Avogadro’s
number, absolute temperature and the solvent viscosity (Stokes-Einstein equation: 𝑫= 𝒌𝑩𝑻
𝟔𝝅𝜼𝑹 )
6. Afterwards, this equation was derived further with considering the time dependence of the
particle motion by another relation between the root mean-square displacement <x2>1/2 and time
t (Einstein-Smoluchowski equation: <𝒙𝟐>𝟏/𝟐=(𝟐𝑫𝒕)𝟏/𝟐) 7. Another derivation of the time
related diffusion coefficient was presented in the Langevin equation 𝒎𝒅𝒗
𝒅𝒕+𝟔𝝅𝜼𝑹=𝒇𝑩(𝒕)
where m is the mass of the particle 8. This equation accounts for the Brownian time constant as
the particles are much bigger than the molecules existing in the media, their response time is
bigger than the applied force, =𝒎
𝟔𝝅𝜼𝑹.
Figure 1.1. Different geometries predicted by the packing parameter.
Chapter 1. Introduction
3
Van der Waals interaction is the attractive force between molecules as well as particles as a
result of the dipole-dipole, dipole-induced dipole and induced dipole-induced dipole (London
dispersion) interactions. These interactions all promote the net van der Waals force and depend
on 1/R6 separation distance of small spherical particles 9. For a distance D of particles longer
than 10 nm, the London dispersion decays faster to 1/R7 which is called retardation effect
because of the long time needed for the transfer of the electric field of the dipole from one atom
to another one and its response 10. Hamaker established a connection between additivity derived
from London dispersion by integrating the interatomic Van der Waals pair potentials which
assumes the non-retarded interactions. 𝑨=𝝅𝟐𝑪𝝆𝟏𝝆𝟐, A is the Hamaker constant, C is
coefficient in the interatomic pair potential: W(D) = -CD-6,𝝆𝟏 𝒂𝒏𝒅 𝝆𝟐 are the number of atoms
per unit volume of two interacting particles and D is the particle edge to edge separation
distance. Relating the geometry with the interactions, one can reach the Van der Waals force
between the spherical vesicles as W(D) = AR/12D2, R being the reduced radius 𝑹=𝑹𝟏𝑹𝟐
𝑹𝟏+𝑹𝟐.
Similarly, the force between a sphere and a planar surface is obtained as =AR/6D2.
Additionally, Lifshitz continuum theory brings a different approach by considering the medium
and the particles separately avoiding the additivity problem, and the phases here are interacting
by electrical field fluctuations. The force can be calculated from the dielectric constants,
refractive indices, molecular rotational frequencies and electronic absorption frequencies.
Electrostatic interactions are another important force in colloid science. Surfaces in water are
basically charged with bound or adsorbed ions and these charges make an electrical field which
is attracting the counterions. The layer formed by the charges and the counterions, is defined as
electrical double layer and has been studied intensively. Helmholtz explained this interaction
with a model of a metal surface with adsorbed ions and showed the change of interaction
potential between the surface and the solution. The diffuse double layer idea was suggested by
Gouy-Chapman which is based on the thermal diffusion of ions from the surface to the solution.
The exponential decay of the potential with increasing distance from the surface can be obtained
from Poisson-Boltzmann equation. Moreover, Stern presented another model to explain the
charged surface and the diffuse layer of the interface. Due to his model the potential depends
on both, the distance from the surface, which causes a linear decay, and the exponential decay
in the diffuse layer.
On the other hand, two charged surfaces in a media is also another interesting point to be
examined. In this case both the electric double layer and Van der Waals interactions are taken
into consideration. The (Derjaguin, Landau, Verwey, and Overbeek,) DLVO theory basically
Chapter 1. Introduction
4
explains this interaction considering the attractive Van der Waals and repulsive electric double
layer forces both are additively stabilizing the dispersion. The energy per unit area can be
summarized as Wtotal = WvdW + Wedl therefore, the DLVO force can be approximated with 𝐹=
2𝜋𝑅𝜎2𝑒−𝜅𝐷
κε𝜀0−𝐴𝑅
12𝜋𝐷2 , were κ is the inverse Debye length, A is the Hamaker constant, ε and ε0 are
the dielectric constant of the medium and permittivity constant of vacuum, respectively, D is
the distance between the spherical particles, 𝜎 is the surface charge density and R the radius 10.
The DLVO force dependence on distance and ion concentration shows characteristic behavior,
depicted in Figure 1.2. For small distances between the particles, the Van der Waals attraction
becomes larger than the electrical double layer repulsion because it increases exponentially
faster. Additionally, Van der Waals is effective at high salt levels or small surface charge
densities (lower electrostatics) while for the opposite case, repulsion is more dominated. In
Figure 1.2, DLVO change from a to b can be seen when the surface potential decreases with
increasing electrolyte concentration.
Figure 1.2. DLVO interaction energy vs. distance
profile.
Chapter 1. Introduction
5
The Repulsive Hydration Force is an additional force beyond the DLVO model, which
describes interactions between the particles at short distance even closer than a few nanometers.
The repulsive forces here arise from the water layer which is bound to the hydrophilic surface
tightly and by this way can be stabilized even at high ionic strength 11,12. Aggregation occurs
from the Van der Waals interaction, because the stabilizing surface charges are shielded by the
electrolyte. Equation 𝑾=𝑾𝟎𝒆−𝑫/𝝀𝟎 describes the hydration repulsion between two
hydrophilic surfaces with D distance where λ0 ~ 1 nm and W0 depends on the hydration of the
surfaces and usually is in the range of 3-30 mJ/m2 at T = 25°C 9,13.
Concentration and the size of the ions have significant effects on hydration forces wherefore
they directly affect the H-bonding network in water which is the main reason of this type of
forces 10,14,15.
The Attractive Hydrophobic Force is prevailing when the water molecules interact with the
hydrophobic molecules. To reduce the total free energy and shift to an energetically favorable
state, water attracts to other water molecules while causing an attraction between the
hydrophobic ones. This attraction has an important role to understand the particle fusion
especially the vesicle-vesicle interactions. It is stronger than the Van der Waals, since the
hydrocarbon surfaces attracts stronger 9. The hydrophobic interaction energy between two
hydrophobic surfaces in water is expressed as 𝑾=−𝟐𝜸𝒊𝒆−𝑫/𝝀𝟎 where 𝜸𝒊 is between 10-50
mJ/m-2 and 𝝀𝟎 being 1-2 nm 10.
1.1.2 Micelles
Amphiphilic molecules in water form aggregates termed as micelles, with the arrangement of
their non-polar tail and polar head group. Micelle formation concept was suggested at earlier
1900 and evolved from starch solutions to soap and detergency with the work of McBain who
described the micelle formation from monomeric molecules 16,17. While surfactants at low
concentration in water are existing as monomers at high concentrations they form micellar
aggregates. Basically this critical concentration for micelle formation is called CMC (critical
micelle concentration) and was introduced by Davies and Bury in 193018. The CMC is an
important characteristic of surfactants; above the CMC micellar aggregates are forming in
equilibrium. Below CMC, typically already at much lower concentration, the surfactants are
adsorbed at the water-air interface and form a densely packed film there. Different parameters
as temperature, pH, pressure, and electrolytes have effects on the CMC, for instance pressure
cause an increase of volume and temperature mainly changes the ΔHmic0 19–21.
Chapter 1. Introduction
6
Another important phenomenon is the Krafft temperature or point, where the solubility
increases drastically and above it, surfactants form micelles. Thus, the CMC meets the solubility
curve 22,23 and below the Krafft point, surfactants are present as monomers.
Many studies were established to develop different models for predicting micellization. In
addition to Israelachvili’s packing parameter model described above, Tanford suggested the
free energy model where the energy is related with transferring the surfactant molecule from
the water to the aggregate state 24.
(Δ𝜇𝑔
0)=(Δ𝜇𝑔
0)𝑇𝑟𝑎𝑛𝑠𝑓𝑒𝑟+ (Δ𝜇𝑔
0)𝐼𝑛𝑡𝑒𝑟𝑓𝑎𝑐𝑒+(Δ𝜇𝑔
0)𝐻𝑒𝑎𝑑 𝑔𝑟𝑜𝑢𝑝 (1.2)
First term describes the negative free energy from the transfer of the unfavorable contact of the
hydrophobic tail from water; second term is a positive value of free energy that arises from the
unavoidable interaction of the surface of the tail and water interface and the last term comes
from the positive contribution from the repulsive interaction of headgroups of the amphiphile.
The interfacial free energy was defined as a combination of interfacial tension, σ, and surface
area, a. Repulsion of the headgroups increases with decreasing distance and is inverse to a.
Accordingly, the change of the chemical potential evaluates to (Δ𝜇𝑔
0)=(Δ𝜇𝑔
0)𝑇𝑟𝑎𝑛𝑠𝑓𝑒𝑟+
𝜎𝑎+𝛼/𝑎 where 𝛼 is the headgroup repulsion parameter. In thermodynamic equilibrium,
𝜕
𝜕𝑎(Δ𝜇𝑔
0)=0 here 𝜎−𝛼
𝑎2=0 and 𝑎=(𝛼
𝜎)1/2. The aggregation number Nagg, is then related
with area per molecule 𝑎; 𝑁𝑎𝑔𝑔∝1/𝑎. To summarize, this model provides information about
the basics of micellization, the tail transfer which is responsible for aggregation, affects the
CMC not the size and shape of the structures, the residual contact is the reason for growing of
the aggregates and the headgroup repulsion controls the size growth 24.
Later, this approach was developed further by Nagarajan and Ruckenstein 25. According
Tanford’s theory, the surfactant headgroup controls the aggregation. However, the role of the
tail is mainly neglected. Nagarajan and Ruckenstein extended this theory considering the
packing entropy of tail. Thus, the headgroup area is related with the tail length and the final
shape. One end of the tail is fixed at the water-surfactant interface while the other end locates
flexibly in the core. It deforms non-uniformly to fit in the core and behaves in compliance with
packing and uniform density constrains. The free energy observed from this conformational
constrain is termed as tail deformation energy. For spherical micelles, it is expressed by:
(Δ𝜇𝑔
0)𝑑𝑒𝑓
𝑘𝑇 = (9𝑝𝜋2
80 )(𝑅𝑠2
𝑁𝐿2) where Rs is the core radius, p is packing factor, L segment length of
tail, N is the number of segments in the tail (N=ls/L, ls is the extended length of tail). Adding
Chapter 1. Introduction
7
this into Tanford’s equation, shape transitions are related with tail length as well as the head
group area.
(Δ𝜇𝑔
0)=(Δ𝜇𝑔
0)𝑇𝑟𝑎𝑛𝑠𝑓𝑒𝑟+ (Δ𝜇𝑔
0)𝐼𝑛𝑡𝑒𝑟𝑓𝑎𝑐𝑒+(Δ𝜇𝑔
0)𝐻𝑒𝑎𝑑 𝑔𝑟𝑜𝑢𝑝+(Δ𝜇𝑔
0)𝑃𝑎𝑐𝑘𝑖𝑛𝑔 (1.3)
Different shape geometries such as spheres, cylinders or ellipsoids have different advantages
based upon the application fields, for instance rod-like micelles have interesting rheological
properties and viscoelastic behavior 26,27. These structures can be induced either by the
amphiphile’s molecular structure of some other additives such as additional amphiphiles, oils
or cosurfactants. The latter is a surface-active compound which does not form micelle alone,
however, when cooperating with the surfactants, its small headgroup gives rise to the packing
parameter and therefore new geometries can be formed.
In micellar systems solubilization has a significant importance. Due to the IUPAC definition
micellar solubilization means, the incorporation of solubilizate into or on the formed micelles
28. The interior of micelle is a good host for hydrophobic compounds and small molecules can
be solubilized more easily than the larger ones 29. However, the distribution of solubilizate is
affected by the head group of amphiphile and can be located either in interfacial or in the interior
site of the micelle. When a non-polar molecule is added with an increasing amount to a constant
concentration of surfactant, micelles swell with increasing its curvature until a certain point. At
that point micelles become large enough that the solubilizate captured inside seems similar to
its pure bulk. The new transparent, stable system is called as microemulsion, consisting of
spherical droplets with size in a range of 3 – 30 nm 30,31.
1.1.3 Vesicles
Among the structures described up to now, especially vesicles have attracted much attention
because of their interesting hollow spherical geometry. These closed bilayers are analogs as
biological membranes either for transport of hydrophobic compounds in the lipid shell or
hydrophilic ones in their aqueous core. Many investigations on vesicles have been done in terms
of understanding the behaviour of the system. Furthermore, these aggregates have a wide
application area from cosmetics and detergency formulations to nanoreactors, or for energy
conversion and pharmaceutical applications 32–35.
In general, vesicles can be formed from different type and classes of compounds. When aqueous
dispersions of phospholipids form vesicles, the formed kinetically stable structures are called
liposomes 36,37. Glycolipids known as the main component of the cell membranes are another
Chapter 1. Introduction
8
class that can form vesicles 38,39 and successfully being used as drug carriers40 in biotechnology
41. Catanionic vesicles are vesicles formed from oppositely charged surfactant mixtures such
systems consisted of sodium dodecyl sulfate and cationic dodecyltrimethylammonium bromide
42; cetyltrimethylammonium tosylate and anionic sodium dodecylbenzene sulfonate 43; or
cetyltrimethylammonium bromide and anionic sodium octyl sulfate 44. Interestingly, at
equimolar compositions of both surfactants precipitation takes place, while for an excess of
either cationic or anionic surfactant thermodynamically stable vesicles are formed 45–48.
Additionally, different studies have shown that zwitterionic surfactants, polyelectrolytes, short
chain alcohols and copolymers can play a role in the formation of vesicles 49–51.
Vesicles can be divided in two classes depending on having either one single bilayer or more.
The first class is called unilamellar vesicles and consists of only one bilayer. This class also can
be subdivided in categories based upon their size. For instance, small unilamellar vesicles have
radii of 5 – 50 nm and large unilamellar vesicles have the radii of 50 nm – 10 µm (Figure 1.3).
The second type of vesicles are multilamellar vesicles which have various shells of vesicles like
onion shape 52,53. While unilamellar vesicles are found in dilute systems; multilamellar vesicles
often are observed in concentrated systems.
Mainly, the surfactant’s molecular structure influences the final shape in addition to the
environmental factors such as temperature, pH or ionic strength. As described above, changing
Figure 1.3. Different vesicle types.
Chapter 1. Introduction
9
the packing parameter, one observes the transition in the geometry and for vesicles packing
parameter lies between ½ and 1. Tanford presented the critical chain length of hydrocarbon
which is the effective chain length in the liquid state lc = (0.154+ 0.1265n) nm for chains with
n hydrocarbon atoms 1. Additionally, the volume of hydrocarbon chain is defined as vc = (27.4
+ 26.9n) x 10-3 nm3 1. For vesicles with N number molecules, outer vesicle radius R0, and
thickness t0; the outer volume V0 can be calculated by: 𝑉0 =N 𝑣𝑐 =4
3𝜋[𝑅03−(𝑅0−𝑡0)3]; and
the outer surface area is given as: 𝑆0=4𝜋𝑅02 . From these equations the area per head group
then can be written as 𝑎=4𝜋𝑅02
𝑁0. This equation can be changed for the ratio between the actual
and optimal area: 𝑎
𝑎0=3(𝑣𝑐
𝑎0𝑙𝑐)𝑙𝑐𝑅02
[𝑅03−(𝑅0−𝑡0)3] and for vesicle formation the head group area is
needed to approach an optimal value 54. As seen from the equation, the packing parameter given
in equation 1.1 𝑝= 𝑣𝑐
𝑎0𝑙𝑐 relates the head group area to the chain length, outer vesicle radius and
outer layer thickness. When the p and 𝑙𝑐 are fixed, the ratio can change depending on the radius
and thickness. The minimum radius thereof can be reached once thickness becomes equal to
the chain length and 𝑅𝑚𝑖𝑛=3+[3(4𝑃−1)]1/2
6(1−𝑃)
𝑙𝑐 where p refers to the packing parameter 54. It is
noticeable that the minimum size of the vesicle is depending on two factors: the packing
parameter and the chain length.
It has to be noted that the vesicles prefer a value for packing parameter closer to ½ and planar
bilayers are formed at p = 1. Surfactants with small headgroup and bulky tail are required for
changing the packing conditions to such range. Thus, double-chain surfactants, or perfluoro
surfactants, or addition of cosurfactants such as medium-chain alcohols can be used for this
purpose 51,55.
Figure 1.4. Geometrical parameters of vesicle: S0: outer surface area, t0:
thickness of the outer layer, R0: outer vesicle radius.
Chapter 1. Introduction
10
Another important term for vesicle formation is the bending elasticity of the bilayer which is
fundamentally described by two parameters: the mean bending modulus κ, and the Gaussian
modulus κ. Hence, the free bending energy of the bilayer is expressed by the formula 𝐹𝑏=
∫𝑑𝐴[(κ/2)(𝑐1+𝑐2−2𝑐0)2+κ𝑐1𝑐2], where 𝑐1and 𝑐2 are the principal curvatures while 𝑐0 is
the bilayer’s spontaneous curvature and A is the surface area 56,57. When the Gaussian modulus
κ <<0; geometry shifts from planar bilayers to vesicles 58. With the help of this approximation,
the energy needed for the formation of different geometries from the surfactant layer can be
calculated.
In the case of mixed surfactant bilayers, Safran et al presented that energetically stable vesicles
can form through the interactions between two species 59. This induces an asymmetry of
composition which is coupled between the local curvature. In mixed surfactant bilayers, an
exchange of the amphiphiles occurs from lower to upper monolayer. When the coupling is
rather strong, geometry evolves to spontaneous bending 59. Later on, Ligoure and Porte
theoretically defined the bending characteristics of mixed vesicles consisting of two surfactants
not at fixed composition but at fixed chemical potential 60. They showed the size of this so-
called enthalpically stabilized vesicles depends on the relative concentration of both surfactant
types. In another work, the effect of chain length on bending rigidity and spontaneous curvature
was calculated theoretically revealing that the addition of short chain amphiphiles to the
vesicles consisted of long chain lipids lowers the bending rigidity 61.
1.1.3.1 Preparation of vesicles
Different preparation methods had been developed to provide sufficient conditions for vesicle
preparation. However, the most important point taken into consideration here, is the properties
expected from the final product. Size, polydispersity, permeability, surface charge potential,
long time stability are some important factors that depends on the preparation methods. Below
we briefly summarize the more general methods for vesicle preparation.
One of the most classical way is the sonication of lipid dispersions for homogenizing the
amphiphilic compound, in such case Vortex mixing can provide enough mechanical power and
has been used extensively 62–64. However the final product does not have uniform size
distribution and reproducibility is a big problem for this method 65.
Thin-film hydration is another method where a solution of surfactant is prepared in a volatile
solvent. A thin film of amphiphile is prepared by evaporating the solvent and vesicles are
formed by dissolving the remained film in water 64.
Chapter 1. Introduction
11
Additionally, by the way of high-pressure extrusion through the membrane filters with a certain
pore size, leads to the formation of highly monodisperse vesicles 66. The microfluidization
method is when the high-pressure extrusion is applied to lamellar phases, the lamellar
structures+ are demolished by the shear forces and consequently forms vesicles 67.
In some studies vesicles were also formed from the way of reverse evaporation 68. This means
an aqueous phase is dispersed in an organic solvent. Therefore, a water-in-oil emulsion is
formed by sonification. Later on, the solvent is removed under the pressure. The remaining part
is centrifuged then filtered to obtain the vesicles with sizes depending on the filtration pore
sizes. Also, nowadays supercritical carbon dioxide is reported being used as an alternative to
organic solvents 69.
It was shown that polyelectrolytes can be used to form hollow nanospheres which are very
similar to vesicles. Step-by-step deposition of oppositely charged polyelectrolytes on a
spherical surface can basically shape as a hollow sphere. Subsequently the substrate is removed
and the final structure forms vesicles 70.
1.1.3.2 Formation of vesicles
One of the key issues in the preparation of vesicles that effects the formation process is the need
of input of external energy. Studies have shown that in general the formation can be done in
two ways: the shear induced or the spontaneous formation of vesicles.
Shear-induced formation indicates that as an external energy, shear force can be applied to the
planar bilayers and induces a transition to vesicle structures. There have been many studies in
the literature on this type of vesicles formation. For instance, SDS/dodecane/pentanol/water
system as well as AOT/brine showed this transition where at high sear rates, planar lamellar
structures shifted to multilamellar vesicles 56,71. It was shown that the size of the vesicles is
related with the inverse square root of the shear rate 𝛾. Additionally, along with the vesicle
formation, viscosity increases. The reason for this is that planar lamellar structures do not show
a high flow resistance due to their orientation in the flow field, and accordingly their viscosity
is low. In contrast, vesicles are often densely packed resulting in a remarkable effect on the
viscosity 72.
Another study revealed the shear-induced transition from sponge phase (L3) to vesicles which
is reversible 73. In a similar manner, Candau and Manohar showed in their work that vesicles
can also transform to cylindrical micelles with applied shear 74.
Chapter 1. Introduction
12
Spontaneous formation of vesicles is the other challenging subject in this context. As described
above, an external energy input is necessary for most of the vesicle preparation methods. For a
spontaneous formation, this driving force is not required, however a type of shear is applied to
homogenize the system. One of the first examples of vesicles formed spontaneously, can be
given as vesicles consisted of the didodecyldimethylammonium surfactant with the presence of
hydroxide ions 75,76. Another study reported that anionic carboxylate surfactants can form
spontaneously vesicle together with an acidic cosurfactant as a function of pH 77,78.
Mixtures of cationic and anionic surfactants are interesting systems leading to the formation of
vesicles 45. These catanionic systems require an excess of one of the components for preventing
the precipitation at equimolar ratio. Sodium dodecylsulfate/dodecyltrimethyl-ammonium
bromide, cetyltrimethylammonium bromide/ sodium octyl sulfate or cetyltrimethylammonium
tosylate/sodium dodecylbenzenesulfonate catanionic surfactant mixtures can be presented in
this category 42–44. Alcohols or amine compounds have been used as cosurfactants changing the
packing parameter and taking a part in the formation of vesicles. For instance, the work on
hexanol in tetradecyldimethylamine oxide (TDMAO) and decane system examines deeply the
structural formation and transition of vesicles 51. Using surfactants with polymeric hydrophilic
headgroups such as PEO group, is another way to induce the vesicle formation as well as
enhancing the stability 79. Spontaneous formation can be also applied to non-aqueous systems.
Vesicles have been observed in the system of hexaethyleneglycol hexadecyl ether or sucrose
monoalkanoate in decane 80.
The theoretical basis of spontaneous vesicle formation is still been worked on. Nevertheless, it
is known that the curvature elasticity has a key role in the energetic stabilization. As an example,
in the case of catanionic vesicles, the inner and outer layer composition difference leads to an
effective curvature of equal and opposite signs, whereupon energetically stabilized vesicles are
formed 56,59. For the first time Helfrich introduced the relation between the theoretically
calculated size distribution of vesicles and bending moduli via the equation 81: 𝑓(𝑅)=
8𝑅3
〈𝑅2〉2exp (−2𝑅2〈𝑅2〉2), where 〈𝑅2〉2 is the mean square radius.
Moreover, when the bending modulus is significantly larger than kT, it is also possible that the
unilamellar vesicles are stabilized by the spontaneous curvature C0 82. Such an example can be
given for the system consisted from CTAB and sodium perfluoro-octanoate where the vesicles
were stabilized with low polydispersity 82
Chapter 1. Introduction
13
1.1.3.3 Polymer stabilized vesicles
Much research in recent years has focused on developing methods for avoiding the vesicle
fusion and enhancing their stability. Basically, this can be achieved by supporting the vesicles
either from outside or inside the membrane (Figure 1.5). For instance silica precursor, TEOS
(tetraethyl orthosilicate) or TMOS (tetramethyl orthosilicate) have been used for this aim
forming a silica shell and supporting the membrane from the outside. This method is nowadays
commonly used for the formation of silica hollow nanosphere 83. Steric stabilization can be also
achieved by using block copolymers 84 or inserting hydrophobic moieties into the vesicle
membrane which reduces to mobility, and hence, enhances the stability. As an example, Semple
presented the work where cholesterol was incorporated into the vesicle membrane 85 and
investigated its functionality in the lipid membrane.
A common way for keeping the stability of these submicron structures and the quality of the
permeability, is to strengthen the bilayer with the help of polymers 86. The formed polymer
stabilized hollow spherical nanostructures are termed as polymer nanocapsules (Figure 1.6).
Due to the hollow spherical geometry, their cavity allows to restrain the hydrophilic agents, and
the polymeric membrane can provide a controlled release87,88. Furthermore, the hydrophobic
moieties can also be isolated in the hydrophobic membrane and can be transported by this way.
Different methods to design the polymer nanocapsules are presented so far (Table 1.1),
involving the formation from preformed polymers or the polymerization reactions starting from
the organic monomers. Nanocapsules formed by dispersing preformed polymers can be
prepared using solvent evaporation, nanoprecipitation, dialysis or supercritical fluid
technology. For this purpose, emulsions are prepared by dissolving the polymers in different
solvents 89. In the solvent evaporation method, volatile solvents are used and after the
evaporation of the solvent, emulsion is converted into the nanoparticle suspension. In
Figure 1.5. Representation of vesicle stabilization methods.
Chapter 1. Introduction
14
nanoprecipitation technic, also called solvent displacement method, preformed polymers
precipitate from an organic solvent, which is later displaced with the aqueous phase 89. Dialysis
provides the formation of small and narrow distributed nanoparticles by dissolving the polymer
in an organic solvent placed in the dialysis tube while the outside media is a non-solvent 90. In
this method, nanoparticles are formed via solvent displacement, which leads to the polymer
aggregation. Supercritical fluid technology was developed for providing environmentally safe
preparation method. Instead of organic solvents, here polymer solution is prepared in a
supercritical fluid and thereby, nanoparticles were formed by rapid expansion of the solution
91.
Producing polymer nanocapsules from monomers offers a straight approach to obtain the
designed nanoparticles with objected properties. In addition to industrially used controlled-
radical polymerization, micro-, mini-, and emulsion polymerization methods are commonly
used techniques that allows the formation of nanoparticles with different sizes. In conventional
emulsion polymerization, main components are water, low water soluble monomer, water-
soluble initiator and surfactant 92. Monomer is dispersed in the solution and diffuses through
the micelles, and accordingly reaction occurs when dissolved monomer collides with the
initiator. Unlike the conventional emulsion, mini-emulsion method contains co-stabilizer and
the use of high-shear 93. In micro-emulsion polymerization, reaction starts from the
thermodynamically stable micro-emulsions along with the addition of water-soluble initiator 94.
Table 1.2 summarizes the differences between these polymerization methods and the product
properties.
Table 1.1. Different preparation methods of polymer nanocapsules.
Polymer Nanocapsule
From preformed polymer
From monomer
Solvent evaporation
Controlled radical polymerization
Nanoprecipitation
Emulsion
Dialysis
Miniemulsion
Supercritical fluid technology
Microemulsion
Chapter 1. Introduction
15
The important points to take into consideration are the stability, rigidity, controllability,
permeability and versatility of the final product. Therefore, preparation methods are of great
importance. In addition to above mentioned methods, using polymerizable surfactants or
amphiphilic block copolymers which can support the bilayer membrane from the inner side
provides advantages to control the desired properties of the final product 95–97. It was shown
that vesicles formed and stabilized from amphiphilic block copolymers are stable structures and
their polymer shell properties are controllable by simply changing the block length and polymer
moiety 98. In the work of Liu and Ding, poly(isoprene)-block-poly(2-cinnamoyl methacrylate)
PI-PCEMA diblock copolymer in hexane–tetrahydrofuran mixtures formed stable and water-
soluble polymer nanocapsule with the size of 50-60 nm radius 99. Another work was done by
Meier where vesicles with controllable size of 50 to 500 nm were formed from poly(2-methyl-
oxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyloxazoline) PMOXA-PDMS-
PMOXA triblock copolymers in water.
Later, the polymerization reaction of functional amphiphiles carrying styryl groups in vesicle
membranes was presented, strengthening the network with copolymerization and establishing
a two-dimensional interpenetrating network in the vesicle bilayer 100. Moreover, synthesis of
cationic, anionic, and zwitterionic surfactants with polymerizable chains have attracted
attention, as the permeability of the vesicles can be enhanced by this way 101.
An effective way for polymerization of vesicles can be done by inserting hydrophobic organic
monomers within the bilayers followed by fixating them by polymerization. For the first time
Murtagh and Thomas introduced the polymerization in the vesicle bilayer concept in the 1980s
102 with the cross-linking polymerization of styrene and divinyl benzene where they
successfully stabilized the vesicle size. Following works of non-cross-linked polymerization of
styrene showed that phase separation occurred in the bilayer after the polymerization which
caused the formation of so-called parachute like structures 103. Pulsed-laser polymerization
experiments were used to analyze the mechanism of the parachute morphology 104. This
Table 1.2. Different properties among the emulsion polymerization types 89.
Properties
Emulsion
Mini-emulsion
Micro-emulsion
Particle size
50-300 nm
10-30 nm
30-100 nm
Droplet size
1-10 µm
20-200 nm
~10 nm
Thermodynamic stability
Non-stable
Non-stable
Stable
Polydispersity
Low
Very low
Very low
Chapter 1. Introduction
16
problem became one of the key issues which needs in general to be considered while
stabilization by polymerization.
In other work, polymeric nanocapsules consisting of cross-linked polystyrene or cross-linked
polymethacrylates were synthesised by incorporation of the monomers into the vesicle bilayer
86,105,106. Kaler reported cross-linked polymerized vesicles in mixtures of styrene and
divinylbenzene monomers with the cationic surfactants cetyltrimethylammonium
toluenesulfonate (CTAT) or dodecyltrimethylammonium bromide (DTAB) with the branched
chain anionic surfactant sodium dodecylbenzenesulfonate (SDBS) 106. In another work,
polymerization kinetics of styrene containing surfactants were investigated by Fendler 107. Jung
and co-workers presented the phase separation during the polymerization of styrene in
dioctadecyldimethylammonium bromide (DODAB) vesicles. The formed structures had so
called parachute-like shape and this phenomena was investigated via small angle neutron
scattering (SANS) 108. More recently, Pinkhassik and co-workers reported the formation of
polymer nanocapsules produced by the polymerization of hydrophobic monomers in the
hydrophobic interior of the bilayers of different types surfactant vesicles. Accordingly, they
investigated the encapsulation capacities with entrapping catalysts into the nanocapsules 109–111.
This method drew much interest because of its ease instead of complex synthesis, it allows for
the use of common surfactants and afterwards to the removal of the surfactant templates.
Furthermore, mostly polymerization reactions need rough reaction conditions, which later can
have disruptive effects on self-assembled structures. However, this method can allow mild
reaction conditions for instance using UV initiated polymerization at low temperatures. Finally,
the properties of the nanocapsules produced this way (such as permeability, stability and
strength) can easily be modified 112.
Figure 1.6. Monomer loaded vesicle and polymer stabilized vesicle.
2 Motivation
Vesicles have been important structures formed from lipids or surfactants. These closed bilayer
shells attract great attention due to their hollow spherical geometry which allows for their use
as nanocarrier systems. Surfactant mixtures of different types such as anionic, cationic or
zwitterionic amphiphiles can form vesicles spontaneously without any energy input with an
average size between 50-100 nm. These submicron structures can be stabilized by
polymerization, or in other words they can be used as templates for the synthesis of polymer
nanocapsules.
In recent years, many approaches have been developed for the vesicle templating procedure.
Polymerization within the vesicle membrane is a widely used method. This method is
categorized in two parts; in the first part polymerizable surfactants and amphiphilic block
copolymers are employed, and in the second part hydrophobic organic monomers, i.e. styrene
or acrylates, are used for cross-linked polymerization (by having a certain percentage of cross-
linker present in the monomer mixture) to provide an enhanced stability 102,113. The latter is an
effective way where the vesicle template is not involved directly in the reaction, but at the same
time shapes the finally formed polymer network.
Although it is a convenient approach for controlling the final structure, it is challenging with
respect to the polymerization process. One of the important issues observed is the phase
separation in the bilayer whereby polymerization led to the formation of parachute-like
structures 103. Consequently, products with unintended morphologies can likely be formed.
Additionally, another problem is the rough polymerization reaction conditions such as high
temperature or the need of organic solvents and their direct effects on self-assembled
architecture, therefore the final product.
The aim of this PhD work was to address these open questions and to provide a powerful,
straightforward method of stabilization via polymerization at mild reaction conditions.
Therefore, we choose to template a well-defined vesicle system by inserting hydrophobic
monomers, i.e. styrene and acrylates with different chain lengths, into the membrane.
Afterwards the structures were fixated by UV-initiated polymerization under very mild
conditions, eventually generated monodisperse hollow spherical polymer nanocapsules.
Thus, the vesicle system of choice consisted of nonionic tetradecyldimethylamine oxide
(TDMAO) and anionic lithium perfluorooctylsulfonate (LiPFOS), which had been studied
Chapter 2. Motivation
18
earlier, was chosen as template. It was shown that due to the synergistic interaction between the
surfactant pairs, vesicles are spontaneously formed, since the head groups are interacting
attractively and reducing the joint head group area 114. Stability and size of these vesicles can
be controlled by addition of Pluronic copolymers (EOn-POm-EOn) where L35 attaches on the
rim, stabilizes the structures in a way preventing them to converge. As a consequence small,
monodispersed and kinetically long-time stabilized vesicle are formed with a low polydispersity
PDI of ~ 0.05 115.
In the present study, we used these well-defined, monodisperse vesicles of a TDMAO/LiPFOS
mixture with molar ratio of 55:45 in the presence of 1 mol % Pluronic L35, as a suitable
template for the cross-linked polymerization to produce polymer nanocapsules. For this aim,
hydrophobic organic monomers, which can effectively be inserted into the vesicle membrane,
were introduced into the vesicle membrane. For determining the effect of various monomers
with different hydrophobicity, styrene, butyl-, dodecyl-, isooctyl- and hexyl acrylate monomers
were used and their concentration as well as the ratio of cross-linkers were varied. Concurrent
loading was chosen as the plausible way for dissolving the water-insoluble monomers entirely
and preventing the ageing of the vesicles. Therefore, monomers were initially dissolved in the
micellar 50 mM TDMAO / 0.5 mM L35 stocks. Their influence on the primer micellar
aggregates was of the key importance in the formation of vesicles. Secondly,
TDMAO/L35/monomer stocks were mixed with 50 mM LiPFOS solutions with the molar ratio
of 55:45, leading to monomer loaded vesicles. Latterly, the structural changes of the vesicles
upon incorporation of the different monomers and the cross-linkers were examined structurally
and morphologically. These monomer-loaded vesicles were fixated by UV-initiated
polymerization (Figure 2.1) in water at 18°C. Effect of polymerization on the bilayer membrane
and the degree of cross-linking were examined for optimization and enhancing the
Figure 2.1. Schematic representation of the general synthesis approach.
Chapter 2. Motivation
19
encapsulation efficiency of the finally formed nanocapsules which was tested via loading with
calcein as a water-soluble fluorescence dye.
The thesis and the obtained results are organized in the chapters as follows:
Chapter 3: Materials and methods
The materials and methods used in this study with the experimental preparation of samples and
polymerization methods are explained.
Chapter 4: TDMAO/L35/Styrene System
Chapter 4.1 describes the unusual behavior of increasing amount of styrene monomer in
TDMAO/L35 system and the results from the analyses of turbidity, dynamic and static light
scattering, (DLS, SLS), small angle neutron scattering (SANS) measurements.
In chapter 4.2, polymerization of styrene in TDMAO/L35 leading to viscous hybrid system is
investigated in terms of viscosity and rheology, small angle neutron scattering (SANS) and
NMR measurements.
Chapter 5: TDMAO/L35/LiPFOS/Styrene System
Chapter 5.1 explains loading the styrene monomer into the vesicle template of
TDMAO/LiPFOS/L35 and the obtained results regarding its effect on well-defined vesicle
system.
In part 5.2, we present the effect of polymerization of styrene in TDMAO/LiPFOS/L35 vesicle
membrane.
Chapter 6: The effect of acrylate monomers
In chapter 6, we summarized the effects of acrylate monomers on the studied vesicle systems.
Primarily, different chain length of acrylates tested on TDMAO/L35 mixtures in part 6.1 and
results are presented. Secondly, (in chapter 6.2) by mixing this solution with LiPFOS, monomer
loaded vesicles are formed and analyzed structurally in detail. Consequently (part 6.3), the
optimized system of TDMAO/LiPFOS/L35/ hexyl acrylate is polymerized and investigated by
means of phase behavior, scattering, NMR, neutron spin echo, cryo-TEM methods, and the
encapsulation efficiency was tested with fluorescence spectroscopy.
Chapter 7: Summary and Outlook
In chapter 7, results are generally summarized and the key points for stabilizing vesicles via
polymerization are indicated. Additionally, future studies about improving this method, such as
Chapter 2. Motivation
20
its compatibility with biodegradable systems are suggested for future work and discussed in
this chapter.
3 Materials and Methods
3.1 Methods
3.1.1 Density
Densities were measured at 25°C by using a capillary
oscillating density meter DMA 4500 Anton Paar,
based on electronically measuring the oscillation
period of a known volume fraction of sample in a U-
shaped borosilicate tube with the relation of 𝜌=
𝐴𝜏2+𝐵 where the density 𝜌 is related with the
oscillation period 𝜏, A and B are device constants.
3.1.2 Refractivity Measurements
Refractivity measurements have been done with an Abbe 2WAJ refractometer at 25°C for 630
nm wavelength and values are averaged for three repeated measurements.
3.1.3 Refractive Index Increment Measurements
Refractive index measurements were performed on the device from Orange Analytics 19” dndc
on the wavelength of 620 nm. The refractive index increments were derived from concentration
series at constant sample composition up to the 100-mM content and used for evaluating the
data from turbidity and light scattering measurements. For the higher styrene content, dn/dc
values were estimated via:
Measured values of dn/dc are listed in Table A1, Table A3, Table A4, Table A5 (see Appendix).
𝑑𝑛
𝑑𝑐=𝑥.𝑑𝑛
𝑑𝑐𝑠𝑡𝑦𝑟𝑒𝑛𝑒+(1−𝑥)𝑑𝑛
𝑑𝑐 (𝑡𝑑𝑚𝑎𝑜
𝐿35 )
(3.1)
Figure 3.1. Capillary oscillating
density meter.
Chapter 3. Materials and Methods
22
3.1.4 Viscosity
Viscosity measurements were performed on calibrated Schott Ubbelohde micro Ostwald
viscometers, type I (517 10) and Ic (517 13). Capillary constants are given for I, 0.01151 mm2
s-2 and for I, 0.02942 mm2 s-2. Zero shear viscosity 𝜂0 values were calculated from the flow time
t of the samples using the equation 3.2 related with density 𝜌 and capillary constants Κ :
Flow time t were registered automatically on Ivisc (Lauda-Brinkmann) and are averaged values
of six measurements.
3.1.5 Light Scattering
Light scattering is one of the fundamental methods in the characterization of colloids. In
principal, it can be described as the redirection of an incident light beam due to its interactions
with a sample material. When the electromagnetic radiation interacts with the material, it
induces an oscillation of the electrons in the molecule and leads a secondary radiation which is
the scattering from the sample (Figure 3.2).
In scattering experiments, intensity over time is dependent on the magnitude of the scattering
vector, q, which is angle dependent and given by 𝑞=(4𝜋𝑛
λ)sin (𝜃
2) where n is the refractive
index, λ the wavelength, and θ is the scattering angle.
𝜂0=𝜌Κ𝑡
(3.2)
Figure 3.2. Schematic representation of light scattering experimental setup.
Chapter 3. Materials and Methods
23
3.1.5.1 Dynamic Light Scattering
Dynamic light scattering (DLS) is based on the time dependence of fluctuations of the scattering
intensity. The collective diffusion coefficient can be observed due to the fluctuations of particles
undergoing Brownian motion. In such scattering experiment, Figure 3.2. light passes through a
scattering volume and is scattered due to inhomogeneities. The intensity is measured and
detected at a certain angle by a photomultiplier from the correlation function of the photon
count rate of the detector. Thus, dynamic information about the particles can be obtained during
the measurement. In particular, DLS allows to determine the particle radius using the scattered
light to measure the rate of diffusion of the particles and polydispersity.
The intensity autocorrelation function g2(τ) for a given delay time τ (termed as t at DLS curves
given thereinafter) is obtained by:
𝑔2(𝜏)=〈𝐼(𝑡)𝐼(𝑡+𝜏)〉
〈𝐼(𝑡)〉2
(3.3)
Where I(t) and I(t+ 𝜏) are the intensities of the scattered light at times t and (t+ 𝜏).
Based on the Siegert relation the intensity autocorrelation function g2(τ) can be converted to the
field autocorrelation function g1(τ) via:
𝑔2(𝜏)=1+𝐵|𝑔1(𝜏)|2
(3.4)
The field correlation function decays exponentially in the solutions for monodisperse particles:
𝑔1(𝜏)=𝑒−𝛤𝜏 where the decay rate is Γ=D𝑞2. For polydisperse samples g1(τ) is presented as
a sum of a distribution of decay rates G(Γ).
The diffusion coefficient of the particles can be determined from the intensity correlation
function g2(τ). By plotting g2(τ)-1 as a function of τ, data shows an exponential decay and fitting
the exponential decay one can obtain the diffusion coefficient D. Using the Stokes-Einstein
relation hydrodynamic radius Rh can be estimated via:
𝑅ℎ=𝑘𝐵𝑇
6𝜋𝜂D
(3.5)
In the equation T is the temperature, η the viscosity, kB the Boltzmann constant, and D the
diffusion coefficient.
As mentioned above, for polydisperse systems g1(τ) is given as a sum of a distribution of decay
rates G(Γ). Cumulant method is one precise way to characterize the decay rates in such a case,
which determines the size and polydispersity extending the logarithm of g1(τ) with regards to
Chapter 3. Materials and Methods
24
cumulants of the distribution 116. In this work, intensity correlation functions with monomodal
decays have been analyzed with cumulant method via the second order to the function:
ln[𝑔1(𝜏)]=− Γτ+𝜇2
2𝜏2−𝜇3
6𝜏3
(3.6)
Rh the hydrodynamic radius can be calculated from the cumulant analysis using equation 3.5
and also the polydispersity index can be calculated from second order fitting: 𝑃𝐷𝐼=𝜇2
Γ2.
Light Scattering measurements were performed using an ALV/CGS-3 Compact Goniometer
with an ALV/LSE-5004 multiple tau digital correlator equipped with a He-Ne laser with the
wavelength of = 632.8 nm. All experiments were done in a thermostated bath at 25±0.1 °C
using cylindrical sample cells with a diameter of 8 mm placed in toluene bath. The intensity
autocorrelation function g2(τ) was obtained by averaging three repeat measurements at different
angles (30, 40, 50, 60, 70, 80, 90, 100, 110 and 120°) and performed on all samples at 25 °C.
Static light scattering (SLS) measurements were performed using the same setup as employed
for in Dynamic light scattering (DLS).
3.1.5.2 Static Light Scattering
Static light scattering (SLS) analyses the time-averaged scattering intensity as a function of the
angle In SLS one normalizes the time-average scattered intensity with incoming light. SLS
provides critical information about the radius of gyration Rg, which is a measure of the size of
an object of arbitrary shape and can be obtained from the Guinier approximation 28, and
molecular weights Mw of the aggregates.
The absolute intensities can be obtained by using the equation 3.7. normalizing the measured
intensities of the samples with the solvent and toluene as a standard, then correcting them with
the Rayleigh ratio of toluene Rtoluene(q) toluene as reference, 1.34x10-5 cm-1 at 632 nm 117.
Finally for the cylindrical cells, this value is corrected with the Herman & Levinson factor
(ntoluene/nsolvent)2 118,119.
𝐼(𝑞)=𝐼𝑠𝑎𝑚𝑝𝑙𝑒(𝑞)−𝐼𝑠𝑜𝑙𝑣𝑒𝑛𝑡(𝑞)
𝐼𝑡𝑜𝑙𝑢𝑒𝑛𝑒(𝑞)𝑅𝑡𝑜𝑙𝑢𝑒𝑛𝑒(𝑞)(𝑛𝑡𝑜𝑙𝑢𝑒𝑛𝑒
𝑛𝑠𝑜𝑙𝑣𝑒𝑛𝑡)2
(3.7)
Th forward scattering I(0) is extrapolated by fitting the Guinier approximation on scattering
curves whereby the weight average molecular weights, Mw can be determined 120:
𝑀𝑤=𝐼(𝑞=0)
𝐾𝑐𝑔
(3.8)
Chapter 3. Materials and Methods
25
K in the equation is the optical constant given with the formula as:
𝐾=4(𝜋𝑛𝑜(𝑑𝑛
𝑑𝑐))2
𝑁𝐴𝜆𝑜
4
(3.9)
Where dn/dcg is refractive index increment and cg is the weight concentration of sample content.
3.1.6 Small Angle Neutron Scattering
Small angle neutron scattering (SANS) is one of the most important characterization methods
for microstructural investigations on various materials. SANS allows investigations on the
nanoscale and determines the morphology of particle systems for average particle sizes or
shapes. Small angle scattering was first discovered by Guinier in 1930s during X-ray diffraction
experiments and 30 years later SANS experiments were developed with the work of Stuhrmann
on contrast variation experiments 121,122. Neutrons are produced by two types of sources, first
steady-state reactors where neutrons are produced by fission processes and secondly spallation
sources, where heavy nuclides are subjected to spallation by protons arriving form a high-power
accelerator, often working in pulse mode. Being non-destructive, neutrons do not to alter or
destroy the materials therefore SANS has a great contribution in several areas of the basic and
applied research, for instance in polymer science, biology and materials science 123–125.
Fundamentally the scattering length density, which is a measure of the scattering power of a
material, differences between isotopes especially hydrogen and deuterium, is the main basis of
this method 126.
As described above the scattering length density is the decisive parameter in SANS. For each
sample mixture, SLD values were calculated according to the equation 3.10 using the Scattering
Length Density Calculator of the NIST web page 127.
𝑆𝐿𝐷=∑𝑏𝑖
𝑛
𝑖=1
𝑣𝑚
(3.10)
bi is the sum of scattering length contributions from each species of n atoms in the molecule
and divide by the molecular volume, 𝑣𝑚.The scattering length density of aggregates in the
model fits was calculated as an average of all SLDs of components and weighted by the
corresponding volume fractions.
In a statically isotropic system, the amplitude scattered by different particles is given as:
𝐴(𝑞)=∫ 𝜌(𝑟)𝑒−𝑖𝑞𝑟𝑑𝑟
𝑣
(3.11)
Chapter 3. Materials and Methods
26
In the equation 𝜌(𝑟)is the distribution of length densities in the particle related with the
composition. For averaging the fluctuations 𝜌(𝑟) can be split in two parts 𝜌(𝑟)=〈𝜌〉+𝛿𝜌(𝑟)
and equation 3.11 is modified to 𝐴(𝑞)=∫𝛿𝜌(𝑟)𝑒−𝑖𝑞𝑟𝑑𝑟
𝑣, for q>0 average term is null. The
measured intensity per unit volume is the absolute square of the amplitude and defined as:
𝐼(𝑞)=𝐴(𝑞)𝐴∗(𝑞)
𝑉=1
𝑉∫∫𝛿𝜌(𝑟)𝛿𝜌(𝑟′)
𝑉𝑉 𝑒−𝑖𝑞(𝑟−𝑟′)𝑑𝑟𝑑𝑟′
(3.12)
For a two-phase system 𝜌 becomes to 𝜌p and 𝜌s then equation 3.12 evolves to:
𝐼(𝑞)=1
𝑉(𝜌𝑝−𝜌𝑠)2∫∫𝑒−𝑖𝑞(𝑟−𝑟′)
𝑉𝑝
𝑉𝑝𝑑𝑟𝑑𝑟′=1
𝑉 Δ𝜌2∫∫𝑒−𝑖𝑞(𝑟−𝑟′)
𝑉𝑝𝑑𝑟𝑑𝑟′
𝑉𝑝
(3.13)
The scattering length density difference of particle and matrix is the Δ𝜌 and for N of particle
number
And equation is reorganized to:
𝐼(𝑞)=𝜙𝑉𝑝Δ𝜌2𝑃(𝑞) , P(q) is particle form factor
(3.16)
Additionally, the interference of neutrons scattering from different particles is termed as
structure factor S(q). In the isotropic solutions S(q) is defined by:
𝑆(𝑞)=1+4𝜋𝑁𝑝∫ [𝑔(𝑟)−1]sin (𝑞𝑟)
𝑞𝑟 𝑟2𝑑𝑟
∞
0
(3.17)
𝑔(𝑟) is the pair correlation function of the scattering objects. While the form factor term is
describing the shape and size of the scattering object, structure factor determines the correlation
between particle mass centres. Equation 3.16 is rewritten as
A typical example setup of neutron scattering instrument can be seen in Figure 3.3. After the
production of neutrons from the reactor source, the required wavelength is selected by a velocity
selector. Since neutrons cannot be easily focused, the coming neutron beam is collimated in the
next part and defined with an aperture depending on the cell geometry which is providing a
𝐼(𝑞)=𝑉𝑝2
𝑉𝑁𝑝Δ𝜌2[𝐹(𝑞)]2
(3.14)
𝐹(𝑞)=1
𝑉𝑝∫𝑒−𝑖𝑞𝑟𝑑𝑟
𝑉𝑝
(3.15)
𝐼(𝑞)=𝜙𝑉𝑝Δ𝜌2𝑃(𝑞)𝑆(𝑞)
(3.18)
Chapter 3. Materials and Methods
27
better focus on the sample cell. Later on, the beam interacting with the sample has been
scattered and detected at certain distances, which is called as sample to detector distance (SD).
The choice of configuration in terms of wavelength, collimation and detector combinations, is
important to address the need of information at different q-ranges.
In this study, SANS experiments were performed on 2 different instruments. For all
measurements, quartz cuvettes with path lengths of 1 or 2 mm were used as sample cells and
measured at 25 oC. For simulation and modelling of the data SASfit software was used 128.
Details are described below.
KWS 1, Germany: Measurements were performed on the instrument KWS 1,FRM II, Jülich
Center for Neutron Science (JCNS) at MLZ, Munich, Germany 129. Scattering intensities were
measured with a position-sensitive 6Li scintillation detector of Anger type has 128 x 128 pixels
with a 5.25 x 5.25 mm special resolution. A 50 x 50 mm beamstop is located in the middle of
detector with a small window for a 3He counter, which determines the intensity of the direct
beam for transmission measurements. Measurements were done at a wavelength of 0.6 nm, the
spread of wavelength in our case was given by a FWHM (full width at half maximum) of 9 %,
and three sample-detector distances of 1.2, 7.7, and 19.7 m with corresponding beam
collimation lengths of 8.0, 8.0, and 20.0 m respectively. Transmissions were measured at 8 m
distance with the attenuated direct beam. Water was used as standard to determine the absolute
intensities and detector efficiency. Data reduction was done using the QtiKWS software
developed by JCNS 130 and as background the scattering of the empty cell was subtracted.
D11, France: Spectra were recorded using instrument D11 at the Institute Laue Langevin (ILL)
in Grenoble, France 131 on a 2-dimensional 3He-detector. A wavelength of 0.6 nm (FWHM of
9%), three sample-detector distances of 1.2, 8.0, and 39.0 m with corresponding beam
collimation lengths of 5.5, 8.0, and 40.5 m were employed, respectively. Transmissions were
Figure 3.3. Schematic representation of neutron scattering instrument general setup.
Chapter 3. Materials and Methods
28
measured at 8.0 m distance with the attenuated direct beam. Data reduction was done using the
Lamp software developed by ILL 132. Background the scattering of the empty cell was
subtracted and sample transmissions and dead-times were considered. Scattering of H2O in a 1
mm cuvette was used for obtaining the absolute units.
3.1.6.1 Model Independent Analysis
The scattering vector is reciprocal length of the sample and radiation source. Simply, in a small
angle scattering curve, q scale can be described by three ranges, which are low, middle and
large q. Thus, we consider gaining information of bigger structures at small q values while for
smaller structures, they arise from the large q regime. Depending on the intensity decay, I(q) ∝
q-α, structural geometries of the aggregates can be described and Figure 3.4 illustrates the
geometrical assumptions. In the intermediate q, one predicts for α = 2 discs of two dimensions
and α =1 cylinders of one dimension.
Guinier Approximation is applied independently from the particle structure at very low q. In
the range of qr < 1, P(q) form factor is generalized to a form which described by the forward
scattering I(0) and the radius of gyration Rg 133:
𝑃(𝑞) ~ 𝜙𝑉𝑝𝑒𝑥𝑝(−(𝑞𝑅𝑔)2
3) , (3.19a) lim
𝑞→0𝐼(𝑞)=𝐼0𝑒𝑥𝑝(−(𝑞𝑅𝑔)2
3)
(3.19b)
Figure 3.4. Schematic representation of neutron scattering instrument general setup.
Chapter 3. Materials and Methods
29
Rg, can be obtained from the slope of a plot of ln(I(q)) vs q2. The radius of gyration of a
homogenous sphere of radius R is: 𝑅𝑔2=3
5𝑅2, for the thin spherical shell of radius R, 𝑅𝑔2=
𝑅2and for a cylinder with R radius and length of 𝑙, 𝑅𝑔2=𝑅2
2+𝑙2
12 126. It also has to been kept in
mind that Guinier approximation is limited to low angles (qRG << 1) and valid when the
interparticle interactions are negligible 134.
Porod Law is valid for two-phase systems with sharp interfaces. When the q vector is larger
than the curvature of interfaces, the interfaces appear flat and intensity is proportional to the
surface area, S. Due to the Porod limit at high q range, intensity decays with q-4 135.
𝐼(𝑞)= 2𝜋𝑆∆𝜌2𝑞−4
(3.20)
Kratky-Porod Plot is derived from the intermediate-q Guinier approximation and used for
different geometries in the form of 134,136:
𝐼(𝑞)= 𝐼(0)𝑞−𝛼exp (−𝑞2𝛽)
(3.21)
α has different forms such as 0 for spherical particles, -1 for rod-likes and -2 for flat bilayer.
While 𝛽 is the space average radius of gyration and 𝛽 is Rg2/3 for spherical particles, Rt2 for flat
bilayers. As described above in the part of Guinier approximation, Rg differs for different radii
and thicknesses. For flat bilayers with thickness t, Rt2 = t2/12 and intermediate q Guinier
approximation has the form of:
𝐼(𝑞)= 𝐼(0)𝑞−2exp (−𝑞2𝑡2
12 )
(3.22)
When we plot the ln(q2I(q)) vs q2 middle q range give a straight line with the slope -t2/12, thus
membrane thickness can be extracted from the slope.
3.1.6.2 Model Dependent Analysis
Scattering intensity described above with the Equation 3.18 has two important parameters
which are termed as form and structure factor. While the form factor P(q) describes the
interference of neutrons scattered from different parts of the same object; structure factor, S(q)
represents the interference of neutrons scattered from different objects meaning interparticle
interactions and for very dilute systems S(q) = 0. The form factor, P(q) describes the size and
shape of the scattering object and can be derived for common geometries for instance spheres
Chapter 3. Materials and Methods
30
or spherical shells from analytical expressions. Following part presents the form factor
expressions of different geometries applied for modelling the scattering curves in the present
work.
3.1.6.2.1 Form factor of sphere
The form factor of homogenous spheres and globular objects with radius of R is given as 137:
𝑃(𝑞.𝑅)=[𝐹(𝑞.𝑅)]2, 𝐹(𝑞,𝑅)= [3sin(𝑞𝑅)−(𝑞𝑅)cos (𝑞𝑅)
(𝑞𝑅)3]
(3.23)
3.1.6.2.2 Form factor of cylinders
The form factor P(q) for cylinder with length L and radius R is given as 138:
𝐼𝑐𝑦𝑙=16(𝜋𝑅2𝐿)2∆𝑆𝐿𝐷𝑠2∫[𝐽1(𝑞𝑅√1−𝑥2)
𝑞2𝑅sin (𝑞𝐿𝑥
2)
√1−𝑥2𝐿𝑥]2𝑑𝑥
1
0
(3.24)
where J1 is the first order Bessel function of the first type.
3.1.6.2.3 Form factor of spherical shell
Spherical shell is described with R0 as inner radius, R1 as outer radius (giving 𝐷=𝑅1−𝑅0 as
a shell thickness), and ∆𝑆𝐿𝐷𝑖 is the scattering length density difference between shell and
matrix. It has to be noted that the mean vesicle radius, Rves was defined as the sum of R0 and D,
i. e. the outer vesicle radius.
𝑃(𝑞,𝑅𝑖,∆𝑆𝐿𝐷𝑖)= (∑ 𝐹(𝑞,𝑅𝑖,∆𝑆𝐿𝐷𝑖)
1
𝑖=0 )2 (3.25)
𝐹(𝑞,𝑅𝑖,∆𝑆𝐿𝐷𝑖)=4
3𝜋𝑅𝑖3∆𝑆𝐿𝐷𝑖(3sin (𝑞𝑅𝑖)−𝑞𝑅𝑖𝑐𝑜𝑠(𝑞𝑅𝑖)
(𝑞𝑅𝑖)3) (3.26a)
Chapter 3. Materials and Methods
31
The scattering intensity was calculated by an integrating over the contained distribution of
particle sizes as described with a log-normal distribution.
𝐼= 𝑁 ∫𝐿𝑁(𝑅0,𝑅𝑣𝑒𝑠,𝜎)𝑃(𝑞,𝑅0,𝐷,∆𝑆𝐿𝐷𝑖)𝑑𝑅0+𝐼𝑖𝑛𝑐
∞
0
1 (3.26b)
3.1.6.2.4 Log-normal size distribution
Since our system consists of different aggregate types, for a detailed understanding we
employed different form factors of different geometries taking into consideration their
polydispersity by assuming a log-normal distribution function which was described as:
𝐿𝑁(𝑅0,𝑅𝑣𝑒𝑠,𝜎)= 1
𝑅0𝜎√2𝜋𝑒𝑥𝑝(−(ln(𝑅0)−(𝑅𝑣𝑒𝑠−𝐷))2
2𝜎2) (3.27)
σ is the variance. Additionally, in the SASfit software 128, the volume fractions fp has been
implemented in the log-normal size distribution by integrating over the particle volume 𝑉𝑝:
𝑓𝑝=∫𝐿𝑁(𝑟,𝜇,𝜎)𝑉𝑝(𝑟)𝑑𝑟
∞
0 (3.28)
Volume fractions of samples Φ, were calculated from the total concentration of amphiphilic
materials compounds used in each sample Ctot multiplied by the sum of the molar mass Mi and
the density 𝜌i of each component i:
Φ= 𝐶𝑡𝑜𝑡∑𝑀𝑖
𝜌𝑖 (3.29)
Figure 3.5. Spherical shell model.
Chapter 3. Materials and Methods
32
3.1.6.2.5 Instrumental resolution
The experimental smearing was accounted in the intensities I, by the resolution function
assuming a Gaussian function 139:
𝐼(𝑞)=1
√2𝜋𝜎q(𝑞)∫𝑒𝑥𝑝
+∞
−∞ (−[ 𝑞′−𝑞
√2𝜋𝜎q(𝑞)]2)𝐼 (𝑞′)𝑑𝑞′
(3.30)
The standard deviation σ(q)/q was taken into account for the instrument resolution via:
(𝜎(𝑞)
𝑞)2=(𝜎(𝜆)
𝜆)2+(𝜎(𝜃)
𝜃)2
(3.31)
q is defined as the magnitude of scattering wave vector q = (4π/λ)sin(𝜃/2) with 𝜃 being the
scattering angle. In equation 3.30 first contribution is the q dependent wavelength spread Δqλ =
(Δλ/λ) q and was related to the FWHM (full width at half maximum).
3.1.6.2.6 Structure factor
In equation 3.18 S(q) defines the inter-particular interaction. We used the structure factor of
hard sphere to describe the scattering curves. It can be solved in the Percus−Yevick
approximation as closure relation to solve the Ornstein-Zernike equation to yield an analytical
expression 140. Here the S(q) determines a pair interaction potential U(r) with the repulsion
radius r.
𝑈(𝑟)={∞,0<𝑟<𝜎
0, 𝑟>𝜎 , 𝑆𝐻𝑆(𝑞, 𝑅𝐻𝑆,𝑓𝑝)= 1
1+24𝑓𝑝𝐺(𝑞,𝑅𝐻𝑆,𝑓𝑝)
𝑅𝐻𝑆𝑞
(3.32)
The expansion of the structure factor can be explained as
𝐺(𝑞)=𝛼sin𝐴−𝐴cos𝐴
𝐴2+𝛽2𝐴sin𝐴+(2−𝐴2)cos𝐴−2
𝐴3+
𝛾−𝐴4cos𝐴+4[(3𝐴2−6)cos𝐴+(𝐴3−6𝐴)sin𝐴+6]
𝐴5
(3.33)
𝛼=(1+2𝑓𝑝)2
(1−𝑓𝑝)4, 𝛽=−6𝑓𝑝(1+𝑓𝑝
2)2
(1−𝑓𝑝)4, 𝛾=𝑓𝑝𝛼
2 and 𝐴=2𝑅𝐻𝑆𝑞 (3.33a)
Practically this approximation depends on the hard sphere radius RHS and hard sphere volume
fraction𝑓𝑝. 𝑓𝑝. In our system the cylindrical particles occupy larger hard sphere volume. We
considered the RHS a sum of the particle diameter and two hydrophilic chain lengths (-EO11-) of
L35 attached on the aggregate. According to de Gennes approximation, the area occupied by
single PEO chain was calculated before with assuming the PEO chains of L35 form mushroom-
Chapter 3. Materials and Methods
33
like conformations when they attach on the surface of the aggregate 115,141. The interactions
were determined by fitting this model.
3.1.7 Neutron Spin-Echo Spectroscopy (NSE)
Neutron spin echo is a type of time-of-flight spectrometer whereby one can determine the
energy changes along with the scattering. Although it is an inelastic neutron scattering method
invented by Mezei 142,143, it differs from other conventional techniques in the way of measuring
singular incident and scattered neutron velocities considering the Larmor precession of the
nuclear neutron spin in a magnetic field. This spin vector works like an internal clock which
relates the individual neutron to its velocity, and by this way one measures directly the velocities
before and after the scattering.
In general, while the incident neutron beam (generally with a wavelength interval of 10-20%
width) is polarized through the guide direction, it is subjected with a spin flip of 𝜋/2 which
leads the polarization to a vertical direction via Larmor precession. However, because of the
different neutron velocities thereof the flight times, the final beam reaching to the sample loses
the polarization. When the beam hits the sample, it passes through a 𝜋 flipper and goes towards
the second precession field, which thereafter is applied for aligning the position of different
spin via another 𝜋/2 flipper. Finally, it restores the initial polarization again. Figure 3.6
represent a general scheme of neutron spin echo spectrometer.
This technique allows decoupling the energy resolution of the experiment from the
monochromaticity of the beam, which allows to detect energy transfers as small as a few
nanoelectonvolts 144. This provides highest energy resolution and differently it measures the
intermediate scattering function S(q,t) which makes it useful to investigate the dynamic
measurements, and slow relaxations. The scattering functions are related to the coherent or
incoherent intermediate scattering function by Fourier transformation 144 and by this way the
dynamic structure function S(q,ω), can be converted to S(q,t):
𝑆(𝑞,𝜔)=∫exp(𝑖𝜔𝑡)𝑆(𝑞,𝑡)𝑑𝑡
(3.34)
The spin manipulations of the NSE spectrometer serve to detect miniscule energy transfers
during scattering in a way that finally yields the intermediate scattering function which is related
to the time-dependent coordinates of the nuclei in the system 145:
Chapter 3. Materials and Methods
34
𝑆(𝑞,𝑡)=1
𝑁∑exp (−𝑖𝑞(𝑟𝑖(0)−𝑟𝑗(𝑡)
𝑁
𝑖,𝑗 ))
(3.35)
Details of the neutron spin echo method can be found in the literature 142,146–148.
The NSE experiments in this thesis were performed on the instrument IN15 of the ILL, which
provides highest resolution with longest Fourier time worldwide, at Grenoble (France). Samples
are measured in quartz cuvettes (Hellma) with path lengths of 1 mm and measured at 25 oC.
The Fourier time was ranging up to 532 ns by varying the wavelength of 14, 10 and 6 Å thereof
covering a q-range from 0.014 to 0.14 1/Å.
3.1.8 Cryogenic Transmission Electron Microscopy (cryo-TEM)
Transmission electron microscopy (TEM) measures the structures in real space providing a high
resolution of submicron structures. Fundamentally, an electron beam is focused on the vitrified
sample while the light passes through it, the transmitted beam forms the image of the aggregate.
TEM consists of illumination and imaging part. The first part consists of an electron source
where electrons are emitted from a heated filament, and the electron density can be controlled
via an applied voltage. A high voltage field accelerates the emitted electrons through the
condenser lens. The imaging part provides the representation of the sample under high vacuum
to prevent the electron gas interaction. This part is formed from objective, projector lens and
corresponding apertures, the phosphor viewing screen and the photographic film. When the
transmitted electron beam is focused on the objective lens, an image forms on the phosphor
image screen and with the help of the generated light, image can be seen.
Figure 3.6. Schematic representation of neutron spin echo instrument general setup.
Chapter 3. Materials and Methods
35
Since this technique needs specific preparations for instance on a grid made of copper, gold or
nickel, it does not allow to image samples in their native environment. Thus, cryogenic
Transmission Electron Microscopy (cryo-TEM) has been developed for investigating the
nanostructured materials in aqueous solutions.
3.1.8.1 Sample Preparation
Cryo-TEM images were taken from two different measurements. Imaging of the polymerized
vesicle sample was performed at Technion, Haifa (Israel). Here, the samples were prepared in
controlled environment vitrification system (CEVS) at 25°C and humidity at saturation to
prevent evaporation from the specimen. Prior to specimen preparation, the grids were plasma
etched in a PELCO EasiGlow glow-discharger (Ted Pella Inc., Redding, CA) to increase their
hydrophilicity. Inside the CEVS chamber, a carbon-coated perforated polymer film, supported
on a 200 mesh TEM grid was held by tweezers. A drop of the sample was placed on the film
and blotted by a filter paper-covered metal strip to remove excess solution and form a thin film
of liquid. The specimen was then vitrified by quickly plunging it into liquid ethane at its freezing
point. After vitrification, the sample is kept in liquid nitrogen until transfer into the TEM for
imaging. Imaging was performed using a FEI Tecnai T12 G2 transmission electron microscope.
The microscopes are equipped with LaB6 electron gun and operates at 120kV. Specimens were
equilibrated in the microscope below -178 °C in Gatan 626 cryo-holders and imaged using a
low-dose imaging procedure to minimize electron-beam radiation damage (where the electron
exposure in a recorded micrograph is less than 1000 nm. A Gatan US1000 high-resolution
cooled-CCD camera recorded the images at exposure time of 1 second using the
DigitalMicrograph software package.
Images of the unpolymerized sample were taken at TEM laboratory of MLZ (JCNS) Munich,
Germany. Similar as described above, the specimen was vitrified by plunging the sample into
liquid ethane and prior to vitrification it was maintained at 20 °C and 80% relative humidity.
Here, the sample is deposited on holey carbon grids and grids were replaced to a Gatan 910
multi-specimen holder.
3.1.9 Rheology
Rheological properties of self-assembling systems provide crucial information for their
potential applications as new materials 149–151. In general, dilute surfactant systems usually
behave as Newtonian fluid, which has a linear relationship between shear stress and shear rate,
Chapter 3. Materials and Methods
36
thus the viscosity remains constant and related to the concentration and shape of aggregates. In
contrast, for non-Newtonian fluids the viscosity of the fluid changes when shear is applied.
Some surfactant solutions show more complicated rheological behaviour being elastic and
viscous at the same time and depending on the shear rate. Oscillatory measurements provide
detailed information about the viscoelastic properties by determining storage (elastic) modulus
G' and loss (viscous) modulus G''. The two moduli can be described by the Maxwell model 152:
𝐺′(𝜔)= 𝜔2𝜏𝑅
2
1+𝜔2𝜏𝑅
2𝐺0 and 𝐺′′(𝜔)= 𝜔𝜏𝑅
1+𝜔2𝜏𝑅
2𝐺0
(3.36)
Rheological measurements were performed using a Malvern Bohlin Gemini 200 HR nano
rheometer. Measurements were done at 25°C with the cone-plate- geometry at the cone angle
of 4° and the cone diameter of 40 mm. The gap size between cone and plate was set to 150 μm.
A pre-shear of 1 s-1 was given to the sample for 30 seconds, subsequently the sample was left
to rest for 3 minutes before measurements. For the shear experiments, shear rate was varied
from 1 – 70 s-1 and the experimental data was analysed with Carreau-Yasuda model (equation
3.37) 153:
𝜂=𝜂0[1+(𝜏𝛾)𝑎]𝑛−1
𝑎
(3.37)
η0 is the zero-shear viscosity, the relaxation time τ, the constant a, and n is the power law
exponent (for n < 1 one observes shear thinning). The relation (equation 3.38) between zero
shear viscosity and relaxation time leads to the shear modulus G0, corresponding the plateau of
loss modulus G’.
From the oscillatory experiments, the viscoelastic response of polymerized sample was
obtained by means of the loss (G') and storage (G'') modulus; therefore, a frequency sweep was
applied between 0.006-10 Hz.
3.1.10 Nuclear Magnetic Resonance Spectroscopy (NMR)
Polymerization was followed by using 1H NMR for confirming the full conversion of monomer
with vanishing the proton signals of monomers. A Bruker Avance II 400 spectrometer operating
at 400 MHz was used to recording the spectra. As the solvent, D2O was used to obtain the
chemical shifts and Tetramethylsilane (TMS) was used as reference agent.
𝐺0=𝜂0
𝜏
(3.38)
Chapter 3. Materials and Methods
37
3.1.11 UV-vis Spectrometry
UV-vis spectroscopy has been used to measure the transmissions of the samples for obtaining
the turbidity values. Measurements were performed on a Varian Cary 50 UV-vis
spectrophotometer. Rectangular Hellma quartz cuvettes with a thickness d= of 1 mm were used
for measurements and data was scanned over a range of 350-800 nm wavelength. For each
sample, measurements were repeated three times at 25 °C.
3.1.11.1 Turbidity and Transmission
Turbidity values are calculated from the transmission T observed from UV-vis measurements
according to the equation:
𝜏=−ln(𝑇)
𝑑
(3.39)
The following relations define the relation between the transmission and the molecular weight
of the aggregates:
𝐾=32𝜋3𝑛𝑜
2(𝑑𝑛
𝑑𝑐𝑔)2
(3𝑁𝑎𝑣λ4)
(3.41)
Here, λ is the wavelength, n the refractive index of the solvent, NAv is the Avogadro constant,
c the weight concentration of the samples, dn/dcg the refractive index increment, and ρ the
density of the aggregates.
3.1.12 Fluorescence Spectroscopy
Fluorescence is the emission of light by a substance (fluorophores) which is absorbing light.
When the species are absorbing photons of sufficiently high energy, they become excited from
the ground electronic state to an excited electronic state. After staying short time in this excited
state, the molecule returns to its stable ground state with transferring its energy in the form of
emitting light of longer wavelength than that of the initial light. Typical fluorophores are mostly
aromatic molecules. Most important characteristics of a fluorophore is the lifetime and quantum
yield. The lifetime of the fluorophore is the duration of the excited state of the fluorophore
before returning to its ground state. Moreover, the quantum yield is the ratio of the number of
emitted protons to the number of absorbed photons.
𝑀𝑤𝑒𝑓𝑓=𝜏
𝐾∗𝑐𝑔
(3.40)
Chapter 3. Materials and Methods
38
Quenching is a process that reduces the fluorescence intensity of a substance. Different
molecular interactions such as molecular rearrangements, complex formation, energy transfer,
or collision can be result with quenching. Molecular oxygen 154, metal ions 155,156 are common
quenchers have been used widely in the literature. Stern-Volmer equation 3.42 describes the
collisional quenching theoretically 157:
𝐹0
𝐹=1+𝑘𝑞𝜏0[𝑄]=1+ 𝐾𝐷[𝑄]
(3.42)
Where 𝐹0 and 𝐹 are the fluorescence intensities before and after the addition of quencher,
respectively, 𝑘𝑞 is the bimolecular quenching constant, 𝜏0 is the lifetime of the fluorophore and
Q is the concentration of the quencher. 𝐾𝐷 is the Stern-Volmer constant which signs the
sensitivity of the fluorophore to the quencher. Depending of the variety of quenchers, the
fluorophore-quencher combinations can be determined for a desired aim.
Steady-state fluorescence measurements were performed using a fluorescence
spectrophotometer Hitachi FL 4500. Fluorescence intensities were recorded in the region from
490 to 600 nm with an excitation wavelength of 495 nm and an emission wavelength of 515
nm in the scan mode of emission. The excitation and emission slits were set at 2.5 nm and scan
speed was chosen as 240 nm/min. For each sample, measurements repeated three times (in10
mm × 10 mm × 45 mm quartz cell) at 25 °C.
Figure 3.7. Absorption and emission of light schematically for
two shifted potential energy surfaces.
Chapter 3. Materials and Methods
39
3.2 Materials and Experimental
3.2.1 Chemicals
Table 3.1 present the surfactants used in this thesis, and details will be explained below.
Tetradecyl(dimethyl)amine oxide (TDMAO, cmc = 0.12 mM) was kindly provided by Stepan
company (Illinois, USA) as a 25% TDMAO solution in water named as Ammonyx M. This
solution was freeze-dried and used without any further purification. Pluronic L35 (L35,
EO11PO16EO11, Mw = 1900 g/mol, on average but as a polymer being polydisperse with respect
to its molecular composition) was kindly given by BASF SE (Ludwigshafen, Germany).
Lithium perfluorooctylsulfonate (LiPFOS, cmc = 6.3 mM) was purchased from TCI Europe
Pluronic L35
Tetradecyl(dimethyl)amine oxide
Lithium perfluorooctylsulfonate
Figure 3.8. Chemical structures of surfactants used in this study.
Table 3.1. Surfactants used in this thesis, with their common name, abbreviation,
molecular formula, molecular weight, density and scattering length densities.
Chapter 3. Materials and Methods
40
(purity > 96%). Samples for small-angle neutron scattering (SANS) experiments were prepared
using D2O (D content > 99.9 %) from Eurisotop company (France). All the stock solutions used
in this work were prepared by dissolving the proper mass amount of the compounds either in
Millipore or D2O.
Additionally, compounds used for polymerization reactions such as monomers, cross-linkers
and photo-initiators are tabulated in Table 3.2. In details, styrene (C8H8, >99% GC, contains
0.005% 4-tert-butylcatechol as stabilizer), hexyl acrylate (98%, contains 100 ppm
hydroquinone as inhibitor), dodecyl acrylate (technical grade 90%, contains 60-100 ppm
monomethyl ether hydroquinone as inhibitor), isooctyl acrylate (>90%, contains 75-125 ppm
monomethyl ether hydroquinone as inhibitor,) monomers, 1,6-hexanediol diacrylate (technical
grade 80%, 100 ppm monomethyl ether hydroquinone as inhibitor), and divinylbenzene
(technical grade 80%, 1000 ppm p-tert-butylcatechol as inhibitor) as cross-linking agents were
all purchased from Sigma-Aldrich. All monomers and cross-linkers were washed through
inhibitor remover column before usage and stored dark in the fridge. Therefore, flash columns
with 20 cm height and 3 cm diameter size were produced in-house, filled with initiator remover
(Sigma Aldrich- CAS:311332) up to 5 cm height. While monomers were washing through it,
the column was covered with aluminum folio and worked in the dark.
Chapter 3. Materials and Methods
41
Benzoyl peroxide, 2,2-Dimethoxy-2-phenylacetophenone (99%) and Azoisobutyronitrile
(98%) were used as water insoluble photo-initiator (Figure 3.10) and received from Sigma-
Aldrich. These compounds were used without further purification.
Additionally, calcein (λex 470 nm; λem 509 nm at pH 7.4) was used as water-soluble
fluorescence dye and Cobalt (II) chloride (anhydrous) as the quenching agent. Both were
purchased from Sigma-Aldrich.
Figure 3.9. Chemical structures of monomers and cross-linkers used in this study.
Chapter 3. Materials and Methods
42
Figure 3.10. Chemical structures of photo-initiators used in polymerization reactions
in this study.
Figure 3.11. Structural presentation of top) poly(styrene-co-divinylbenzene); bottom)
poly (hexyl acrylate-co-1,6-hexanediol diacrylate).
Chapter 3. Materials and Methods
43
Compounds Synonym Molecular
formula Mw/ (g/mol) / (g/ml)
at
25 C
Water
Solubility/
at
25 C
(g/L)
SLD /
(1010cm-1)
Styrene - C8H8104.15 0.906 0.3 1.21
Divinylbenzene DVB C10H10 130.19 0.914 0.052 1.224
Hexyl acrylate HA C10H10 156.22 0.888 0.4 0.397
1,6-hexanediol
diacrylate HDODA C12H18O4226.27 1.01 <0.1 0.623
Butyl acrylate BA C7H12O2128.17 0.89 0.01 0.554
Dodecyl acrylate
Lauryl
acrylate C15H28O2254.41 0.86 0.0015 0.144
Isooctyl acrylate IOA C11H20O2184.28 0.88 0.001 0.286
Benzoyl peroxide BPO C14H10O4242.23 1.33 <1 2.60
2,2-Dimethoxy-2-
phenylacetophenone DMPA C16H16O3256.3 1.122 insoluble 1.68
Azobisisobutyronitrile
AIBN C8H12N4164.21 1.1 <0.1 1.84
Table 3.2. Monomers, cross-linkers, photo-initiators used in this thesis, with their
common names, abbreviations, molecular formula, molecular weights, density,
water solubility and scattering length densities.
Chapter 3. Materials and Methods
44
3.2.2 Sample Preparation
The basic model system in our work composed of nonionic TDMAO, pluronic L35 (1 mol %
with respect to TDMAO) and anionic LiPFOS surfactants. Vesicles were formed from these
mixtures with the final concentrations of 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM
LiPFOS.
TDMAO/L35/Monomer
Dissolving the hydrophobic monomers into the vesicle membrane was the challenging issue
during this study since the outside media was water. To prevent aging and provide better
dissolving, concurrent loading was efficient. Therefore, concurrent loading of monomers was
chosen in which case monomers (in the case of polymerization, details will be described below)
were dissolved in 50 mM TDMAO / 0.5 mM L35 stocks prior to vesicle preparation. 50 mM
TDMAO / 0.5 mM L35 stocks / varying concentrations of monomers were prepared in millipore
water or in D2O for SANS experiments. Samples were equilibrated by stirring for 3 days at
room temperature in the dark.
TDMAO/L35/Monomer/LiPFOS
Concurrent monomer loaded vesicles were prepared by mixing monomer loaded TDMAO /
L35 stock solutions described above with 50 mM LiPFOS stocks at the molar ratio of 55:45.
Consequently, monomer loaded vesicles with the final concentration of 27.5 mM TDMAO /
0.275 mM L35 / 22.5 mM LiPFOS / different monomer concentrations were freshly prepared
before experiments.
Polymerization of vesicles from TDMAO/L35/Monomer/LiPFOS
Polymerized samples were prepared by dissolving 3% mol water-insoluble initiator (varying
due to the total monomer concentration) in monomer/cross-linker mixtures. The cross-linker
ratio was adjusted to three different values 0.1, 0.2 and 0.4 with respect to the monomer
concentrations. These mixtures were added to 50 mM TDMAO / 0.5 mM L35 and homogenized
by stirring for 3 days at room temperature in the dark. Then the solution was taken into a three-
neck round bottom flask and was degassed with nitrogen for one hour to remove dissolved
oxygen from the system (Figure 3.12). The flask was placed into in-house made thermostated
water bath at 18 °C. As mentioned above, the model system had the molar ratio of TDMAO
and LiPFOS as 55:45. For the preparation, the calculated amount of 50 mM LiPFOS was taken
with the help of syringe and added in one stroke from the side neck into the flask while reaction
Chapter 3. Materials and Methods
45
mixture was stirring vigorously with a PTFE stir bar. Spectroline Quartz Pencil shape UV lamp
with wavelength of 365 nm inserted into the reaction flask from the middle neck up until it is
fully submerged in the reaction mixture but does not touch the stirrer. All these steps were done
in the dark. After adding the LiPFOS, the reaction mixture was let to stir for 1 minute then
immediately the UV lamp was turned on. Reaction was run over the night. Polymerization was
followed by NMR and run 15 hours for polymerization of styrene and 18 hours for the case of
hexyl acrylate under nitrogen atmosphere with moderately stirring at 18 °C in the dark.
Figure 3.12. Representation of polymerization reaction.
Chapter 3. Materials and Methods
46
4 TDMAO/L35/Styrene System
Introduction
Surfactants have a tendency for self-assembly in water to spherical or rod-like micelles, or flat
bilayers or vesicles depending on the packing parameter described in chapter 1.1 3. Especially,
rod-like micelles are of great interest because of their rheological properties and viscoelastic
behavior 27,35 and their potential applications 149,158,159.
Therefore, such rod-like systems were studied where they were used as templates and by
systematically varying the reaction conditions, the dimension of the final aggregates can be
controlled 160,161. Due to the ease of styrene’s polymerization, its solubilization into the micellar
solutions has been preferred usually and studied before 162. Soltero et al. examined the
polymerization of styrene in wormlike micelles of the cationic CTAT surfactant and concluded
the wormlike structures were fixated by polymerization at the same time with the formation of
spheroidal polymer particles depending on the concentration 163. Another study on
microemulsion polymerization of styrene stabilized by DTAB in water yielded the formation
of small nanolattices instead of bigger polymeric particles 164.
In the present chapter, the effect of styrene monomer on the rod-like micelles consisting of
nonionic tetradecyldimethylamine oxid (TDMAO) and Pluronic L35 were investigated. Here,
L35 was included in order to stabilize sterically the former self-assembled structures 114 and
styrene monomer was added in order to serve as starting point for templating via
polymerization. The interaction of different additives with micellar TDMAO was studied
before and have seen that in the presence of medium chain alcohols such 1-hexanol, leads to
the formation of cylindrical aggregates and then to bilayers 165 which can be considered as
cosurfactants. These hydrophobic moieties can either insert into the interior of micellar
aggregates or become incorporated within the palisade layer of self-assembled structures and
by this way the packing parameter can be changed.
In our study, we investigated the structural effect of styrene monomer on the micellar
TDMAO/L35 system, where styrene is at the same time hydrophobic and having a cosurfactant
character (Figure 4.1). Our investigations concluded to an unusual finding that styrene promotes
a transition from micellar to vesicular structures, i.e., here the cosurfactant character dominates
and behaves in a similar fashion as has been observed for medium chain alcohols such as 1-
hexanol 51,166. This phenomenon was supported and confirmed with light scattering, turbidity,
Chapter 4. TDMAO/L35/Styrene System
48
and small angle neutron scattering techniques. For these self-assembled structures, the effect of
subsequent styrene polymerization is explained in section 4.2. and resulted in the formation of
rod-like aggregates with corresponding high viscosity in solution.
4.1 TDMAO/L35 /Styrene
4.1.1 Phase Behaviour
Co-surfactants have an effect on decreasing the spontaneous curvature of interfaces when they
are added to the micellar aggregates. By virtue of their occupied area during the formation of
the aggregates, interfacial curvature changes and ends up with different aggregate types and
geometries 167,168. Basically, system adjusts the two principal curvatures of the micelles to the
same as spontaneous curvature with keeping the bending energy as low as possible. With
increasing amount of additive, then system shifts from micelles to spheres and rods or to
bilayers. These transitions can be simply monitored by phase behaviour of the samples.
In the first step, we investigated the phase behaviour of the system 50 mM TDMAO / 0.5 mM
L35 as a function of the concentration of monomer styrene. Styrene has a low water solubility
of ~ 3 mM 169 and at the same time high volatility. Thus, when styrene was added into the
TDMAO/L35 solution, sample closed firmly, a PTFE magnet added in the bottle and stored in
the dark at room temperature. This was repeated for all samples for increasing amount of styrene
and stirred continuously for 3 days until the entire monomer was dissolved into the solutions.
Finally, after 3 days of stirring they had a homogeneous appearance up to 230 mM styrene
concentration. After this point, a two-phase system formed. These systems were followed by
Figure 4.1. Schematic representation of TDMAO/L35/styrene system.
Chapter 4. TDMAO/L35/Styrene System
49
visual inspection after 1 hour, 7 days and 2 weeks after preparation in terms of homogenization
and ageing.
Firstly, the 50 mM TDMAO / 0.5 mM L35 sample without styrene formed a visually transparent
single-phase solution. When styrene was introduced into the system the transparent, single
phase, isotropic micellar solutions (L1) remained up to 50 mM styrene concentration as seen in
Figure 4.2. From 50 to 120 mM styrene content, system shifted to another phase. The samples
in this range had a bluish tinge, which is typically an indication of the presence of vesicles (Lves)
170,171. This bluish appearance is because of the scattered light by the bigger aggregates 172.
Additionally, vesicle formation in this intermediate styrene range was confirmed by scattering
methods as well.
Secondly, between 120 - 230 mM monomer content, the bluish appearance of previous range
was replaced by a less turbid phase, which may be assigned as L1-phase, simply an O/W
microemulsion. With the addition of more styrene, formed vesicles were ruptured and evaluated
into microemulsion droplets. Moreover, for higher than 230 mM styrene content, the turbidity
increased drastically to white, milk-like appearance. The presence of 2 phase region was started
around 240 mM, the undissolved monomer separated into an upper phase within a few hours
(2Φ), while shaking the samples made the whole sample white turbid again.
We measured the UV-transmissions of the samples 1 hour and 1 week after preparation and the
turbidity values were calculated from these experiments. Figure 4.3. shows turbidity as a
function of styrene concentration. The first part (L1), seen in the figure corresponds to the
Figure 4.2. Sample photographs of 50 mM TDMAO / 0.5 mM L35 / styrene mixtures (20
mM – 475 mM content) at 25 °C.
Chapter 4. TDMAO/L35/Styrene System
50
transparent micellar regime as describe above. The turbidity of the micellar solutions increases
very slightly with introducing styrene into the system, however, for the 50 to 120 mM styrene
content a sharp increase in the turbidity values is observed. Styrene, here, has an effect on
shifting the packing parameter and induces the transition from micelles to vesicular aggregates.
Later on, with the addition of more monomer to the system, turbidity decreases again to the
similar values observed in the first L1 phase, confirming the return to the O/W microemulsion
phase. Measurements were repeated after 1 week from preparation, and we obtained similar
results meaning samples remained stable, almost unchanged.
4.1.2 Light Scattering
Dynamic and static light scattering analyses reveal more detailed information about the nascent
structures and the structural transitions. Varying the amount of styrene, we determined the
hydrodynamic radius Rh, polydispersity index PDI, and molecular weight Mw of the aggregates.
The autocorrelation functions of the samples seen in Figure 4.4 indicate an apparent shift to the
longer relaxation times. The relaxation processes of the curves are mainly monomodal however,
the decay time below 50 mM styrene increases significantly, thereby indicating a growth of the
micelles. Above 50 mM, the characteristic time increases, and the curves become more
stretched. This implies slower particle diffusion i.e., the presence of bigger particles in the
system with broader size distribution confirms the formation of vesicles with high
polydispersity.
Figure 4.3. Turbidity (λ=632 nm) as a function of styrene concentration in 50 mM
TDMAO / 0.5 mM L35/ styrene mixtures at 25°C. (green circles): after 1 hour of mixing;
(orange triangle): after 1 week of mixing.
Chapter 4. TDMAO/L35/Styrene System
51
Upon further increase of the styrene concentration, one shifts back to a monomodal fast decay
for 90 to 150 mM. However, between 150 and 200 mM monomer amount, curves become
stretched, but now having shorter characteristic times. At 200 mM concentration the
characteristic behavior of scattering curve changes completely and having bimodal behavior at
this point. The second mode appears at the concentration close to where the phase behavior
starts to shift from microemulsion (L1) to 2-phase region (2Φ).
Dynamic light scattering of the samples was measured at seven different angles 30, 45, 60, 75,
90, 105 and 120°. The cumulant method was used to analyze the scattering curves for obtaining
the average aggregate size and polydispersity index of their distribution. For the sample without
styrene, a hydrodynamic radius, Rh of 3.1 nm was observed and with introducing monomer into
the system this value increased to 6-7 nm. Clearly, this is because of the swelling of micelles
by loading with monomers which ends up in a more elongated cylindrical structure. For more
than 50 mM styrene, hydrodynamic radii for vesicle reach a value of 70 nm together high
polydispersity. The loaded micelles show polydispersity indices of 0.1-0.2, while for the
vesicles values of ~ 0.2 are observed. Figure 4.5 (left) shows the evolution of hydrodynamic
radii of the aggregates of TDMAO/L35 solutions with different amounts of dissolved styrene
for repeated measurements of 1 week and 2 weeks after preparation. Below 50 mM styrene
concentration, small aggregates with hydrodynamic radius of 6-8 nm were formed. However,
for 50 to 120 mM styrene concentration Rh increased drastically. Evidently, this increase of the
size confirms the transition from smaller structures to relatively big ones, in this case from
Figure 4.4. Intensity autocorrelation function g2(t) measured at θ = 90° of 50 mM
TDMAO / 0.5 mM L35 / styrene mixtures at 25°C.
Chapter 4. TDMAO/L35/Styrene System
52
micelles to vesicles. For more than 120 mM styrene, hydrodynamic radii become smaller again
to 5-6 nm confirming the disruption of vesicular aggregates to the small droplets.
As an alternative way to determine the effective diffusion coefficients, we obtained them from
the initial slopes of the scattering curves. Therefore, we replotted (Figure 4.6) the
autocorrelation functions ln((g2/t)-1) vs time and from the initial slopes (t = 0-0.06 ms) , we
obtained the effective diffusion coefficient via:
𝑔2(𝑡)−1=𝐴exp(−2𝐷𝑞2𝑡)
(4.1)
ln(𝑔2(𝑡)−1)=𝑙𝑛𝐴−2𝐷𝑞2𝑡
(4.2)
Plots showing the correlation functions of the samples above 50 mM styrene do not decay
linearly which is the sign of high polydispersity. Collaboratively, the polydispersity values from
cumulant method seen in Table 4.2 are in good agreement with being ~ 0.2.
For comparison of the results from the DLS and SANS analyses, we calculate the theoretical
diffusion coefficients using the size parameters obtained from SANS with the equation:
𝐷=𝑘𝑇(ln(𝑝)+𝛾)/3𝜋𝜂𝐿
(4.3)
where 𝜂 is the solvent viscosity, and 𝛾=0.312+0.565
𝑝+0.1
𝑝2, 𝑝=𝐿/2𝑅, L being the length of
the cylinder and R is the radius 173.
Figure 4.5. (left): Hydrodynamic radii, Rh, of aggregates in 50 mM TDMAO / 0.5 mM L35 /
styrene solutions at 25°C. (Rh from cumulant method: orange circle:1-week, purple triangle: 2
weeks after preparation). (right): Molecular weight, Mw, of aggregates in 50 mM TDMAO / 0.5
mM L35 /styrene solutions at 25°C.
Chapter 4. TDMAO/L35/Styrene System
53
Diffusion coefficients from initial slopes, cumulant method, and theoretically calculated D0
from the obtained SANS parameters are listed in Table 4.1. As seen in the table, values are
decreasing along with the increase of styrene concentration. The slower diffusion of the
particles indicates the formation of bigger particles. For the first L1 phase, diffusion coefficient
values are in the range of ~10–11 m2s–1, however for the vesicle phase, Lves, corresponding 50
mM -120 mM styrene concentration diffusion coefficient values decreases to the order of ~10–
12 m2s–1, which is much smaller than expected for micelles 174. Furthermore, for the samples
with more than 120 mM styrene, diffusion coefficient values started to increase and reached to
Figure 4.7. Intensity vs q2 for 50mM TDMAO/0.5mM L35/styrene mixtures at 25 °C,
Guinier plots of the SLS data.
Figure 4.6. Autocorrelation functions g2(t) measured at θ = 90° of 50 mM TDMAO / 0.5
mM L35 / styrene mixtures at 25°C (solid lines: linear fit).
Chapter 4. TDMAO/L35/Styrene System
54
the order of ~10–11 m2s–1 again which is in the same range as for the L1 phase, indicating the
presence of smaller aggregates.
From the static light scattering (SL) measurements, the absolute intensities I(q), were obtained
and plotted against q2 (Figure 4.7). Scattering curves were fitted with the Guinier
approximation: 𝐼(𝑞)=𝐼0𝑒𝑥𝑝−(𝑅𝑔𝑞)2
3 to extrapolate the forward scattering I(q=0).
Deduced absolute intensities were used for the calculation of apparent molecular weights via
equation 3.8 and listed in Table 4.2. Similarly, the molecular weights, Mw of the turbid samples
was calculated from equation 3.40 relating it to the turbidity measurements, and they are
compared to SLS results in Figure 4.5 (right). Due to the increase in size, molecular weight
increases until the end of vesicular regime up to 120 mM styrene content. For higher styrene
concentrations, the molecular weights decreased according to the formation of droplets of
smaller sizes. Results are summarized in Table 4.2.
To compare the outcome from the scattering analyses, we calculate the theoretical radii of
vesicles considering the membrane thicknesses gained from SANS analyses therefore
estimating the vesicular volume for the aggregate densities (see Appendix 9.1.3). We observe
Styrene
amount
(mM)
Deff (m2/s)
from
initial
slope
Deff
(m2/s)
from
cumulant
D (m2/s)
from
SANS
0
4.05x10-11
5.6x10-11
6.8x10-11
10
5.59x10-11
5.6x10-11
3.5x10-11
20
1.92x10-11
3.0x10-11
2.6x10-11
30
1.53x10-11
2.6x10-11
2.3x10-11
40
1.47x10-11
1.9x10-11
1.8x10-11
50
4.02x10-12
5.1x10-12
-
60
7.44x10-12
7.5x10-12
-
70
4.9x10-12
3.9x10-12
-
80
6.1x10-12
5.1x10-12
-
90
1.29x10-11
9.0x10-12
-
100
8.87x10-12
7.9x10-12
-
120
5.21x10-12
5.9x10-12
-
150
4.82x10-11
5.2x10-11
-
180
3.62x10-11
4.9x10-11
-
200
3.36x10-11
3.7x10-11
-
Table 4.1. Diffusion coefficient values deduced from: Deff: linear fits from initial slopes of
figure 4.6, Deff: from cumulant analyses, and D: theoretically calculated values by using the
size parameters observed from SANS analyses.
Chapter 4. TDMAO/L35/Styrene System
55
that the results are rather close to the radii obtained from light scattering, confirming the
accuracy of the analyses. Similarly, theoretical lengths of the micelles were determined and
are presented in Table 4.2.
Styrene
amount
(mM)
Rh
(nm)
Rves,th
(nm)
lth
(nm)
PDI
Mwapp
(g/mol)
Nagg
from
SLS
0
3.11
-
7.23
0.1
7.17x104
262
10
4.77
-
23.3
0.12
1.9x105
879
20
5.25
-
30.2
0.15
2.0x105
886
30
7.9
-
45.8
0.16
2.9x105
1370
40
8.2
-
56.5
0.25
8.4x105
4230
50
72.0
89.6
-
0.38
1.1x107
58100
60
64.2
68.2
-
0.43
8.2x106
45200
70
87.3
89.1
-
0.40
1.6x107
92800
80
80.7
77.5
-
0.41
1.4x107
88100
90
49.2
45.3
-
0.28
6.3x106
37100
100
52.3
50.5
-
0.19
8.8x106
51800
120
70.1
-
-
0.27
2.4x107
99700
150
6.9
-
-
0.21
5.5x105
3570
180
7.1
-
-
0.18
5.9x105
3960
200
8.2
-
-
0.17
6.5x105
4430
Table 4.2. Results from the SLS and DLS measurements of 50 mM TDMAO / 0.5 mM L35 and
added styrene mixtures at 25°C. Given are the hydrodynamic radius Rh from the cumulant
method, Rves,th theoretically calculated vesicle radius from Mw, lth theoretically calculated
cylinder length from Mw, polydispersity index PDI, the apparent molecular weight Mwapp, and
aggregation number Nagg (of TDMAO molecules).
Chapter 4. TDMAO/L35/Styrene System
56
4.1.3 Small Angle Neutron Scattering (SANS)
For a more detailed characterization of nanostructures and complementary to the investigation
by light scattering, we analysed these aggregates by small angle neutron scattering. SANS
measurements were performed on the instrument KWS1 Jülich Center for Neutron Science
(JCNS) at MLZ, Munich, Germany, at a wavelength of 0.6 nm, with a FWHM (full width at
half maximum) of 9 %, and three sample-detector distances of 1.2, 7.7, and 19.7 m with
corresponding beam collimation lengths of 8.0, 8.0, and 20.0 m respectively. Transmissions
were measured at 8 m distance with the attenuated direct beam. These experiments were done
in D2O as solvent for having a better contrast. Samples with various amounts of styrene
monomer mixed with 50 mM TDMAO / 0.5 mM L35 were measured at 25 °C.
Figure 4.8 shows the general view for the neutron scattering curves of samples with increasing
amount of styrene. At first glance, one observes initially the scattering of small micelles that
grow in size with higher scattering intensity along with the increasing styrene content. These
samples in the range of L1 phase up to 50 mM show rod-like shape of TDMAO/L35 micelles and
for pure TDMAO, the presence of short rod-like micelles was shown by SANS before 175.
With increasing styrene content, we noticed that the q-1 behavior extends to lower q thereby
indicating an elongation of these structures, being in good agreement with the light scattering
results (see Table 4.2). At 50 mM styrene amount, a rather abrupt change of the scattering pattern
Figure 4.8. SANS intensity patterns of 50 mM TDMAO / 0.5 mM L35 and increasing amount
of styrene mixtures at 25°C. (styrene contents (mM): red:0, dark blue:20, magenta:30, green:40,
navy:50, violet:60, purple:70, wine:80, dark yellow:90, blue:100, light blue:120, gold:140, light
green:170, light magenta: 200.
Chapter 4. TDMAO/L35/Styrene System
57
takes place, the intensity becomes much larger and a q-2 scaling is observed in the middle q region
between 0.1 to 0.7 nm-1, which is the signature of formation of bilayers. This characteristic
behavior remains similar between 50 to 140 mM styrene content evidencing the formation of
vesicles which clearly has been induced by the styrene likewise cosurfactants 166. Similar
behavior has been seen before for the addition of medium chain alcohols as cosurfactants to
TDMAO 176.
As seen in the Figure 4.8, the drastic increase of intensity at low q implies the formation of larger
objects in the system. Additionally, for the samples between 70 and 100 mM styrene we observed
oscillations denoting more well-defined structures, i.e. vesicles with well-defined radii of 50-60
nm. Above 120 mM styrene content, the scattering pattern changes qualitatively, with lowering
the intensity at low q regime. These curves point out the presence of spherical droplets with radii
of ~6 nm eventuated from the form factor minimum around q = 0.75 nm-1.
Styrene
amount
(mM)
Φ
Volume
Fraction
R1
(nm)
l (nm)
Rves
(nm)
D
(nm)
PDI
R2
(nm)
Mw
(g/mol)
Nagg
0
0.0124
1.93
5.79
-
-
0.12
-
3.72x104
136
20
0.0144
1.90
28.7
-
-
0.14
-
1.79x105
798
30
0.0157
2.05
38.6
-
-
0.15
-
2.80x105
1330
40
0.0161
2.21
44.6
-
-
0.14
-
3.78x105
1901
50
0.0171
-
-
70.0
2.82
0.19
-
9.52x106
50300
60
0.0181
-
-
80.2
2.88
0.22
-
9.72x106
53500
70
0.0191
-
-
80.5
2.97
0.28
-
1.08x107
63470
80
0.0199
-
-
72.4
3.00
0.30
-
1.00x107
57200
90
0.0209
-
-
49.6
3.20
0.21
-
5.42x106
32800
100
0.0217
-
-
55.1
3.36
0.17
-
6.99x106
43400
120
0.0238
-
-
-
3.21
0.28
-
-
-
140
0.0258
-
-
-
-
0.18
5.48
3.77x105
2530
150
0.0265
-
-
-
-
0.16
5.75
4.36x105
2970
180
0.0293
-
-
-
-
0.15
6.00
4.95x105
3510
200
0.0314
-
-
-
-
0.15
6.27
5.65x105
4092
Table 4.3. Results from the SANS analysis of 50 mM TDMAO / 0.5 mM L35 and different
styrene content at 25 °C: Φ: Volume fraction, R1: Radius of cylindrical micelles, l: Length of
the cylinders, Rves: Mean vesicle radius, D: Bilayer thickness, PDI: Polydispersity index of
radius (R1, R2, Rves) distribution; R2: Radius of spheres; Nagg: aggregation number (App. 9.2),
Mw: Molecular weight of aggregates. For styrene amounts of 0-40 mM, parameters arise from
the cylindrical model, for 50-100 mM from spherical shell model; for 140-200 mM content
from the sphere model.
Chapter 4. TDMAO/L35/Styrene System
58
SANS curves were analyzed by applying different geometrical models described in section
3.1.6.2. to the curves as can be seen in Figure 4.9. In more details, we applied a cylindrical model
to TDMAO/L35 sample without monomer, and results indicate the presence of small rod-like
aggregates with a length of 5.8 nm and a radius of 1.9 nm as initial structure. We should consider
that these values are shorter than previously observed for pure TDMAO 175. This can be explained
in the way that two PEO chains of Pluronic L35 and its bulky head group stabilizes more curved
structures, i. e., shorter rods. Therefore, the system ends up such shorter rod-like particles.
As it can be predicted from looking at the curves in Figure 4.9, when styrene is introduced into
the system, structures start to be elongated. For the sample with 20 mM styrene, an increase of
the length to 28 nm and a radius of 2.02 nm is obtained from modelling the curve with cylindrical
geometry. Same model was applied for 30 and 40 mM styrene amounts, and the length of the
aggregates already reaches to an average value of 40 nm with a radius of 2.2 nm in combination
with the structure factor of hard spheres, as our system composed from uncharged particles which
may interact via steric repulsion, was fitted to the experimental data (see section 3.1.6.2.6.).
In the system the cylindrical particles occupy larger hard sphere volume. The hard sphere radius,
RHS, was fixed to the sum of the radius of one particle and two hydrophilic chain lengths (-EO11-
) of L35 attached on the aggregate which basically stabilize the particle according to de Gennes
approximation, the area occupied by single PEO chain was calculated before with assuming the
PEO chains of L35 form mushroom-like conformations when they attach on the surface of the
aggregate 115,177. Considering the steric hindrance of PEO chains from two particles approaching
to each other, the length of the two PEO chains were added to the hard sphere radius. Therefore,
Figure 4.9. SANS curves of 50 mM TDMAO / 0.5 mM L35 / styrene mixtures at 25°C.
(solid black line: fitted data). For clarity subsequent data sets were multiplied each with a
scale factor of 3.
Chapter 4. TDMAO/L35/Styrene System
59
RHS becomes a sum of the radius and two chain lengths. The interactions were determined by
fitting with this model and the variables are summarized in Table 4.4.
In Figure 4.9. samples with styrene beyond 50 mM concentration reveal the q-2 behavior at middle
q regime, indicating clearly the formation of bilayers 178. Except the deviation at low q, a shell
model agrees with a high polydispersity of ~ 0.2 for the radius and a thickness around 3-3.3 nm.
Systematical analysis of these curves shows that the size of the particles increases until 90 mM
styrene concentration along with high polydispersity, however above 90 mM the oscillations at
low q become more visible, and the polydispersity drops to a value of ~ 0.1.
The samples in this range of styrene concentration (50-120 mM) are the most interesting part of
this chapter. The evolution from micelles to vesicular structures initiated only by styrene
monomer is confirmed by SANS measurements. The size parameters, radii, bilayer thickness,
Styrene
amount
(mM)
RHS/(nm)
fp
30
10
0.054
40
10
0.061
180
11
0.175
200
11
0.185
Figure 4.10. SANS curves of 50 mM TDMAO / 0.5 mM L35 / styrene mixtures at 25°C.
(solid black line: fitted data). For clarity subsequent data sets were multiplied each with a
scale factor of 3.
Table 4.4. Parameters for structure factor fit
of hard sphere interaction.
Chapter 4. TDMAO/L35/Styrene System
60
polydispersity index, aggregation numbers and molecular weights obtained from the spherical
shell model are summarized in Table 4.3.
The Kratky-Porod plot is an alternative representation of the SANS curves from that one can
obtain the one-dimensional radius of gyration Rg, which is related to the bilayer thickness D using
𝑅𝑔2=𝐷2
12 , 134,136. Therefore, Ln[I(q)q2] vs q2 plots were fitted with: 𝐼(𝑞)𝑞2=𝐼(0)𝑒𝑥𝑝(−𝑞2𝑅𝑔
2
1)
for samples with styrene concentration between 50-100 mM (Figure A1 and A2). The values
from both approaches are in good agreement and results are given in Table 4.5 (for the Kratky-
Porod plots see Appendix 9.1.4).
For the sample containing 120 mM styrene, the SANS curve shows a more complicated pattern
which is mainly indicating that spherical aggregates start to show up together with the previously
formed bilayers. The spherical shell model does not fully agree with this curve, nevertheless as
much as it could be fitted on the curve we were able to obtain the value for the polydispersity of
radius (Rves) which jumps to ~ 0.3. However, defining a radius was not possible from these curves
since the sample contains a mixture of different aggregate types.
Upon the addition of still more styrene, the spherical droplets become more pronounced and a
disappearance of the bilayers is evident as seen from the vanishing q-2 slope at middle q. This is
also visible from the Figure 4.10, droplet radius changes slightly from 5.5 to 6.3 nm. At the same
time a correlation peak becomes dominant. Therefore, here a hard sphere structure factor was
applied to describe the curves of the samples with 180 and 200 mM styrene with keeping the RHS
of 11.5 nm which is relevance for the measured data due to the calculation of volume fractions
(Table 4.4).
Styrene
amount
(mM)
D/(nm)
Spherical
shell
D/(nm)
Krakty-
Porod
50
2.82
2.72
60
2.88
2.87
70
2.97
2.94
80
3.00
3.01
90
3.20
3.32
100
3.36
3.45
Table 4.5. Calculated Thickness for the bilayer from of
spherical-shell model and Kratky-Porod approximation.
Chapter 4. TDMAO/L35/Styrene System
61
Consequently, SANS provides a full picture of the aggregates formed in the presence of styrene
with TDMAO and L35 surfactants. It can be understood and confirmed from these experiments
that styrene induces the formation of bilayers like cosurfactants and by increasing its amount,
structural transition occurs starting from cylindrical micelles to spherical droplets.
Chapter 4. TDMAO/L35/Styrene System
62
4.2 Polymerization of the TDMAO/L35/Styrene System
In this part, we investigate whether the styrene containing samples can be fixated by a photo-
initiated radical polymerization and how the polymerization does affect the vesicle system with
respect to phase behavior, small angle neutron scattering and in particular rheological
properties. The completeness of the polymerization reaction was verified by NMR and the
obtained polymerized samples characterized with respect to their properties and structure.
Figure 4.11. 1H-NMRspectra of unpolymerized (top) and polymerized (bottom) 50 mM
TDMAO / 0.5 mM L35 / 80 mM styrene at 25 °C.
Chapter 4. TDMAO/L35/Styrene System
63
4.2.1 Nuclear Magnetic Resonance (NMR)
Polymerization was followed by using 1H NMR for confirming the full conversion of monomer.
As solvent we used D2O to obtain the chemical shifts and Tetramethylsilane (TMS) as reference
agent. Spectra were recorded on Bruker Avance II 400 spectrometer operating at 400 MHz.
Vanishing of the proton signals of double band at styrene molecule means that the monomer is
fully converted to polystyrene. In the Figure 4.11 at the top given the NMR spectrum of the
sample before the polymerization, however at the bottom one sees the polymerized sample. The
main difference in both is the disapparance of signals at δ 5.02-5.5-6.5 corresponds to CH- of
vinyl group. As expected, after polymerization the signals of the vinyl bond are disappearing
and the CH3 signals on the spectrum mainly becoming more pronounced due to the
polymerization.
4.2.2 Phase Behaviour
As described above, the most interesting
part of TDMAO/L35/styrene system is the
vesicle regime, which corresponds to the
styrene concentration of 50-100 mM.
Therefore, we studied fixating these
samples by UV- initiated polymerization.
The polymerized system showed a different
phase behavior than the systems they
Figure 4.12. Photographs of polymerized
samples of 50 mM TDMAO / 0.5 mM L35 /
styrene mixtures (50 mM – 80 mM content) at 25
°C.
Figure 4.13. Zero-shear viscosity 𝜂0 of 50 mM TDMAO / 0.5 mM L35 / styrene
mixtures at 25°C, measured with a capillary viscometer, except for the polymerized
80 mM styrene sample, which was measured with the Gemini 200 HR rheometer.
Chapter 4. TDMAO/L35/Styrene System
64
derived from. For instance, while the vesicle samples were bluish, slightly turbid, and water
viscous (see Figure 4.2), polymerization led to more transparent and differently viscous
solutions (Figure 4.12).
We measured the viscosity of samples before and after polymerization and concluded with the
result that in general viscosity increased slightly when compared to the unpolymerized case.
However, for the case of the 80 mM styrene sample, the viscosity increased by a factor of 100,
Figure 4.13 summarizes the viscosity values for both cases. The high viscosity of this sample
can be seen in Figure 4.15, after shaking it, air can be trapped in the sample.
4.2.3 Small Angle Neutron Scattering (SANS)
Because of the fact that the behavior of system changes entirely after polymerization, we
investigated the polymerized samples with respect to their detailed structure by means of SANS.
These measurements were all done at KWS1 instrument, Munich, with the same experimental
set up described in section 4.1.3. In Figure 4.14, we plotted the SANS curves, exhibiting largely
different scattering profiles than seen for the unpolymerized samples (Figure 4.9). The q-2 slope
at intermediate q range disappears, which means that bilayers are no longer present. The change
in the scattering patterns indicates that after polymerization, vesicles are essentially replaced by
small ellipsoidal aggregates. Until 80 mM styrene concentration, the length of the aggregates
is 11 nm and the radius 2.04 nm. Additionally, a correlation peak at ~ 0.2 nm-1 becomes more
pronounced along with increasing monomer amount. This peak is the sign for a higher ordering
of the structures in combination with a repulsive interaction of elongated particles.
Figure 4.14. SANS intensity patterns of polymerized 50 mM TDMAO / 0.5 mM L35 / styrene
mixtures at 25°C.
Chapter 4. TDMAO/L35/Styrene System
65
This structural transition can be explained by the packing parameter concept. Styrene with its
double bond is a relatively polar molecule, but after the polymerization the double bond is gone,
and the formed polystyrene is mainly hydrophobic. Accordingly, it behaves like an oil and
becomes solubilized within the micellar core and this morphological change was characterized
by SANS.
For higher styrene concentrations of 120 to 200 mM, which are in the microemulsion phase (for
unpolymerized samples Figure 4.10, for polymerized samples Figure 4.14), polymerization lead
to spherical particles with radii of ~7 nm having the similar inter-particular interactions.
Apparently, the initially formed microemulsion droplets are retained in structure, i. e. one has
a case of precise templating.
4.2.4 Rheology
We have described so far that polymerization noticeably disrupts
the formed vesicles which are induced with the presence of
styrene. As explained in section 4.2.2 phase behavior of the
polymerized samples was quite different from unpolymerized
ones, losing the characteristic bluish tinge of vesicles and being
more viscos. The most interesting sample of the polymerized
series is certainly the one with 80 mM styrene, which led to a
much more viscous phase (Figure 4.15) and can be seen on the
Figure 4.16. Measured shear viscosity of polymerized sample containing 50 mM TDMAO
/ 0.5 mM L35 / 80 mM styrene as a function of applied shear rate (black circles), solid red
line is the model fit of the Carreau-Yasuda model (equation 3.37).
Figure 4.15. Photograph of
polymerized sample of 50
mM TDMAO / 0.5 mM L35
/ 80 mM styrene at 25 °C.
Chapter 4. TDMAO/L35/Styrene System
66
photo that after shaking the sample, air is trapped. It draws interest in terms of the rheological
behavior of the polymerized sample of 80 mM styrene / 50 mM TDMAO / 0.5 mM L35 and
this sample was examined in more detail by means of oscillating and shear experiments.
As known, a non-linear relation between shear rate and shear stress implies the behavior of non-
Newtonian flows 179 which is observed for our case as well. The constant shear experiments
showed a constant viscosity at low shear rates which was followed by a shear thinning behavior
for shear rates above 2 s-1.
Figure 4.16 shows the viscosity as a function of shear rate. The curve was modeled with the
Carreau-Yasuda model (Equation 3.37), to obtain the zero shear viscosity η0 of 0.12 Pas, and
the relaxation time τ of 0.128 s 153. By using these observations, we can calculate the shear
modulus G0 being as 0.93 Pa.
On the other hand, the viscoelastic properties of the sample can be provided by oscillatory
measurements in terms of determining storage (elastic) modulus G' and loss (viscous) modulus
G''. These two moduli are plotted as a function of oscillating frequency in Figure 4.17, and from
the crossover, where both moduli are equal (G'=G'') the relaxation time can be determined via
τ = ω -1. The Maxwell model summarized by equation 3.36 152 describes these two parameters
and fitted on the curves which shown as solid lines in Figure 4.17.
Figure 4.17. Storage G' (black circle) and loss G'' (red square) modulus as a function of
angular frequency ω for polymerized sample of 50 mM TDMAO / 0.5 mM L35 / 80 mM
styrene, solid lines represents fits from Maxwellian model.
Chapter 4. TDMAO/L35/Styrene System
67
The Cole-Cole plot is the presentation of G’ against G" providing more information about
viscoelastic behavior of the systems 150. For a defined Maxwellian behavior this plot fits on a
semicircle and described by the equation: 𝐺′′(𝜔)=[𝐺′(𝜔)𝐺0−𝐺′(𝜔)2]𝑚 (see equation
3.36)
When we look at the Cole-Cole plot in Figure 4.18, the discrepancies between the measured
and fitted data indicates that the Maxwell model is clearly not perfect in this case. This
implication seems considerably parallel to the idea revealed before, while most of the entangled
rod like micellar structures are showing similar behavior to Maxwell model, polymeric systems
with a wide chain length distribution do not comply with it 180. The plateau modulus G0 and the
relaxation time τR obtained from steady shear and oscillatory measurements are summarized in
Table 4.6 and show very good agreement of both experiments.
Carreau-
Yasuda
G’
(ω)
G’’
(ω)
G0/Pa
0.93
0.86
0.73
τR/sec
0.128
0.19
0.19
η0/Pa
0.12
-
-
Table 4.6. Summarized results obtained from fitting with the Carreau-Yasuda
Equation 3.37 and the Maxwell Equation 3.36 models.
Figure 4.18. Cole-Cole plot of loss modulus vs storage modulus for
polymerized sample of 50 mM TDMAO / 0.5 mM L35 / 80 mM
styrene.
Chapter 4. TDMAO/L35/Styrene System
68
4.3 Summary
In this chapter we aimed to investigate the effect of styrene, as a common hydrophobic
monomer, on the mixture of nonionic surfactants tetradecyldimethylamine oxide (TDMAO)
and L35, where the copolymer provides colloidal stabilization in the system. Our main aim was
to fixate the self-assembled structures by polymerization, therefore we varied the monomer
composition until the solubilization limit was reached. Phase behavior, structural evaluations,
then the subsequent polymerization of styrene monomer was studied by means of turbidity,
static and dynamic light scattering (SLS, DLS), as well as small-angle neutron scattering
(SANS). Because of the interesting viscoelastic properties of polymerized system, we probed
the rheological behavior with oscillatory and shear experiments.
First, from the scattering analyses we obtained that with increasing styrene amount, rod-like
micelles of TDMAO/L35 evaluate to more elongated micellar structures. Upon further increase
of styrene, spontaneously vesicles are forming where styrene plays the similar role as
cosurfactants. However, when styrene is in excess, much smaller spherical aggregates are
formed. Apparently, this additional styrene can no longer takes part in vesicle formation and
does not go the micellar interface but goes to the hydrophobic core. Basically, it solubilizes
internally, reaching to the solubilization limit for the ~4.5 molar ratio of styrene to surfactants,
and accordingly forming small microemulsion droplets (oil-in-water (O/W)). Additionally,
visual inspection together with turbidity measurements of the given system confirm these
results.
The interesting part of this chapter is that the vesicle regime corresponds the 50 mM to 120 mM
styrene amount. As described in section 1.1, here the packing parameter plays the key role in
formation of vesicles. Styrene molecules effect the packing parameter not substantially on the
head group area but largely on the volume. This means numerically the packing parameter, p,
increases. This induces the transition from cylinders to a locally flat geometry and the formation
of vesicles 3.
In the second part, we discussed that the polymerization of TDMAO/L35/styrene vesicles lead
to the disruption of the bilayers and formation of more elongated aggregates. This structural
evaluation is also described by the packing parameter. Styrene is a relatively polar molecule
based upon its double bond. However, after the polymerization, the double bond is gone, and
the formed polystyrene becomes more hydrophobic. Accordingly, this hydrophobic polymer
chains are like oil molecules, which can be solubilized in the micellar core. With the help of
SANS we could confirm that this structural conversion ends up with spherical structures with
Chapter 4. TDMAO/L35/Styrene System
69
the radius around ~7 nm which means basically the size of the initial microemulsion droplets
were retained after polymerization. Importantly, a polymerized sample with 80 mM styrene
showed a noticeable viscoelastic behavior that we can explain by the fact of formation of
wormlike micelles which are stiffened and permanent in shape due to the polymerization and
also investigated from rheological side. This chapter presents and confirms that as a common
hydrophobic monomer, styrene takes part in the formation of vesicles with the nonionic
TDMAO/L35 surfactant mixture, like the effect of cosurfactant. When it is polymerized, it
becomes much more hydrophobic and changes the structure back to the smaller rod-like
aggregates. This study presents a new way of vesicle formation by addition of the styrene and
fixating the self-assembled structures by subsequent polymerization.
Chapter 4. TDMAO/L35/Styrene System
70
5 TDMAO/LiPFOS/L35/Styrene System
Introduction
Vesicles have great potential for being used as templates within the aim of producing
nanocarriers 112,181 because of their structural feasibility for encapsulation 3,43,182,183. They can
transport hydrophobic compounds in their lipid shell or hydrophilic moieties in the core.
Various studies have shown that their formation and structural properties can be controlled as
well as an improved stability, thereby vesicles have a wide application area from biomedicine
to electronics 184–188. In addition, these well-defined systems can be formed spontaneously by
proper surfactant types 56,189,190.
Surfactant mixture of zwitterionic tetradecyldimethylamine oxide (TDMAO) and anionic
lithium perfluorooctylsulfonate (LiPFOS) is spontaneously forming monodisperse vesicles
with a PDI ~ 0.05 which had been shown previously 191,192. This formation is explained due to
the synergistic interaction between the surfactant pairs, where the head groups are attractively
interacting thereby reducing the joint head group area and therefore inducing vesicle formation.
Stability and size of these vesicles can be controlled by addition of Pluronic copolymers (EOn-
POm-EOn) 114,115. Consequently, small, monodispersed and kinetically long-time stabilized
vesicles are formed in their mixtures.
This chapter reveals the effect of styrene monomer on this system with the intention of fixating
the vesicles by polymerization. TDMAO/LiPFOS mixture with molar ratio of 55:45 in the
presence of 1 mol % Pluronic L35 was employed, since this ratio is yielding the most well-
defined vesicles 114,115. As chapter 4 defines the effect of styrene on TDMAO/L35 system, with
the present part we conclude its role on the essential vesicle system in this context by focusing
on the subsequent polymerization of this system and thereby its permanent fixation.
Chapter 5. TDMAO/LiPFOS/L35/Styrene System
72
5.1 TDMAO/LiPFOS/L35/Styrene
5.1.1 Phase Behaviour
As a hydrophobic monomer and slightly soluble in water, styrene needs to homogenize into the
vesicle bilayer according to our main objective. Its low water solubility, ~ 3 mM 169, makes the
homogenization difficult, thus we choose to dissolve the styrene into the TDMAO/L35
solutions prior to vesicle preparation. Chapter 4 has described the behaviour of styrene during
the dissolution into the 50 mM TDMAO / 0.5 mM L35 solutions. Mixing them with LiPFOS
leads to formation of vesicles loaded with styrene monomer.
In this step we investigate 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS / styrene
mixtures, for varying amounts of styrene. These samples were created by blending the 50 mM
TDMAO / 0.05 mM L35 / various styrene (prepared 3 days prior to vesicle formation) mixtures
and 50 mM LiPFOS together at room temperature in the molar ratio of 55:45
(TDMAO:LiPFOS).
We gained information on the styrene solubilization, stability and the potential aging process
of the aggregates by turbidity measurements parallel to visual inspection. The TDMAO/L35
samples with dissolved styrene were closed firmly and stirred for 3 days in order to dissolve
the monomer entirely in the solutions at 25 °C. Later on, vesicles were prepared by mixing
Figure 5.1. Turbidity τ (λ=632 nm) as a function of styrene concentration in 27.5
mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS (red diamond); polymerized
samples of 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS (blue triangle)
at 25 °C. Measurements were done after 1 hour of both sample preparation and
polymerization.
Chapter 5. TDMAO/LiPFOS/L35/Styrene System
73
these solutions with 50 mM LiPFOS at aforementioned ratio. After mixing both solutions, the
vesicle phase, Lves, directly was characterized by the bluish appearance as seen in Figure 5.2
171,176. From 0 mM to 65 mM styrene amount, samples had the same slightly turbid appearance,
no precipitation or phase separation was observed. However, for more than 65 mM styrene
amount, this sight changed drastically. Apparently, system was saturated with styrene
monomer, becoming very turbid view and an excess of the monomer was phase separated at
the top pf the solution within an hour (2Φ).
Figure 5.1 shows the turbidity values calculated by UV-transmission measurements of
TDMAO/L35/LiPFOS vesicles with ascending amount of styrene. When the bare vesicles
without styrene and the styrene loaded vesicle samples are compared, it can be seen from the
figure that turbidity values are stayed almost the same. This means styrene loading had no
disruptive effect on vesicles, simply it became dissolved in the hydrophobic bilayer. On the
other hand, after reaching its saturation limit, then turbidity jumps into a higher-level following
by a phase separation. These findings are in good agreement with the visual inspection of the
samples.
Additionally, the inspection of samples revealed that the bluish-one phase appearance of the
samples stayed unchanged for an observation window of one year. After 1 year, samples were
phase separated.
Figure 5.2. Sample photograph after 1 hour of preparation 27.5 mM TDMAO / 0.275 mM
L35 / 22.5 mM LiPFOS with increasing amount of styrene mixtures from 0 to 65 mM at 25
°C.
Chapter 5. TDMAO/LiPFOS/L35/Styrene System
74
5.1.2 Light Scattering
In order to gain more systematic structural information on the effect of styrene, we performed
light scattering measurements.
The size of styrene loaded vesicles was obtained by dynamic light scattering measurement.
Figure 5.3 shows the intensity autocorrelation functions of the TDMAO/LiPFOS vesicles with
different styrene concentrations measured at the scattering angle of 90° at 25°C. These curves
show a monomodal decay for all samples. However, the decay time of the aggregates increases
along with styrene concentration. This means the particle diffusion becomes slower implying
the formation of bigger particles in the system.
The average vesicle size and polydispersity in a distribution was obtained with the cumulant
method. Analyses of the data indicate and confirm that this bluish slightly turbid phase
consisted of vesicular aggregates. The obtained hydrodynamic radii from cumulant method
after the insertion of styrene into the system is depicted in Figure 5.4 together with gyration
radii deduced from static light scattering. Based upon the analyses, vesicle radii increased
systematically up to 30 mM styrene amount, then stagnated until 45 mM concentration. Above
this point, it increased substantially again until the 2-phase region was reached. Styrene
insertion to the bilayer of TDMAO/LiPFOS system, reduces the preferred curvature hence
leading to bigger vesicles. Furthermore, styrene loaded vesicles hold a much wider size
Figure 5.3. Intensity autocorrelation function g2(t) measured at an angle of 90° of monomer
loaded vesicles in 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS / styrene mixtures
at 25 °C.
Chapter 5. TDMAO/LiPFOS/L35/Styrene System
75
distribution than the system without styrene, as seen in Table 5.1, and the PDI value jumps from
0.09 to ~0.3-0.4.
In the light of static light scattering measurements, information about the radii of gyration Rg,
and molecular weights, Mw of the aggregates can be gained from Guinier approximation (see
section 3.1.5.2). Samples were measured at the same set-up as DLS at 10 different angles (30,
40, 50, 60, 70, 80, 90, 100, 110 and 120°). When we look at the Rg, it changes exactly in the
same fashion as the
hydrodynamic radii seen
(Figure 5.4). For vesicles
Rg=Router126, which is in our
case in good agreement and
therefore corroborative to the
presence of vesicles.
The forward scattering, I(0)
values were extrapolated by
fitting the scattering curves (in
Figure 5.5)120 with Guinier
approximation, therefore,
intensities were plotted against
q2. Then the Mw was
Figure 5.4. Intensity autocorrelation function g2(t) measured at an angle of 90° of monomer
loaded vesicles in 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS / styrene
mixtures at 25 °C
Figure 5.5. Intensity vs q2 for 27.5 mM TDMAO/0.275 mM
L35/22.5 mM LiPFOS/styrene mixtures at 25°C.
Chapter 5. TDMAO/LiPFOS/L35/Styrene System
76
determined for the vesicles from the equation 3.8 (Table 5.1). Refractive index increments used
in the equation 3.9 were measured and are listed in Appendix 9.2.1, Table A3. Table 5.1
represents Rh hydrodynamic radii, PDI, polydispersity index, (both from DLS), Mw, apparent
molecular weights, Rg, radius of gyration values and Nagg, the aggregation numbers (from Mw,
and with respect to all molecules contained) for styrene loaded vesicles.
Based upon the analyses so far, we prefer to study the styrene concentrations between 30-45
mM in which range the particle size stagnated after the increase, and just before the saturation
point of the system to the monomer. Coming part below is describing the samples with
mentioned styrene range for a comparative analysis of unpolymerized and polymerized
vesicles.
Styrene
amount
(mM)
Rh
(nm)
Rg
(nm)
Rves
theor.
(nm)
PDI
Mwapp
(g/mol)
Nagg
from
SLS
0
35.6
35.1
30.7
0.09
1.29x107
35000
10
39.4
38.8
33.2
0.38
1.80x107
54400
15
42.1
43.2
36.7
0.37
2.23x107
70300
20
47.7
52.8
42.2
0.42
3.08x107
110700
25
54.3
56.4
51.1
0.39
4.46x107
163500
30
48.9
49.2
45.7
0.29
3.54x107
128000
35
46.2
53.9
47.9
0.44
2.80x107
102000
38
57.8
59.1
53.4
0.39
4.82x107
180000
41
49.6
47.4
44.2
0.41
3.83x107
148000
44
51.5
48.9
41.8
0.43
4.09x107
154000
46
44.6
42.1
36.7
0.41
2.67x107
100600
50
81.2
79.2
63.9
0.47
6.11x107
257000
58
89.1
89.9
67.4
0.45
6.52x107
273000
Table 5.1. Results from the SLS and DLS measurements of styrene loaded vesicles of 27.5 mM
TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS mixtures at 25°C. Given are the hydrodynamic
radius Rh, radius of gyration Rg, theoretically calculated vesicle radius from Mw (from SLS)
and thickness (from SANS) Rves,th, polydispersity index PDI (from DLS), aggregation number
Nagg (from Mw, and with respect to all molecules contained) and the apparent molecular weight
Mw.
Chapter 5. TDMAO/LiPFOS/L35/Styrene System
77
5.1.3 Small Angle Neutron Scattering (SANS)
Small angle neutron scattering curves were recorded on instrument D11 at the Institute Laue
Langevin (ILL) with the wavelength of 0.6 nm (FWHM of 9%), three sample-detector distances
of 1.2, 8.0, and 39.0 m with corresponding beam collimation lengths of 5.5, 8.0, and 40.5 m
respectively.
All the samples were prepared freshly in D2O prior to SANS measurements. Styrene amount
was varied over in the same range described so far. Measured data were fitted using a form
factor of a spherical shell geometry yielding a vesicle radius for neat system around 32.5 nm
and a membrane thickness of 2.73 nm. These results are in good agreement with the values
observed for the same system in the previous work 114,115.
Scattering data of the styrene loaded vesicle samples are presented in Figure 5.6. What is more
apparent at first look is that, the significant oscillations at low q (0.08-0.09 nm-1), become
flattened with incorporating styrene into the system. This means the narrow size distribution of
the formed particles becomes wider, namely the polydispersity index is increased. Nevertheless,
the q-2 slope observed in the middle q region, in the range of 0.2 to 0.8 nm-1, is the signature of
the bilayers and this behavior remains similar for all the samples.
Upon addition of styrene into the vesicles, in the beginning the position of the first minima
shifts to the lower q, indicating clearly an increase of the vesicle size. Looking at the scattering
curves of higher styrene concentrations, the intensity at low q increases drastically, evidencing
the presence of larger particles in the sample.
Figure 5.6. SANS curves of 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS / styrene
mixtures at 25 °C. (Colored lines: measured data; solid black line: fitted data). For clarity
subsequent data sets were multiplied each with a scale factor of 3.
Chapter 5. TDMAO/LiPFOS/L35/Styrene System
78
In detail, the modeled curves in Figure 5.6 exhibit an interesting behavior for the samples below
30 mM styrene amount. Here, the vesicle size slightly decreases from 32.5 nm to 26 nm with
the insertion of styrene into the system. For 10 mM sample, this is different from light scattering
result, where bigger and more polydisperse vesicles were observed. This difference can be due
to the light scattering method is sensitive to the collective diffusion in the sample. However,
above this point, similar trend was obtained. Vesicles start to grow to the radius of ~45 nm,
together with an increase of the bilayer thickness to ~2.9 nm by means of the incorporation of
the styrene within the bilayer. The polydispersity reaches a value of ~0.3 confirming the light
scattering results (Table 5.1).
Additionally, Mw, molecular weights were calculated by using the vesicle radii and bilayer
thickness obtained from SANS analyses. When we compare the result listed in Table 5.2 with
Mw from SLS (Table 5.1), both results are in good agreement.
The aggregation number Nagg were calculated for surfactant by using the formula:
where V is the volume, NA Avogadro’s number, 𝜌 is the density, and Mwi is a sum of the
molecular weight of components due to the molar fractions 𝑥, in each sample. Results are listed
in Table 5.2.
Styrene
amount
(mM)
Φ
Volume
Fraction
Rves
(nm)
SANS
PDI
D/nm
Spherical
shell
D/nm
Kratky
Porod
Mw
(g/mol)
Nagg
0
0.0144
35.2
0.10
2.73
2.78
1.32x107
35000
10
0.0156
26.1
0.29
2.71
2.77
1.56x107
47100
20
0.0169
28.9
0.31
2.70
2.80
1.75x107
59000
30
0.0182
46.1
0.34
2.72
2.84
2.80x107
112000
38
0.0188
46.4
0.31
2.84
2.87
2.75x107
109000
41
0.0191
46.7
0.29
2.92
2.94
2.94x107
115000
44
0.0196
48.3
0.30
2.94
2.95
3.03x107
120500
46
0.0202
47.9
0.33
2.95
2.97
3.00x107
118800
𝑁𝑎𝑔𝑔=𝑉.𝜌.𝑁𝐴
𝑀𝑤𝑖
(5.1)
Table 5.2. Results from the SANS analysis of 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM
LiPFOS and styrene mixtures at 25°C. Given are the vesicle radius Rves (Rves=R+ ∆R),
polydispersity index PDI, aggregation number Nagg, molecular weight Mw, bilayer thickness D
(D=∆R) from spherical shell model, bilayer thickness D (D=∆R) from Kratky- Porod analysis
(see Appendix 9.2.2 and Figure A4-A5, and the volume fractions Φ of dispersed material.
Chapter 5. TDMAO/LiPFOS/L35/Styrene System
79
5.2 Polymerization of the TDMAO/LiPFOS/L35/Styrene System
So far, in the previous part the well-defined TDMAO/LiPFOS/L35 vesicles were loaded with
styrene monomer and investigated by systematically varying the monomer amount. The scope
of this part is to elucidate the fixating process of these styrene loaded vesicles with photo-
initiated radical polymerization by means of several methods such as turbidity measurements,
static light scattering (SLS), dynamic light scattering (DLS), small angle neutron scattering
(SANS) and by nuclear magnetic resonance (NMR) technic. Particularly we choose the
monomer range of 30-45 mM for further polymerization step, which can be explained with the
basis of dynamic light scattering measurements in section 5.1.2. As described in
aforementioned part, monomer loaded vesicles in this range showed a stagnated size evolution.
The size of the vesicles in this monomer range are more stable, therefore, aimed to be used for
polymerization.
5.2.1 Phase Behaviour
Cross-linked polymerized samples of 27.5 mM TDMAO / 22.5 mM LiPFOS / 0.275 mM L35
with styrene concentration varying from 35 mM to 46 mM / 0.1 molar ratio of DVB as cross-
linker (with respect to the monomer concentration) showed similar visual appearance as before
polymerization. The bluish, slightly turbid view of vesicles retained after polymerization. No
phase separation, coagulation or precipitation as observed. Figure.5.7 depicts the sample photos
taken after 1 hour from the end of the polymerization which was proceeded for 15 hours.
Turbidities of the polymerized samples were obtained in the same way as unpolymerized ones
via measuring the transmissions then converting them to the turbidity values via the equation
3.39. The values are presented in the same plot together with the values for unpolymerized
Figure 5.7. Sample photograph after 1 hour from the end of polymerization
for 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS / varying amount
of styrene mixtures (with 0.1 molar ratio of cross-linker with respect to the
styrene amount) at 25 °C.
Chapter 5. TDMAO/LiPFOS/L35/Styrene System
80
samples in Figure 5.1. Although both results are almost in the same range, a very slight drop in
turbidities for polymerized samples with styrene amount more than 40 mM, is recognizable.
Presumably, it could be the result of the fact that polystyrene has low refractive index and
therefore the contrast decreases. Another assumption is due to the polymerization, the dissolved
styrene in the mixture arranges. Both lead to a slight decrease in turbidity for high monomer
concentrations. However, it has to be noted that the visual appearance of the samples remained
after the polymerization, which is an indication of preserving the vesicle structures.
5.2.2 Light Scattering
Light scattering is a powerful way to compare the structural variation and size distribution for
both cases. Figure 5.8 presents time dependent autocorrelation functions of the scattered light
intensities for polymerized samples measured at the scattering angle of 90° and performed at
25°C.
At first look, they seem all monomodal and the decay times are close to each other. However,
in detail one can see the autocorrelation curves of the polymerized samples shifted to the
slightly shorter decay times when monomer amount increases.
Figure 5.8. Intensity autocorrelation function g2(t) measured at an angle of 90° of
polymerized vesicles in 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS / styrene and
0.1 ratio of cross-linker mixtures at 25 °C.
Chapter 5. TDMAO/LiPFOS/L35/Styrene System
81
The analysis of the measured data with cumulant method allows the determination of the
average particle diameter and size distribution. According to the analyses, the high
polydispersity of the unpolymerized samples in section 5.1.2 remains in the same range.
Hydrodynamic radii are depicted in Figure 5.9 together with the Rh of unpolymerized vesicles.
The values show a very slight increase for each polymerized sample in the similar trend as in
the unpolymerized case. It can be noted that during the polymerization process, the radius of
the vesicles was retained around 40-60 nm. This implies that these small vesicles can
successfully be fixed in the templating process by polymerization.
Styrene
amount
(mM)
Rh
(nm)
Rg
(nm)
Rves
theor.
(nm
PDI
Mwapp
(g/mol)
35
50.1
47.3
52.6
0.41
4.43x107
38
52.3
54.3
58.2
0.42
4.67x107
41
48.2
46.3
52.0
0.35
4.01x107
44
47.4
54.0
53.1
0.37
3.98x107
46
38.7
43.3
40.7
0.30
2.85x107
Table 5.3. Results from the SLS and DLS measurements of polymerized vesicles of 27.5 mM
TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS mixtures at 25 °C. Given are the hydrodynamic
radius Rh, radius of gyration Rg, theoretically calculated vesicle radius from Mw (from SLS)
and thickness (from SANS) Rvestheor, polydispersity index PDI (from DLS), and the apparent
molecular weight Mw.
Figure 5.9. Hydrodynamic radii before and after polymerization of vesicles in 27.5 mM
TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS / styrene and 0.1 ratio of cross-linker mixtures
at 25 °C.
Chapter 5. TDMAO/LiPFOS/L35/Styrene System
82
Additionally, from the static light scattering measurements, the information on molecular
weights and radius of gyration was provided. Thereby, we calculated the theoretical vesicle
radii from molecular weights obtained by SLS with assuming the bilayer thicknesses from small
angle neutron scattering measurements. Results listed in Table 5.3, show good agreement with
the hydrodynamic radii from DLS confirming the accuracy of the templating process.
5.2.3 Small Angle Neutron Scattering (SANS)
For the aim of small angle neutron scattering measurements, polymerization reactions took
place in D2O as solvent. Measurements were done in the similar set up as for unpolymerized
samples and data were analysed in the same manner. Figure 5.10 illustrates the neutron
scattering curves of polymerized vesicles with 0.1 molar ratio of cross-linker. Noticeably, these
curves show similar scattering patterns as unpolymerized ones, holding the q-2 slope is in the
middle q region of 0.2 to 0.8 nm-1, indicating the presence of bilayer structures. This means that
polymerization did not have a destructive effect on the monomer-loaded vesicles, conversely it
sustained the preformed structures by stabilizing their structures.
When we look at the curves
closely, as a result of the
polymerization, the form
factor minima which is
around 0.06-0.07 nm-1 first
shifted to higher values
(0.04-0.05 nm-1), and
afterwards tended to go to
smaller q again. This
means the vesicles ended
up with smaller radii and
also a higher polydispersity
which is noticeable from
the more flattened form
factor minimum.
Figure 5.10. SANS curves of cross-linked polymerized
samples of 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM
LiPFOS / styrene mixtures at 25°C. (Colored lines: measured
data; solid black line: fitted data). For clarity subsequent
data sets were multiplied each with a scale factor of 3.
Chapter 5. TDMAO/LiPFOS/L35/Styrene System
83
These curves were fitted with a spherical-shell model similarly as unpolymerized samples and
yielded values in the same range changing from 36 to 43 nm in radius for varying styrene
concentrations. Structural parameters obtained for polymerized vesicles are summarized in
Table 5.4. As seen from the analyses, the bilayer thicknesses of the cross-linked polymerized
samples did not change, indicating a robust cross-linked polymer shell was formed within the
bilayers. This interesting finding was analyzed precisely further by the help of Kratky-Porod
plots (see Appendix 9.2.2, Figure A6). The thicknesses deduced from both analyses differ
somewhat from each other, however revealing basically the accuracy of the analyses and are
summarized in Table 5.4.
5.2.4 Nuclear Magnetic Resonance (NMR)
Polymerization was confirmed with 1H NMR measurements. The conversion of monomer and
the end of polymerization was determined with vanishing the proton signals of the double bond
of the styrene molecule. A Bruker Avance II 400 spectrometer operating at 400 MHz was used
to record the spectra. D2O was used as solvent and tetramethylsilane (TMS) was used as
reference agent.
Figure 5.11 shows the 1H-NMR spectra of vesicles before and after the polymerization. Signals
at δ 5.0-5.5 and δ 6.47 (C-H of vinyl group) are visible before polymerization while after
polymerization, these signals had disappeared (Figure5.11 bottom). This evidently shows the
full conversion of styrene monomers to the polystyrene.
Styrene
amount
(mM)
Rves
(nm)
SANS
PDI
D/nm
Spherical
shell
D/nm
Kratky
Porod
Polymer
shell
thickness
Mw
(g/mol)
35
38.8
0.37
2.81
2.87
1.18
2.72x107
38
43.1
0.38
2.82
2.84
1.24
3.14x107
41
44.9
0.39
2.82
2.87
1.30
3.48x107
44
40.8
0.41
2.83
2.90
1.39
2.83x107
46
33.2
0.40
2.84
2.92
1.50
2.11x107
Table 5.4. Results from the SANS analysis of cross-linked polymerized samples of 27.5 mM
TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS / styrene mixtures at 25 °C. Given are the vesicle
radius Rves, polydispersity index PDI, molecular weight Mw, bilayer thickness D from spherical
shell model, bilayer thickness from Kratky- Porod analyses (see Appendix 9.2.2 and Figure A6),
and calculated thickness for the polystyrene shell.
Chapter 5. TDMAO/LiPFOS/L35/Styrene System
84
Additionally, proton signals of the aromatic group appear between δ 7.0-7.8 vanishes or became
reduced, and the peak integrals of the aromatic group decreased by half (compared to the proton
signals of the surfactant molecule between δ 3.0-3.7). This can be explained by the fact that
polymerization within the hydrophobic bilayer of the vesicles restricts the motion of the formed
polymeric network protons therefore their signals are no longer visible or pronounced.
Figure 5.11. 1H-NMR spectra of vesicles before and after cross-linking
polymerization at 25 °C. The molar ratio of divinylbenzene to styrene was 0.1
with a total concentration of monomer of 35 mM. Red arrows: signals of the
vinylic protons.
Chapter 5. TDMAO/LiPFOS/L35/Styrene System
85
5.3 Summary
Kinetically stable, monodisperse vesicles consisted from zwitterionic TDMAO and anionic
LiPFOS surfactants with the presence of Pluronic L35 copolymer, were used as model system
for fixation by polymerization. Such polymer stabilized vesicles are important structures due to
their geometrical feasibility for encapsulation which allows them for being used as nanocarriers.
In this chapter, we performed the concurrent loading of styrene monomer into the hydrophobic
vesicle bilayer and accordingly its polymerization. In the first part, monomer loading capacity
of vesicles were investigated for increasing monomer concentrations with the help of turbidity,
static and dynamic light scattering (SLS, DLS), and small-angle neutron scattering (SANS)
measurements. Scattering experiments provided information on the presence of vesicles due to
the monomer loading, at the same time demonstrated remarkable increase of the size and
polydispersity index. In chapter 4, we showed that styrene monomer has an effect on forming
vesicles already only with TDMAO/L35 surfactants, like a typical cosurfactant. Adding the
LiPFOS surfactant, the model vesicle system was retained. However, styrene loading caused a
drastic shift from monodisperse to polydisperse structures. The reason of this sharp change can
be explained with the fact that styrene as a cosurfactant, may speed up the equilibration.
Second part explained the effects and results of polymerization on vesicles. In general, vesicles
became slightly smaller in comparison to unpolymerized ones. Vesicles were structurally and
morphologically retained after polymerization. Scattering measurements, in particular SANS
experiments, show that vesicle formation was not affected by polymerization and showed
identical scattering patterns either before or after polymerization. The increase of polydispersity
due to the styrene loading stayed similar for polymerized vesicles as well, however, is still in
the range of theoretical polydispersity value which is calculated for equilibrium vesicles 193.
This chapter elaboratively revealed that a model, well-defined vesicle system can be used as
template for fixation by polymerization under mild reaction conditions and structures
successfully retain their initial form after polymerization. By this way, it is possible to produce
polymeric nanocapsules in the size range of 40-100 nm in a straightforward way which enables
for further investigations with different monomers, cross-linkers to different vesicle systems.
Chapter 5. TDMAO/LiPFOS/L35/Styrene System
86
6 The Effect of Acrylate Monomers
Introduction
Polymerization is an effective way for fixating vesicles that results in the formation of
polymeric nanocapsules. These nanostructures can be used as nanocariers in different research
areas from material science to the nanomedicine 87,88,194. Stabilization of the vesicles by
inserting hydrophobic monomers into their bilayers and subsequently polymerizing them,
should increase the bilayer rigidity and should reduce its permeability. Thereby one would exert
control over these important properties of these shell-structured colloids. Therefore, different
studies have been made in recent years in terms of solubilizing traditional hydrophobic
monomers in the bilayer to produce hollow polymer nanocapsules 86,106,195–197.
Although the polymerization is a convenient approach, it can have several challenges. It was
shown before that due to the polymerization, a phase separation can take place in the bilayer
with formation of parachute-like structures 103. Additionally, polymerization mostly needs high
temperature or using organic solvents, which can have crucial effects on the final structure.
In this chapter, we examine acrylate monomers of different alkyl chain lengths with different
hydrophobicity to determine appropriate conditions for an effective templating and
polymerization for the further formation of polymer nanocapsules. As the polymerization will
take place within the few nanometer wide hydrophobic bilayer, the most important points to
take into account are the chain length and hydrophobicity of the acrylate monomers.
Accordingly, we varied the hydrophobicity of the acrylate monomers in a systematic fashion
by using butyl, hexyl, isooctyl and dodecyl acrylate.
The core vesicle system consisted of nonionic tetradecyldimethylamine oxide (TDMAO),
Pluronic L35 and anionic lithium perfluorooctylsulfonate (LiPFOS) surfactant 114,115,192, has
been concurrently loaded with alkyl acrylates, and templated via polymerization under quite
mild conditions, where the reaction was taking place in water at 18 °C (the polymer surfactant
L35 was included in this formulation in order to bring in additional steric stabilization for the
formed colloidal assemblies). For dissolving the water-insoluble monomer and preventing
vesicle aging, concurrent loading was preferred, and primarily varying amount of hydrophobic
monomer as well as the cross-linker were dissolved in micellar TDMAO/L35 solutions. In the
first part of this chapter, the effect of acrylate monomers on TDMAO/L35 micelles is described.
The second part presents the investigation on the formation of acrylate monomer loaded
Chapter 6. The Effect of Acrylate Monomers
88
TDMAO/L35/LiPFOS vesicles and their characterization. In the last part, particularly the effect
of hexyl acrylate monomer on the model vesicle system and its polymerization in the bilayer
were studied. Later on, the encapsulation efficiency of the final hollow polymer nanocapsule
product was studied by using calcein as a water-soluble fluorescence dye.
6.1 TDMAO/L35/Acrylate System
In this section, we investigate the dissolution behaviour of different acrylate monomers, i.e.
butyl acrylate, hexyl acrylate, isooctyl acrylate and dodecyl acrylate in 50 mM TDMAO / 0.5
mM L35 mixtures. Different amounts of monomer were dissolved in samples of constant
concentration of TDMAO/L35 mixture and stirred for 3 days in the dark. Sample vials were
closed firmly for preventing any evaporation of the monomer.
6.1.1 Phase Behaviour
As a first step, the phase behaviour of surfactant solutions of constant concentration was
studied for dissolving increasing amounts of monomer. Starting from the short chain alkyl
acrylates, we studied the dissolution of butyl acrylate. Figure 6.1 depicts the sample photos after
3 days of stirring. Since butyl acrylate has a high water solubility of ~ 1.4 g/L 198, the mixtures
up to 130 mM butyl acrylate concentration could dissolve in micelles and formed transparent
single micellar phase L1. For high butyl acrylate concentration of 180 mM, the system became
saturated with monomer and a more turbid, oily phase separated from solution.
Figure 6.1. Sample photograph after 3 days of stirring 50 mM TDMAO / 0.5 mM L35 / varying
amounts of butyl acrylate mixtures at 25 °C.
Chapter 6. The Effect of Acrylate Monomers
89
Hexyl acrylate is another acrylate monomer having a medium chain length and an intermediate
water solubility of 0.4 g/L 199 which is appropriate for the polymerization within the
hydrophobic vesicle membrane amongst other acrylate monomers (Figure 6.2). Our
investigations showed that hexyl acrylate can dissolve in TDMAO micelles up to 120 mM
concentration. Above this point, the micellar phase shifts to a turbid phase indicating that an
emulsion of monomer droplets is formed. For higher than 200 mM hexyl acrylate amount,
samples were turbid with a whitish appearance and separated into 2 phases within an hour (2Φ).
A third monomer that we studied was; isooctyl acrylate. Its solubility in micellar solution
differed from butyl and hexyl acrylate due to the low water solubility of ~ 0.01 g/L at 25 °C 200.
Samples (Figure 6.3 top) up to 90 mM monomer concentration, showed a clear single phase of
micellar solutions, L1. Afterwards samples turned to white turbid solutions and the excess of
monomer phase separated after one hour at the top of the vial (2Φ).
Figure 6.2. Sample photograph after 3 days of stirring 50 mM TDMAO / 0.5 mM L35 / varying
amounts of hexyl acrylate at 25 °C.
Figure 6.3. Sample photograph after 3 days of stirring 50 mM TDMAO / 0.5 mM L35 / varying
amounts of top: isooctyl acrylate, bottom: dodecyl acrylate mixtures at 25 °C.
Chapter 6. The Effect of Acrylate Monomers
90
The forth monomer was dodecyl acrylate with a water solubility of <<0.01 g/L at 25 °C 201. The
solubility of dodecyl acrylate monomer in TDMAO micelles was quite low, and a micellar L1
phase was only observed up to 75 mM concentration. Then for higher monomer amounts,
samples immediately turned to white turbid solutions, and phase separated at the top in an hour
(2Φ), as seen before for the case of isooctyl acrylate.
As known from the previous works 115,202, TDMAO/L35/LiPFOS vesicle has a bilayer thickness
of ~2.7 nm. Therefore, hexyl acrylate which has a medium chain length in comparison with
other acrylates, was preferred to use for further polymerization. Apart from that, its water
solubility is in the moderate range, thus this monomer can still be mixed in the solution and at
the same time effectively being entrapped into the hydrophobic membrane. Moreover, hexyl
acrylate enables to work in a wide concentration range for further vesicle formation and
subsequent polymerization.
6.1.2 Light Scattering
To confirm that the monomers incorporate in vesicles as suggested above, the
acrylate/TDMAO/L35 mixtures were characterized by dynamic light scattering. The increasing
hydrodynamic radius upon increasing the monomer amounts was followed for all acrylate
monomers to find optimal conditions for subsequent polymerization.
In the butyl acrylate case, intensity correlation functions were all monomodal (Figure 6.4 left).
With introducing the monomer into the system, the decay time stayed nearly constant for
monomer amounts up to 90 mM. For 90 and 130 mM amount it slightly shifted to higher decay
Figure 6.4. Intensity autocorrelation function g2(t) measured at an angle of 90° of 50 mM
TDMAO / 0.5 mM L35 / left) butyl acrylate right) isooctyl acrylate mixtures at 25 °C.
Chapter 6. The Effect of Acrylate Monomers
91
times, corresponding to the increasing size of micelles. Similar trend can be seen from the phase
behaviour confirming it.
The presence of isooctyl acrylate did not have significant effects on changing the scattering
behaviour up to 55 mM concentration. Monomodal curves with fast decay time are depicted in
Figure 6.4 (right) and hydrodynamic radius varied from 3.9 to 5.5 nm (see Table 6.3). However,
at 75 mM monomer concentration, the curve looked completely different, being bimodal and
shifting to the higher decay time. As a comparison to the previous part 6.1.1, 75 mM is the
boundary concentration where the sample appearance changes (see Figure 6.3). Clearly, DLS
confirms the shift from L1 phase of monomer dissolved micelles and presence of bigger
aggregates, presumably emulsion droplets of undissolved excess monomer.
DLS analysis of samples with dodecyl acrylate monomer shown in Figure 6.5, revealed
different characteristic than others. Dodecyl acrylate’s very low water solubility 201 made it
difficult to dissolve in TDMAO/L35 micelles, therefore different sizes of ~ 4.2 to 6.8 nm (see
Table 6.2) were observed from the intensity correlation curves and none of them was
monomodal. In general, this trend of lowered solubilisation with increasing chain
length/hydrophobicity of oil molecules is typically observed in surfactant systems 203,204.
Figure 6.5. Intensity autocorrelation function g2(t) measured at an angle of 90° of 50 mM
TDMAO / 0.5 mM L35 / dodecyl acrylate mixtures at 25 °C.
Chapter 6. The Effect of Acrylate Monomers
92
As expected from the visual inspection, hexyl acrylate monomer showed more consistent
behaviour in micellar solutions (Figure 6.6). Autocorrelation curves for increasing amounts of
monomer, looked almost similar. As for the butyl acrylate monomer, the decay time stayed
almost constant up to 80 mM monomer content, however later on with increasing monomer
addition, it noticeably shifted to slower decay time. In all cases, the monomodal decay for the
samples confirms that between 10-100 mM hexyl acrylate addition to 50 mM TDMAO / 0.5
mM L35 samples, only the micellar L1 phase exists.
The curves were analysed with the cumulant method (see section 3.1.5.1) for correlation times
from (10-5–10-1) s (Eq. 3.6), and weighted with using the errors, in order to obtain the
hydrodynamic radii and polydispersity index. Cumulant analyses of DLS measurements for
hexyl acrylate/TDMAO/L35 mixtures demonstrated the size of the micelles varied over the
range of 3.1 nm to 4.8 nm along with monomer addition. As 50 mM TDMAO / 0.5 mM L35
forms short rod-like micelles (see chapter 4), we calculated the theoretical length of the micelles
assuming the similar geometry from the molecular weights, Mwapp, which are deduced by SLS,
and end up in a range of 7.3 to 8.1 nm depending on the monomer amount. Results are listed in
Table 6.1. Another important point gained from light scattering is the information of
polydispersity. As seen in Table 6.1 that unlike styrene monomer (see section 4.1.2), hexyl
acrylate/TDMAO/L35 micelles show low PDI values, are being more monodisperse.
Figure 6.6. Intensity autocorrelation function g2(t) measured at an angle of 90° of 50 mM
TDMAO / 0.5 mM L35 / hexyl acrylate mixtures at 25 °C.
Chapter 6. The Effect of Acrylate Monomers
93
6.1.3 Small Angle Neutron Scattering (SANS)
In order to obtain further detailed insight into the structural analyses upon addition of the
different acrylate monomers to the micellar TDMAO/L35 solution, SANS experiments were
performed for increasing addition of monomer with longer alkyl chain and low water solubility.
Since butyl acrylate does not meet the expectation with rather short chain and high water
solubility, only dodecyl-, isooctyl- and hexyl acrylate monomers were analysed in terms of
small angle neutron scattering experiments. SANS experiments performed at KWS1 (Munich),
at 0.6 nm wavelength (with FWHM of 10 %), and three sample-detector distances of 1.2, 7.7,
and 19.7 m with corresponding beam collimation lengths of 8.0, 8.0, and 20.0 m respectively.
Neutron scattering curves of TDMAO/L35mixtures with dodecyl acrylate monomer are
presented in Figure 6.7. We described in section 4.1.3. that 50 mM TDMAO / 0.5 mM L35
mixture without monomer presents short-rod like micelles with a radius of 1.9 nm and length
~6 nm.
Hexyl
acrylate
amount
(mM)
Rh
(nm)
lth
(nm)
PDI
Mwapp
(g/mol)
Nagg
from
SLS
0
3.1
7.34
0.12
7.21x104
275
20
3.2
7.26
0.13
7.34x104
250
30
3.4
7.21
0.14
7.64x104
275
50
4.0
7.34
0.12
8.21x104
301
60
4.3
7.37
0.17
9.38x104
350
70
4.7
7.29
0.16
1.31x105
460
80
4.8
8.10
0.18
1.45x105
550
90
4.8
8.06
0.15
2.07x105
746
Table 6.1. Results from the SLS and DLS measurements of 50 mM TDMAO / 0.5 mM L35 /
hexyl acrylate mixtures at 25 °C. Given are the hydrodynamic radius Rh: from the cumulant
method, lth: theoretically calculated cylinder length from Mw, polydispersity index PDI, the
apparent molecular weight Mwapp, and aggregation number Nagg (with respect to all molecules
contained).
Chapter 6. The Effect of Acrylate Monomers
94
With the addition of dodecyl acrylate, one observes similar short-rod like aggregates with an
increase of the size.
c(dodecyl
acrylate)
/ mM
Φ
Volume
fraction
R
(nm)
L
(nm)
PDI
Mw
(g/mol)
Nagg
from
SANS
Rh
(nm)
Rsphere
(nm)
Rswelling
0
0.0150
1.97
6.02
0.12
3.91x104
143
3.10
2.60
2.76
20
0.0210
2.84
6.40
0.10
7.47x104
283
3.83
3.39
3.85
25
0.0227
2.78
6.72
0.12
6.03x104
230
4.02
3.40
4.08
35
0.0248
4.11
-
0.13
1.26x105
483
4.49
-
4.60
45
0.0284
4.51
-
0.16
1.98x105
770
-
-
5.16
55
0.0291
4.45
-
0.15
1.73x105
673
-
-
5.45
75
0.0359
5.20
-
0.14
2.49x105
983
-
-
6.76
Table 6.2. Results from the SANS analysis of 50 mM TDMAO / 0.5 mM L35 / dodecyl acrylate
mixtures at 25°C. Given are the Φ: Volume fraction, R: cylinder radius, L: cylinder length, PDI:
Polydispersity index of radius distribution, Mw: Molecular weight of aggregates, Nagg:
aggregation number (with respect to all molecules contained), Rh: Hydrodynamic radius
obtained from DLS via cumulant analysis, Rsphere: theoretically calculated spherical radius
(from R and L obtained by SANS), Rswelling: theoretically calculated droplet radius from the
swelling law assuming all the monomer goes into the micellar core.
Figure 6.7. SANS curves of 50 mM TDMAO / 0.5 mM L35 / dodecyl acrylate mixtures at 25
°C. (Colored lines: measured data; solid black line: fitted data (for monomer amounts of 0-25
mM: cylindrical model and for 35-75 mM: spherical model was employed)). For clarity,
subsequent data sets were multiplied each with a scale factor of 5.
Chapter 6. The Effect of Acrylate Monomers
95
For monomer content up to 35 mM, we applied a cylindrical model with a log-norm distribution
of the radius to the scattering curves. Analyses indicated that the radius increased from 1.9 to
2.8 nm and the length from 6 to 6.7 nm upon the addition of monomer and have seen that the
polydispersity is around ~0.1, which is lower than the case of styrene (PDI~0.14). SANS curves
of the samples with monomer concentration higher than 35 mM were analysed applying a
spherical model with log-normal distribution of radius, which can expediently define the
formed microemulsion droplets as indicated in the previous part by light scattering and turbidity
increase. With the addition of 75 mM monomer, the radius of the droplet reaches 5.2 nm. At
the same time for this sample one observes a marked increase of scattering intensity at low q
which indicates the presence of emulsion droplets. The results obtained from both models are
presented in Table 6.2. For samples with monomer amount of 0-25 mM, we theoretically
calculated spherical radius Rsphere (by using the parameters R and L obtained from SANS) and
compare them with Rh from DLS measurements in the same table. Results indicate that Rh (from
DLS) is slightly different from theoretically calculated radius Rsphere. It has to be noted that the
values of Rh determined from DLS can be expected being different since one obtains the
collective diffusion of particles (DLS curves in Figure 6.5 show non-monomodal behaviour) in
DLS measurements.
Figure 6.8. SANS curves of 50 mM TDMAO / 0.5 mM L35 / isooctyl acrylate mixtures at 25
°C. (Colored lines: measured data; solid black line: fitted data (for monomer amounts of 0-20
mM, cylindrical model and for 30-75 mM, spherical model was employed)). For clarity
subsequent data sets were multiplied each with a scale factor of 5.
Chapter 6. The Effect of Acrylate Monomers
96
Accordingly, assuming that all the added monomer locates into the micellar core, one ends up
a theoretical swelling radius, Rswelling. We calculated Rswelling via 𝑅=3∗[(𝑉𝑠
𝑎ℎ)+(𝑉𝑚
𝑎ℎ)∗(𝑛𝑚
𝑛𝑠)],
with 𝑎ℎis the head group area, 𝑉𝑠 and 𝑉𝑚 are the volumes of surfactant and monomer, and 𝑛𝑠
and 𝑛𝑚 are the numbers of surfactant and monomer molecules, respectively. These values are
listed together with the sphere radius, Rsphere from SANS and hydrodynamic radius Rh from
DLS, in Table 6.2. The values for the swelling radius and experimental values are close to each
other up to 25 mM monomer content, which indicates almost all of the added monomer goes
into the micellar core. For concentrations higher than 35 mM, values started to deviate. The
experimental values are lower than the theoretical swelling radius. This means that monomer
cannot anymore penetrate into the micelles. The excess of the monomer forms emulsion
droplets in the outside medium, which leads the scattering intensity increase at low q regime in
Figure 6.7.
Similarly, samples with isooctyl acrylate monomer exhibit SANS curves with same scattering
patterns as dodecyl acrylate. SANS curves for lower monomer content up to 30 mM were
interpreted with a cylindrical model, revealing an increase of the radius from 1.97 to 2.6 nm.
For concentrations higher than 20 mM isooctyl acrylate monomer, a spherical model was
applied with log normal distribution of the radius to determine the size parameters of the
microemulsion droplets (Table 6.3). Scattering curves of isooctyl acrylate mixtures (Figure 6.8)
have the same intensity rise at small q for the samples above 40 mM monomer amount, however
c(isooctyl
acrylate)
/ mM
Φ
Volume
fraction
R
(nm)
L
(nm)
PDI
Mw
(g/mol)
Nagg
from
SANS
Rh
(nm)
Rsphere
(nm)
Rswelling
0
0.0150
1.97
6.02
0.12
3.91x104
143
3.10
2.60
2.76
20
0.0195
2.62
6.21
0.11
6.21x104
250
3.94
3.17
3.58
30
0.0210
3.43
-
0.15
8.12x104
336
3.96
-
3.99
40
0.0235
3.92
-
0.13
1.25x105
533
4.22
-
4.38
55
0.0271
4.56
-
0.14
1.95x105
861
4.50
-
5.04
75
0.0300
5.02
-
0.14
2.79x105
1260
5.51
-
5.74
Table 6.3. Results from the SANS analysis of 50 mM TDMAO / 0.5 mM L35 / isooctyl
acrylate mixtures at 25°C. Given are the Φ: Volume fraction, R: cylinder radius, L: cylinder
length, PDI: Polydispersity index of radius distribution, Mw: Molecular weight of aggregates,
Nagg: aggregation number (with respect to all molecules contained), Rh: Hydrodynamic
radius obtained from DLS via cumulant analysis, Rsphere: theoretically calculated spherical
radius (from R and L obtained by SANS), Rswelling: theoretically calculated droplet radius
from the swelling law assuming all the monomer goes into the micellar core.
Chapter 6. The Effect of Acrylate Monomers
97
not as drastic as dodecyl acrylate samples. As expected from the observations of the previous
parts, isooctyl acrylate monomer has a better solubility in the micellar aggregates when
comparing with dodecyl acrylate monomer therefore we assume it dissolves in the
TDMAO/L35 micelles. Accordingly, when we compare the swelling radius Rswelling, and
experimental radius in Table 6.3, values are rather close and for high monomer concentration
the deviation is relatively small. This implies that for isooctyl acrylate case considerable amount
of monomer locates into the micelles in comparison to dodecyl acrylate monomer.
Lastly, we investigated the effect and solubilisation of hexyl acrylate monomer in the micellar
TDMAO/L35 aggregates. Figure 6.9 shows the scattering intensity of the samples for increasing
monomer amount. Similar to the other two acrylate monomers presented so far, hexyl acrylate
has the neutron scattering curves with same patterns. The analysis of the curves indicate that
micelles were elongated from 1.97 to 2.7 nm (Table 6.4). For monomer amounts above 30 mM
the same model as for the other acrylate monomers, i.e. a spherical model with the log-norm
distribution of the radius was applied and pointed out the increase of the droplet size from 3.4
to 4.4 nm. Unlike the other long chain acrylate monomers, hexyl acrylate samples in general
did not show a significant intensity increase at small q regime. This implies and confirms that
up to 75 mM hexyl acrylate concentration, monomer can easily be dissolved in the micelles and
is not present in form of additional bigger aggregates. In the same table, we have included the
values of swelling radius Rswelling, assuming all the added monomer located into the micelles.
By comparison of the results from other acrylate monomers from Table 6.2 and Table 6.3, these
Figure 6.9. SANS curves of 50 mM TDMAO / 0.5 mM L35 / hexyl acrylate mixtures at 25
°C. (Colored lines: measured data; solid black line: fitted data (for monomer amounts of 0-
30 mM, cylindrical model and for 40-75 mM, spherical model was employed)). For clarity
subsequent data sets were multiplied each with a scale factor of 3.
Chapter 6. The Effect of Acrylate Monomers
98
values are almost identical to the experimental radius. This indicates that, TDMAO/L35
micelles can solubilize high concentrations of hexyl acrylate monomer, and only less amount
of monomer remains insoluble into the outside media.
Eventually small angle neutron scattering results exhibit a general trend that the structures
become bigger with the insertion of hydrophobic acrylate monomers into the micelles. Starting
from rod-like micelles, structures evolved to that of spheres, i. e. microemulsion droplets are
formed. From the Porod approximation, one can determine the surface to volume ratio of the
particle due to the monomer addition via lim
𝑞→∞𝐼(𝑞)𝑞4=2𝜋∆𝑆𝐿𝐷2(𝑆
𝑉) where 𝑆/𝑉 is the specific
surface of the system 205–207. When we plot the 𝐼(𝑞)𝑞4 vs 𝑞4, it reaches a limit for large q values
and this value is proportional to the specific surface 𝑆/𝑉 of the system. Thereby, we can
investigate the effect of the additive on the micellar structure in a precise way. The 𝐼(𝑞)𝑞4 vs
𝑞4 plots (so called Porod-Debye plots) are presented in Appendix 9.3.3.3. The specific surface
𝑆/𝑉 was obtained from the intercept of these curves and plotted as function of monomer
surfactant ratio in Figure A18. Firstly, we deduced an increase due to the addition of monomer.
Subsequently the value remains constant and for high monomer amounts it slightly decreases.
The first increase is due to the monomer incorporation into the interfacial area of the micelle
c(hexyl
acrylate)
/ mM
Φ
Volume
fraction
R
(nm)
L
(nm)
PDI
Mw
(g/mol)
Nagg
from
SANS
Rh
(nm)
Rsphere
(nm)
Rswelling
0
0.015
1.97
6.02
0.12
3.9x104
143
3.10
2.60
2.76
20
0.018
2.54
6.79
0.11
7.1x104
297
3.20
3.20
3.45
30
0.021
2.69
6.94
0.10
7.7x104
340
3.40
3.35
3.83
40
0.022
3.42
-
0.12
8.4x104
380
-
-
4.09
45
0.023
3.77
-
0.12
9.6x104
450
4.0
-
4.28
55
0.025
3.96
-
0.11
1.2x105
590
4.3
-
4.61
75
0.028
4.36
-
0.12
1.7x105
852
4.7
-
5.30
Table 6.4. Results from the SANS analysis of 50 mM TDMAO / 0.5 mM L35 / hexyl acrylate
mixtures at 25°C. Given are the Φ: Volume fraction, R: cylinder radius, L: cylinder length,
PDI: Polydispersity index of radius distribution, Mw: Molecular weight of aggregates, Nagg:
aggregation number (with respect to all molecules contained), Rh: Hydrodynamic radius
obtained from DLS via cumulant analysis, Rsphere: theoretically calculated spherical radius
(from R and L obtained by SANS), Rswelling: theoretically calculated droplet radius from the
swelling law assuming all the monomer goes into the micellar core.
Chapter 6. The Effect of Acrylate Monomers
99
and then monomer goes to the interior of the aggregate, which is the sign of rod to sphere
transition.
In particular, hexyl acrylate could incorporate within TDMAO/L35 micelles in wide range of
monomer concentration and final structures showed low PDI values and were well-defined.
Thus, hexyl acrylate can be used as a model monomer which is appropriate for further
polymerization in the vesicle membrane effectively.
6.2 TDMAO/LiPFOS/L35/Acrylate Monomers
In this section we study the effect of acrylate monomers on our vesicle template system. For
that mixtures of 50 mM TDMAO / 0.5 mM L35 with increasing amounts of acrylate were mixed
with 50 mM LiPFOS solutions (with the molar ratio of 0.55:0.45). In that way monomer loaded
vesicles were prepared and characterized with the aim of determining optimized conditions for
subsequent polymerization reactions.
6.2.1 Phase Behaviour
TDMAO / L35 / LiPFOS vesicles loaded with acrylate monomers were studied regarding the
phase behaviour after mixing the two micellar stock solutions. Vesicle phases are generally
slightly turbid, and have a characteristic bluish tinge. The photo seen in Figure 6.10 top, is the
dodecyl acrylate samples, which presented the described vesicle appearance up to 40 mM
monomer concentration (Lves). With increasing monomer amount, the turbidity increased
Figure 6.10. Sample photograph after 1 hour of preparation of 27.5 mM TDMAO
/ 0.275 mM L35 / 22.5 mM LiPFOS top: dodecyl acrylate bottom: isooctyl
acrylate mixtures at 25 °C.
Chapter 6. The Effect of Acrylate Monomers
100
drastically and later on, the excess of the monomer separated building a second phase to the top
of the vial in a few hours (2Φ).
In the same figure bottom picture displays the samples with isooctyl acrylate monomer. Here
the vesicle phase (Lves) only appeared for very low monomer concentrations below 20 mM,
afterwards samples had very turbid appearance and, as in the dodecyl acrylate case, phase
separation occurred in a few hours.
The formation of hexyl acrylate loaded vesicles can be perfectly seen at Figure 6.11 from the
presence of usual vesicle view (Lves) for samples below 25 mM hexyl acrylate concentrations.
For the samples with high monomer amount than 25 mM, turbidity increased noticeably, and
for even further monomer concentrations, samples turned to white and an excess monomer
phase separated in an hour (Figure 6.12).
Figure 6.11. Sample photograph after 1 hour of preparation of 27.5 mM TDMAO / 0.275
mM L35 / 22.5 mM LiPFOS hexyl acrylate mixtures at 25 °C.
Figure 6.12. Turbidity τ (λ=632 nm) as a function of monomer concentration in 27.5 mM
TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS at 25 °C. Measurements were done after 1 hour
from sample preparation.
2φ
Lves
Chapter 6. The Effect of Acrylate Monomers
101
6.2.2 Light Scattering
First, monomer loaded vesicles were studied with static and dynamic light scattering (SLS,
DLS) in terms of gaining structural information of the size and size distribution. Measurements
were done in 1 hour after the preparation at 7 angles (30, 45, 60, 75, 90, 105 and 120°) at 25
°C.
Autocorrelation functions of the samples with dodecyl acrylate monomer (Figure 6.13, left)
show in general monomodal decay. The fast decay in the autocorrelation functions observed for
TDMAO/L35/acrylate monomers was described in section 6.1.2 (Figures 6.4, 6.5 and 6.6)
indicating the presence of small micelles. Here the decay time shifted to higher values compared
to the micellar case due to the formation of vesicles as expected, which confirms the increase
of particle size. In detail, the scattering behaviour obtained by DLS (Figure 6.13 left) reveals
that by introducing the dodecyl acrylate monomer, vesicles became smaller. The decay time
shifted very slightly to the larger values upon the addition of more monomer. From the phase
study of dodecyl acrylate loaded vesicles, we have seen that Lves extends up to 40 mM monomer
concentration. Above 40 mM, vesicle system was saturated with monomer. High turbidity of
these samples (see Figure 6.10) causes multiple scattering, which does not allow analysing their
light scattering behaviour precisely.
Similarly, autocorrelation functions of the isooctyl acrylate loaded vesicles (Figure 6.13 right)
shifted to smaller decay times along with increasing the monomer amount. However, it is not
as drastic as in dodecyl acrylate case, indicating here isooctyl acrylate loaded vesicles are still
Figure 6.13. Intensity autocorrelation function g2(t) measured at an angle of 90° of 27.5 mM
TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS left) dodecyl; right) isooctyl acrylate mixtures
at 25 °C.
Chapter 6. The Effect of Acrylate Monomers
102
slightly bigger. The Lves phase boundary for isooctyl acrylate was ~25 mM concentration (see
Figure 6.10).
DLS measurements of hexyl acrylate monomer proved the presence of vesicles as for the other
acrylate monomers. Here (Figure 6.14) the size evolution is evident from the shortening of the
decay times. Similar as the longer chain acrylates, the size becomes smaller. Analyses of the
scattering curves via cumulant method yielded first a decrease of the vesicle radius from 36 to
20 nm. However, with further increasing monomer amount, the radius shifted slightly to 27 nm.
For hexyl acrylate, we determined the vesicle phase Lves below 25mM.
Static light scattering analyses were performed at the same setup as DLS. From the intensities,
we calculated the radius of gyration, Rg and molecular weights of the monomer loaded vesicles.
Similar as described in section 5.1.2, the molecular weights were calculated from the equation
3.8 using the refractive index increments (see Appendix 9.3.3.1, Table A5) in the equation 3.9.
In table 6.5, the Rh values determined from DLS analyses are compared with radius of gyration,
Rg deduced by SLS. As known from the literature, Rg ≅ Rves for vesicles 126 and the results
agreed well for Lves phase (< 25 mM). Furthermore, we theoretically determined vesicle radius
Rves,theor, from the molecular weight obtained by SLS (considering the bilayer thickness from
SANS) and are in good agreement with Rh.
Figure 6.14. Intensity autocorrelation function g2(t) measured at an angle of 90° of
27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS / hexyl acrylate mixtures
at 25 °C.
Chapter 6. The Effect of Acrylate Monomers
103
6.2.3 Small Angle Neutron Scattering (SANS)
So far, the effect of acrylate monomers on the vesicle formation and information about
structural changes during the monomer loading were obtained from turbidity and light
scattering measurements. Further insight to the structural progression was gained with SANS
measurements for different acrylate monomers of increasing amounts. SANS experiments were
performed at KWS1 (Munich), at a wavelength of 0.6 nm, the spread of wavelength was given
by a FWHM (full width at half maximum) of 10 %, and three sample-detector distances of 1.2,
c(hexyl
acrylate)
/ mM
Rh
(nm)
Rg
(nm)
PDI
Mwapp
(g/mol)
Nagg
from
SLS
Rves,theor
(nm)
0
36.6
38.6
0.09
1.73x107
39300
32.5
7
20.5
21.8
0.13
9.9x106
25700
24.4
10
23.7
18.3
0.14
1.2x107
33500
27.3
20
26.6
21.1
0.18
1.4x107
37500
29.2
Figure 6.15. SANS curves of 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS /
dodecyl acrylate mixtures at 25 °C. (Colored lines: measured data; solid black line: fitted
data with spherical shell model). For clarity subsequent data sets were multiplied each with
a scale factor of 3.
Table 6.5. Results of the SLS and DLS measurements of 27.5 mM TDMAO / 0.275 mM L35
/ 22.5 mM LiPFOS / hexyl acrylate mixtures at 25 °C. Shown are the hydrodynamic radius
Rh, radius of gyration Rg, polydispersity index PDI (from DLS), apparent molecular weight
Mw (from SLS) and Nagg aggregation number (with respect to all molecules contained), Rves,
theo: theoretically calculated vesicle radius from Mw (from SLS) and thickness (from SANS).
Chapter 6. The Effect of Acrylate Monomers
104
7.7, and 19.7 m with corresponding beam collimation lengths of 8.0, 8.0, and 20.0 m
respectively.
Previous studies of our template vesicle system consisted from 27.5 mM TDMAO / 0.275 mM
L35 / 22.5 mM LiPFOS demonstrated a vesicle radius of 36 nm with PDI of 0.055 114. In a
similar manner, we investigated the model system with SANS and obtained 36.3 nm for the
vesicle radius and 0.06 for polydispersity index, which are very close to the literature values
(see Table 6.8).
First, we studied the effect of dodecyl acrylate monomer on the vesicle system with small angle
neutron scattering. Figure 6.15 depicts the SANS measurements of monomer loaded vesicles
as a function of dodecyl acrylate concentration at 25 °C. From the curves, one observes an
explicit form factor bump around q= 0.17 nm-1, which corresponds to a mean vesicle radius of
~18-19 nm. The linear decay at intermediate q range (0.3-0.8 nm-1) revealed a slope of q-2 and
at high q (1.15-1.7 nm-1) one of q-4, together confirming the presence of bilayers. Additionally,
for the samples above 20 mM, intensity increased noticeably at low q regime pointing out the
presence of bigger particle along with the vesicles in the system. Another implication from the
curves is the less pronounced form factor peaks at high monomer concentrations, which is
related with the size distribution of the formed aggregates.
c(dodecyl
acrylate)
/ mM
Φ
Volume
fraction
Rves
(nm)
PDI
D/nm
Spherical
shell
D/nm
Kratky
Porod
D Swelling
/nm
Mw
(g/mol)
Nagg
from
SANS
0
0.0145
36.3
0.06
2.68
2.72
-
1.83x107
48300
10
0.0176
20.9
0.21
2.89
2.91
3.19
7.35x106
20900
15
0.0186
21.0
0.21
2.88
2.90
3.36
6.97x106
20100
20
0.0197
21.3
0.22
2.89
2.91
3.54
1.52x107
44800
25
0.0217
21.5
0.24
2.94
3.03
3.88
1.93x107
61100
30
0.0221
21.9
0.24
2.93
3.06
3.94
2.16x107
65800
40
0.0258
23.4
0.21
2.91
3.10
4.56
2.62x107
79300
Table 6.6. Results from the SANS analysis of 27.5 mM TDMAO / 0.275 mM L35 / 22.5
mM LiPFOS / dodecyl acrylate mixtures at 25°C. Given are Φ: Volume fraction, vesicle
radius Rves (Rves=R+D), polydispersity index PDI, bilayer thickness D from spherical shell
model, bilayer thickness from Kratky-Porod analyses (see section 3.1.6.1 and Appendix
9.3.1), Dswelling: theoretically calculated bilayer thickness from the swelling law assuming all
the monomer incorporated within the bilayer, molecular weight Mw, and Nagg aggregation
number (with respect to all molecules contained).
Chapter 6. The Effect of Acrylate Monomers
105
The scattering curves were analyzed by using a spherical shell model applying a log-normal
distribution of radius. Our investigations resulted that the vesicle radius decreased from 36 nm
to 20 nm when we introduced the dodecyl acrylate monomer to the system. For 40 mM the
radius increased slightly to 22-23 nm with the addition of more monomer, however different
size of particles started to show up and therefore the polydispersity index also increased slightly.
In general, the PDI is much higher for the monomer-loaded vesicles than for the pure one.
The model vesicle system has a bilayer thickness of 2.7 nm and our model yielded a thickness
of 2.9 nm irrespective of the monomer content (Table 6.6). For a better understanding, we
determined the bilayer swelling assuming all of the monomer is incorporated within the bilayer
and the interfacial area remains unchanged. Thereby, the swelling bilayer thickness, Dswelling,
was theoretically calculated and included in the same table. For low monomer concentrations
theoretical values are rather close to the experimentally deduces ones. Especially for high
monomer concentrations, the discrepancy is high. In general, this deviation can be explained
with the loading capacity of the bilayer. After some time vesicle bilayer is saturated with the
monomer thereof, the excess of the monomer starts forming bigger droplets in the outside
media. This results in an increase of intensity at low q regime.
On the other hand, our assumption above is based on the unchanged interfacial area, which
considers all the monomer are located in the middle part of the bilayer. In such a case, bilayer
thickness increases drastically while the overall size does not change too much. On the contrary,
Figure 6.16. SANS curves of 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS /
isooctyl acrylate mixtures at 25 °C. (Colored lines: measured data; solid black line: fitted
data with spherical shell model). For clarity subsequent data sets were multiplied each
with a scale factor of 3.
Chapter 6. The Effect of Acrylate Monomers
106
when monomer is distributed homogenously within the bilayer, the thickness stays constant but
the overall size increases 208. With the help of Porod-Debye plots (described in section 6.1.3)
we determined the specific surface 𝑆/𝑉 of these samples (Figure 6.18b). The first increase of
𝑆/𝑉 up to 20 mM concentration reveals that monomer is located close to the head groups of
surfactant pairs. Then along with the monomer addition it slightly shifts to the hydrophobic part
of the bilayer which leads to the decrease of surface volume ratio, 𝑆/𝑉.
Isooctyl acrylate showed similar scattering properties (Figure 6.16) in vesicle solutions as
dodecyl acrylate. The slope of q-2 (at q range of 0.35-0.80 nm-1) and q-4 (at high q range of 1.20-
1.6 nm-1) confirmed the vesicle formation was successful at the same time with the monomer
insertion. Scattering curves were analyzed with the spherical shell model, similar as dodecyl
acrylate. In this case, we obtained the vesicle radius of 23 nm. Upon the addition of monomer,
the vesicle radius did not show a significant increase. On the other hand, we observed the
expansion of the bilayer thickness from 2.7 to 2.95 nm. Similar as above, Dswelling was calculated
and compared with the experimental thickness values in Table 6.7. Differently, the thickness of
the isooctyl acrylate vesicles are increasing in a systematic way, while for dodecyl acrylate it
deviates depending on the monomer addition.
c(isooctyl
acrylate) /
mM
Φ
Volume
fraction
Rves
(nm)
PDI
D/nm
Spherical
shell
D/nm
Kratky
Porod
DSwelling
/nm
Mw
(g/mol)
Nagg
from
SANS
0
0.0145
36.3
0.06
2.68
2.72
-
1.83x107
48300
10
0.0168
26.2
0.16
2.79
2.84
3.10
1.25x107
36400
15
0.0176
26.6
0.16
2.82
2.89
3.24
1.41x107
44300
20
0.0190
26.7
0.15
2.83
2.90
3.48
1.48x107
44400
25
0.0199
26.2
0.17
2.95
3.09
3.64
1.37x107
43250
30
0.0210
26.8
0.18
2.93
3.05
3.79
1.54x107
52800
40
0.0226
26.9
0.18
2.95
3.08
4.11
1.70x107
55900
Table 6.7. Results from the SANS analysis of 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM
LiPFOS / isooctyl acrylate mixtures at 25°C. Given are Φ: Volume fraction, vesicle radius
Rves (Rves=R+D), polydispersity index PDI, bilayer thickness D from spherical shell model,
bilayer thickness from Kratky- Porod analyses (see section 3.1.6.1 and Appendix 9.3.2),
Dswelling: theoretically calculated bilayer thickness from the swelling law assuming all the
monomer incorporated within the bilayer, molecular weight Mw, and Nagg aggregation
number (with respect to all molecules contained).
Chapter 6. The Effect of Acrylate Monomers
107
The specific surface area (S/V) of isooctyl acrylate loaded vesicles, presented in the same plot
with other monomers in Figure 6.18b, shows same trend as dodecyl acrylate monomer. Surface
volume ratio (S/V) increases with increasing monomer amount until 20 mM concentration. The
decrease above this point indicates the surface area of bilayer does not change in parallel with
the volume growth. Namely, monomer does not localize anymore into the interfacial area of
the bilayer and large amount of it moves in the interior part of the membrane.
In the last step, we examined the hexyl acrylate behavior in the vesicle formation with SANS
experiments. Scattering curves (Figure 6.17) were analyzed by using the same model above as
spherical shell with log-normal distribution of the radius. The individual fit parameters from
the model are presented in Table 6.8. Similar to other acrylate monomers, we observed a
decrease in vesicle radius to from 36 nm to 23-24 nm when the monomer was introduced. We
had observed the hydrodynamic radius of 20-26 nm from light scattering measurements for
these samples, which confirms that both results are identical. Interestingly, in contrast to other
acrylate monomers, here we obtained low polydispersity values of ~0.1for hexyl acrylate loaded
vesicles. The bilayer thickness varied in the range of 2.7 to 2.9 systematically within the
addition of monomer. For further comparison, the thickness gained from plotting ln(q2I(q)) vs
q2 yielded the values around 2.80 nm (see appendix 9.3.3.2).
Figure 6.17. SANS curves of 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS / hexyl
acrylate mixtures at 25 °C. (Colored lines: measured data; solid black line: fitted data with
spherical shell model). For clarity subsequent data sets were multiplied each with a scale
factor of 3.
Chapter 6. The Effect of Acrylate Monomers
108
The reduction of the initial size along with the monomer addition can be explained with the
rearrangement of surfactants to form a new and more favored equilibrium structure of small
vesicles. On the other hand, swelling of the vesicles is also possible and was shown before with
CTAB/SDBS vesicles, which grew when styrene monomer was added 106. Similarly in our
study, acrylate laden TDMAO/L35/LiPFOS vesicles grew along with increasing the monomer
concentration. We calculated the increase of the size regarding to the bilayer thickness assuming
the added volume of the monomer located into the hydrophobic bilayer. The mass of a vesicle
with bilayer thickness of t + 2b (t is the thickness of the alkyl chain and b is the thickness of the
head group region) was calculated via: 𝑚𝑣=4𝜋𝑀𝑤(𝑅𝑣𝑒𝑠
2+(𝑅𝑣𝑒𝑠−𝑡−2𝑏)2
𝑎ℎ𝑁𝐴 where 𝑀𝑤 is the average
molecular weight of the surfactants: 𝑀𝑤=𝜒𝑇𝐷𝑀𝐴𝑂𝑀𝑇𝐷𝑀𝐴𝑂+𝜒𝐿35𝑀𝐿35+𝜒𝐿𝑖𝑃𝐹𝑂𝑆𝑀𝐿𝑖𝑃𝐹𝑂𝑆; 𝜒𝑖 is
the mole fraction and Mi is the molecular weight, 𝑎ℎ is the average area per head group for the
surfactant mixture, 𝑅𝑣𝑒𝑠 is the outer vesicle radius, and 𝑁𝐴 is the Avogadro’s number. The
volume of the hydrophobic chain region is 𝑉0=4𝜋
3 [(𝑅𝑣𝑒𝑠−𝑏)3−(𝑅𝑣𝑒𝑠−𝑡−𝑏)3]. Upon the
addition of monomer, we assume the volume of the bilayer changes as: 𝑑𝑉=𝑓𝑚𝑣/𝜌, here 𝜌
is the monomer density and 𝑓 is the weight fraction of monomer to surfactant in each sample.
When the monomer only locates in the hydrophobic chain region, the thickness of this region
c(hexyl
acrylate) /
mM
Φ
Volume
fraction
Rves
(nm)
PDI
D/nm
Spherical
shell
D/nm
Kratky
Porod
D Swelling
/nm
Mw
(g/mol)
Nagg
from
SANS
0
0.0145
36.3
0.06
2.68
2.72
-
1.83x107
48300
10
0.0165
24.8
0.10
2.69
2.52
3.00
1.03x107
34000
15
0.0175
25.6
0.11
2.75
2.66
3.18
1.05x107
35200
20
0.0183
25.9
0.12
2.77
2.70
3.30
1.08x107
35700
25
0.0190
26.1
0.16
2.81
2.83
3.41
1.09x107
36800
30
0.0198
26.3
0.17
2.87
2.91
3.55
1.23x107
44000
40
0.0214
26.5
0.18
2.92
2.95
3.82
1.34x107
47000
Table 6.8. Results from the SANS analysis of 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM
LiPFOS / hexyl acrylate mixtures at 25°C. Given are Φ: Volume fraction, vesicle radius Rves
(Rves=R+D), polydispersity index PDI, bilayer thickness D from spherical shell model, bilayer
thickness from Kratky- Porod analyses (see section 3.1.6.1 and Appendix 9.3.3.2), Dswelling:
theoretically calculated bilayer thickness from the swelling law assuming all the monomer
incorporated within the bilayer, molecular weight Mw, and Nagg aggregation number (with
respect to all molecules contained).
Chapter 6. The Effect of Acrylate Monomers
109
should then increase by a factor of ~(1+𝑑𝑉
𝑉0)1/3. Accordingly, then the radius of the monomer
loaded vesicle (theoretically calculated swelling radius) can be written as: 𝑅=(𝑅𝑣𝑒𝑠−
𝑏)(1+𝑑𝑉
𝑉0)1/3+𝑏. The thickness t was taken from the neat TDMAO/L35/LiPFOS vesicles.
For 𝑎ℎ we assumed a value of 0.5 nm2 and for b 0.3 nm 51.
In Figure 6.18a, outer vesicle radii Rves are presented as a function of monomer concentration
which were obtained from the spherical shell model applied to SANS curves and from our
calculation assuming all the monomer accommodates into the bilayer thereby the swelling
radius of the structures. Vesicle with 10 mM monomer content was taken as reference, and then
swelling radii were calculated for other samples. For 15 mM monomer, both values are in good
agreement. However, later the difference between both models suggest that monomer does not
only incorporate into hydrophilic part of the bilayer but also swells into the interior part, thereby
we end up an almost stagnated bilayer thickness. This assumption is corroborated with
determining the specific surface area change of the system. Figure 6.18b expresses the surface
volume ratio S/V of the system, which increases systematically as a function of the monomer
amount. As distinct from the other acrylate monomers, it rises gently until 30 mM hexyl acrylate
amount. This implies here the monomer is distributed more homogenously in the interfacial and
Figure 6.18. a) Outer vesicle radius Rves (of 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM
LiPFOS / hexyl acrylate vesicles black circle: obtained from SANS experiments, red square:
theoretically calculated swelling radius 𝑅 by assuming all monomer was taken into the
bilayer. b) The specific surface area (S/V) as the function of monomer concentration for
dodecyl-, isooctyl-, and hexyl acrylate.
a)
b)
Chapter 6. The Effect of Acrylate Monomers
110
interior areas of the bilayer. However, above 30 mM it tends to stay in the interior part,
consequently does not change the surface area S, but enhances the volume V.
As our main focus is to stabilize the model vesicle system by polymerization, the results of
hexyl acrylate case comply with the idea of retaining the well-defined vesicle system. When we
compare all acrylate cases, the progression of the system is more stable and allows working in
a wide concentration range of monomer. Since the model system consisted of small, unilamellar
well-defined vesicles, the results yielded from the hexyl acrylate case fitted ideally.
Chapter 6. The Effect of Acrylate Monomers
111
6.3 Polymerization of the TDMAO/LiPFOS/L35/Hexyl Acrylate System
These studies were done for gaining an insight into the detailed control of polymer nanocapsule
formation and for optimizing the conditions for stabilization of the vesicles by polymerization,
as it is relevant for potential future application such as their use as delivery vehicles.
From the studied systems above, the hexyl acrylate system was chosen for further
polymerization. First, its phase behavior is more promising in comparison to the other acrylates
that allows to study in a wide concentration range. Secondly, its chain length is moderate
enough to fit in the few nanometer wide hydrophobic bilayer. Besides that, its loading into the
vesicles does not have significant change on the formation of well-defined vesicles. One can
afterwards still effectively retain small, unilamellar highly monodisperse monomer loaded
vesicles than other acrylate monomers. Therefore, it is appropriate for further polymerization
in the vesicle membrane. Initially, the vesicle system was varied over 10 to 30 mM monomer
concentrations and the samples within this monomer range polymerized. Then for monomer
concentrations of 15, 20 and 25 mM three molar cross-linker ratios of 0.1, 0.2 and 0.4 were
employed, with respect to the total monomer concentrations. Samples were polymerized via
UV-initiated polymerization for 18 hours at 15 °C and the reaction was ended by removing the
nitrogen atmosphere and having oxygen quench the reaction (for details see section 3.2.2).
6.3.1 Phase Behaviour
In this study polymerization was done as an
effective way for fixation of the vesicle system.
Thereof, we expect not to see any difference
between the unpolymerized and polymerized
samples in terms of visual appearance, size and
structural progression. Figure 6.19 displays the
polymerized vesicle solutions without cross-
linker. Sample photos were taken after 1 hour
from the end of the reaction. Comparing with
the unpolymerized vesicles (Figure 6.11),
polymerized samples retained the typical vesicle tinge consisting of bluish turbid appearance.
Samples showed no precipitation, separation or any other phase alteration and stayed stable for
1 month. Later than 1 month, presumably some polymer chains settled down and formed a very
Figure 6.19. Photos of polymerized 27.5
mM TDMAO / 0.275 mM L35 / 22.5 mM
LiPFOS / hexyl acrylate mixtures taken 1
hour after the end of polymerization.
(Labels: monomer concentrations in mM).
Chapter 6. The Effect of Acrylate Monomers
112
small smoky cloudy phase which can be easily dispersed into the solution by simply shaking.
The formed polymer chains are not bound to a certain vesicle and will over a longer time simply
associate.
Additionally, we tested the cross-linked polymerized samples of hexyl acrylate. Figure 6.20
displays the sample photos after 1 hour from the end of polymerization reactions. These samples
were varied with respect to the cross-linker/monomer ratios. For instance, for 15 mM total
monomer concentration the cross-linker (1, 6-hexanediol diacrylate) molar ratio was varied
over 0.1, 0.2 and 0.4 with respect to the hexyl acrylate amount but keeping the total
concentration constant. As seen from the sample photo, they all look identical to the
unpolymerized vesicle solution. The only difference encountered was for the highest cross-
linker content with 0.4 molar ratio for 20 and 25 mM samples. These cross-linked polymerized
samples were more turbid than others. However, in general all cross-linked polymerized
samples were homogenous, uniform and none of them showed phase separation or precipitation.
Unlike the uncross-linked polymerized vesicles described above they did not show the smoky
polymer phase in the course of time. This can be explained such that by cross-linking one can
expect to just have one cross-linked polymer molecule in the vesicle shell, which then cannot
diffuse out of it.
6.3.2 Nuclear Magnetic Resonance (NMR)
Nuclear magnetic resonance (NMR) can provide crucial information and 1H NMR experiments
were done before and after the polymerization. For NMR the preparation of the monomer
loaded vesicles and subsequent polymerization was done in deuterium oxide (D2O), and the
Figure 6.20. Sample photo of cross-linked polymerized 27.5 mM TDMAO / 0.275
mM L35 / 22.5 mM LiPFOS / hexyl acrylate / 1, 6-hexanediol diacrylate mixtures
after 1 hour from the end of polymerization. (Labels: total monomer concentration
in mM/cross-linking ratio).
Chapter 6. The Effect of Acrylate Monomers
113
measurements were performed with a Bruker Avance II 400 spectrometer operating at 400 MHz
was used to record the spectra and tetramethylsilane (TMS) was used as reference agent.
Polymerization confirmed the complete conversion of monomer via the vanishing proton
signals of the vinyl bond on acrylate molecule. Figure 6.21 shows the 1H NMR spectra for hexyl
acrylate loaded vesicles (consisted of 27.5 mM TDMAO / 0.275 mM L35 / 00.25 mM LiPFOS)
before (top) and after polymerization (bottom). Disappearance of the 3 signals at δ 5.8, 6 and
6.3 (C-H vinyl groups) implies the full conversion from monomer to polyhexyl acrylate. After
Figure 6.21. 1H-NMR spectra of vesicles before (top) and after (bottom) cross-linking
polymerization at 25 °C. The molar ratio of 1, 6-hexanediol diacrylate to hexyl acyrlate was
0.1 with a total concentration of monomer of 35 mM. The signals of the vinylic protons are
marked with red arrows.
Chapter 6. The Effect of Acrylate Monomers
114
polymerization the peak intensity of the signals at δ 3.6, 3.1, and 0.78 decreased by a ratio of
0.4, and at δ 3.4, and 1.1 decreased by half. The restricted motion of polymer protons is the
main reason of this decrease or having less pronounced peaks.
6.3.3 Light Scattering
From the visual inspection of polymerized samples in the previous section it was assumed that
the vesicle structures were retained successfully after the polymerization reaction. For more
quantitative information, we investigated the polymerized samples by light scattering method
in order to obtain realistic detailed structural information and for confirming the assumptions.
In Figure 6.22 the autocorrelation functions of polymerized samples are presented. The
scattering curves on the left side of the figure belong to the polymer samples without cross-
linker. The hydrodynamic radii and polydispersity values of these polymerized vesicles were
obtained by fitting the scattering curves with cumulant method and parameters from the model
are listed in Table 6.9.
Figure 6.22. Intensity autocorrelation function g2(t) measured at an angle of 90° of left)
polymerized vesicles of 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS / hexyl
acrylate; right) cross-linked polymerized vesicles of 27.5 mM TDMAO / 0.275 mM L35 /
22.5 mM LiPFOS / hexyl acrylate / 1, 6-hexanediol diacrylate mixtures at 25 °C.
Chapter 6. The Effect of Acrylate Monomers
115
When comparing the vesicle size before and after the polymerization, light scattering yielded a
small but noticeable increase in vesicle radii along with the polymerization. While before
vesicles had a radius of ~23 nm, after the polymerization this value increased to ~27-29 nm
depending on the monomer concentration. At the same time, polymerized vesicles showed a
somewhat higher polydispersity and the PDI increased from 0.1 to 0.16-0.2, increasing with the
monomer amount.
On the right side of Figure 6.22, we present the autocorrelation curves of cross-linked
polymerized samples of hexyl acrylate loaded vesicles. Mostly these curves have similar
scattering properties to the curves on the left side. Only for the samples containing 20 and 25
mM hexyl acrylate and having a 0.4 molar ratio of 1, 6-hexanediol diacrylate curves shifted to
the right, meaning the formation of bigger particles with slower decay times. This result is in
good agreement with the visual appearance of the samples seen in Figure 6.20.
Cumulant analysis of the scattering curves of cross-linked polymerized samples confirmed this
increase in size with yielding radii of 30.2 and 33.7, respectively (Table 6.10). In Table 6.10
one observes a very good agreement between the size changes observed via Rh and Rg and the
Mwapp seen in SLS. Unlike the uncross-linked polymerized samples above, cross-linked
polymerized vesicles have smaller sizes for low cross-linking ratios and low polydispersity
c(hexyl
acrylate)
/ mM
Rh
(nm)
Rg
(nm)
PDI
Mwapp
(g/mol)
10
28.7
22.8
0.16
1.7x107
15
27.9
28.0
0.19
1.5x107
20
29.7
29.2
0.21
2.4x107
25
25.4
24.9
0.20
1.1x107
30
26.2
30.5
0.22
1.4x107
Table 6.9. Results from the SLS and DLS measurements of polymerized samples of 27.5 mM
TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS / hexyl acrylate mixtures at 25 °C. Given are
the hydrodynamic radius Rh, radius of gyration Rg, polydispersity index PDI (from DLS),
apparent molecular weight Mw (from SLS) and Nagg aggregation number (with respect to all
molecules contained).
.
Chapter 6. The Effect of Acrylate Monomers
116
index. This change can be explained by the effect of cross-linking. Cross-linked polymer chains
entrapped in the vesicle membrane, basically tighten the particle and make them more robust.
c(hexyl
acrylate)
/ mM
Rh
(nm)
Rg
(nm)
PDI
Mwapp
(g/mol)
15/0.1
22.6
23.2
0.16
1.5x107
15/0.2
23.5
28.0
0.18
1.6x107
15/0.4
23.9
24.5
0.20
1.8x107
20/0.1
28.1
28.0
0.23
2.2x107
20/0.2
28.4
28.5
0.20
2.2x107
20/0.4
30.2
31.5
0.21
2.6x107
25/0.1
24.0
26.7
0.22
1.9x107
25/0.2
27.3
26.2
0.22
2.1x107
25/0.4
33.7
29.7
0.26
3.4x107
Table 6.10. Results from the SLS and DLS measurements of cross-linked polymerized samples
of 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS / hexyl acrylate / 1, 6-hexanediol
diacrylate mixtures at 25 °C. Given are the hydrodynamic radius Rh, radius of gyration Rg,
polydispersity index PDI (from DLS), apparent molecular weight Mw (from SLS) and Nagg
aggregation number (with respect to all molecules contained).
Chapter 6. The Effect of Acrylate Monomers
117
6.3.4 Small Angle Neutron Scattering (SANS)
Figure 6.23 shows the small angle neutron scattering intensity as a function of the magnitude
of the scattering vector q of polymerized vesicles with 50 mM total surfactant concentration
and for increasing amount of monomer. Measurements were performed at KWS1 (Munich)
with the same setup and configuration as unpolymerized vesicles described in section 6.2.3 for
a direct comparison. From the scattering curves, initially one can see that they are identical to
the scattering curves of unpolymerized samples represented in Figure 6.17 (for comparison see
Appendix 9.3.3.4). The form factor minima are around ~0.1 nm-1 and the linear slope of q-2 at
intermediate q range and q-4 at high q range are visible from the scattering curves.
For a detailed analysis of structural improvement after the polymerization, we applied the same
spherical shell model explained earlier with the log-normal distribution of the radius to the
scattering curves (Figure 6.23). For monomer concentrations lower than 40 mM, scattering
patterns did not show a remarkable change and a constant PDI (Table 6.11). For 40 mM, the
form factor minimum is much less pronounced and the PDI increases to 0.26. When we
compare the bilayer thickness before and after the polymerization, the bilayer of the
polymerized vesicles is expanded slightly from 2.7 to 2.8 nm depending on the monomer
concentration.
Figure 6.23. SANS curves of polymerized samples of 27.5 mM TDMAO / 0.275 mM L35 /
22.5 mM LiPFOS / hexyl acrylate mixtures at 25 °C. (Colored lines: measured data; solid
black line: fitted data). For clarity subsequent data sets were multiplied each with a scale
factor of 3.
Chapter 6. The Effect of Acrylate Monomers
118
The cross-linked polymerized vesicles are another important system to be investigated by
neutron scattering in order to gain information on the effect of cross-linking to the vesicle
stabilization. The scattering curves of these samples displayed in Figure 6.24 showed similar
scattering properties as the monomer loaded vesicles. Their size slightly changed due to the
cross-linking ratio and are presented in Table 6.12. The thickness of the formed polymer shell
hexyl
acrylate
amount
(mM)
Rves
(nm)
SANS
PDI
D/nm
Spherical
shell
D/nm
Kratky
Porod
Polymer
shell
thickness
/nm
Mw
(g/mol)
10
29.8
0.14
2.79
2.83
0.13
1.26x107
15
29.6
0.15
2.81
2.87
0.17
1.22x107
20
28.7
0.15
2.83
2.89
0.23
1.17x107
25
29.6
0.15
2.85
2.91
0.38
1.24x107
40
26.3
0.26
3.21
3.10
0.60
1.28x107
Table 6.11. Results from the SANS analysis of polymerized samples of 27.5 mM TDMAO /
0.275 mM L35 / 22.5 mM LiPFOS / hexyl acrylate mixtures at 25°C. Given are Φ: Volume
fraction, vesicle radius Rves, polydispersity index PDI, bilayer thickness D from spherical shell
model, bilayer thickness from Kratky-Porod analyses (see Figure A15), theoretically
calculated polymer shell thickness, and molecular weight Mw.
Figure 6.24. SANS curves of cross-linked polymerized samples of 27.5 mM TDMAO /
0.275 mM L35 / 22.5 mM LiPFOS / hexyl acrylate / 1, 6-hexanediol diacrylate mixtures at
25 °C. (Colored lines: measured data; solid black line: fitted data). For clarity subsequent
data sets were multiplied each with a scale factor of 3.
Chapter 6. The Effect of Acrylate Monomers
119
in the vesicle membrane was calculated theoretically, details are explained in appendix 9.2.5.
When one compares it with the bilayer thickness obtained from SANS analysis, especially for
low monomer concentrations, we ended up reasonable values that can fit in the bilayer range
(Figure 6.25). However, for high monomer amounts results differ from each other. Calculated
polymer shell is higher than the experimental values. This means above 20 mM concentration,
the added monomer could not entirely insert and therefore polymerize into the vesicle
membrane. For a detailed understanding, from the Porod analyses (I(q)q4 vs q4) we determined
the specific surface S/V of the polymerized samples and plotted as a function of monomer
concentration (see Figure A19). It was found that specific surface, S/V, changes very slightly
for 10 to 25 mM monomer amount, however then it decreases for 40 mM. The decrease at 40
mM content can be attributed to the formed polymer chain locate mostly in the middle
hydrophobic part of the bilayer.
Moreover, when polymerized samples are compared with the unpolymerized ones, they
retained the low PDI values in the range of 0.11 to 0.14. This confirms the effectiveness of the
method on stabilization of the vesicle templates via polymerization resulting monodisperse, and
well-defined nanocapsules.
Figure 6.25. Thickness (t) as function of monomer concentration for polymerized samples
of 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS / hexyl acrylate mixtures. Red
square: bilayer thickness obtained from Kratky-Porod analysis; black circle: bilayer
thickness obtained from SANS model; blue diamond: theoretically calculated polymer shell
thickness.
Chapter 6. The Effect of Acrylate Monomers
120
6.3.5 Cryogenic Electron Microscopy (Cryo-TEM)
An effective way to gain further structural and morphological insights can be done by cryo-
TEM experiments on the vesicle samples before and after polymerization. In Figure 6.26 cryo-
TEM images of unpolymerized and cross-linked polymerized vesicles are represented. These
samples consisted from 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS with 20 mM
total monomer concentration and 0. 1 molar ratio of cross-linker with respect to the total
monomer amount. Images were taken separately before and after polymerization. The image
on the left side belong to the monomer loaded vesicles before polymerization and was taken at
TEM laboratory of MLZ (JCNS) at Munich, Germany. Samples were deposited on holey carbon
grids. The other image on the right side was performed at Technion, Haifa (Israel) using a FEI
Tecnai T12 G2 transmission electron microscope. The details on sample preparation can be
found in section 3.1.8.
The Cryo-TEM images illustrate that both the monomer loaded vesicles and their subsequently
polymerized species have spherical shape. A vesicle radius of approximately 25 nm is obtained
from the images. Nevertheless, different size of particles seen on the images indicate a certain
size distribution of the unpolymerized and polymerized vesicles and the polydispersity deduced
hexyl
acrylate
amount
(mM)
Rves
(nm)
SANS
PDI
D/nm
Spherical
shell
D/nm
Kratky
Porod
Polymer
shell
thickness/nm
Mw
(g/mol)
15/0.1
30.0
0.14
2.82
2.83
0.22
1.21x107
15/0.2
30.4
0.15
2.81
2.85
0.24
1.29x107
15/0.4
29.8
0.14
2.75
2.81
0.20
1.17x107
20/0.1
30.1
0.15
2.78
2.87
0.22
1.29x107
20/0.2
30.9
0.14
2.80
2.89
0.23
1.30x107
20/0.4
31.2
0.14
2.94
2.90
0.24
1.40x107
25/0.1
29.4
0.14
2.82
2.86
0.38
1.05x107
25/0.2
30.1
0.18
2.83
2.87
0.44
1.09x107
25/0.4
30.3
0.14
2.81
2.80
0.35
1.24x107
Table 6.12. Results from the SANS analysis of cross-linked polymerized samples of 27.5 mM
TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS / hexyl acrylate / 1, 6-hexanediol diacrylate
mixtures at 25°C. Given are Φ: Volume fraction, vesicle radius Rves, polydispersity index PDI,
bilayer thickness D from spherical shell model, bilayer thickness from Kratky-Porod analyses
(see Figure A16), theoretically calculated polymer shell thickness, and molecular weight Mw.
Chapter 6. The Effect of Acrylate Monomers
121
is in good agreement with that from the scattering results. We substantially observed small
vesicles with radii in the range of 22 - 35 nm.
The most important result pointed out from the Cryo-TEM measurements is the evidence of the
stabilization of vesicular structures by polymerization. When we compare both images before
and after the polymerization, they look almost identical in terms of shape, size and size
distribution. In detail, parameters such as size and polydispersity index as well as the bilayer
thickness of the vesicle were be obtained from the images. Figure 6.27 shows the size
distribution of vesicles determined from different cryo-TEM images taken before and after
polymerization of ~200 vesicles. When we compare the two distributions, radius of 26 nm is
more pronounced for unpolymerized vesicles however after polymerization this value shifted
slightly to 29 nm. This observation is in accordance with what is observed from SANS and light
scattering ((Rves: outer radius), that along with the polymerization vesicle radius increased very
slightly from 26 nm to 30 nm for the sample composition of 27.5 mM TDMAO / 0.275 mM
L35 / 22.5 mM LiPFOS / 20 mM hexyl acrylate / 0.1 molar ratio of 1, 6-hexanediol diacrylate.
Figure 6.26. Cryo-TEM Images of vesicle system consisted from a) unpolymerized sample
of 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS / 20 mM hexyl acrylate / 0.1
molar ratio of 1, 6-hexanediol diacrylate; b) cross-linked polymerized sample of 27.5 mM
TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS / 20 mM hexyl acrylate / 0.1 molar ratio of 1,
6-hexanediol diacrylate.
a)
b)
Chapter 6. The Effect of Acrylate Monomers
122
The unpolymerized vesicles revealed a monodisperse system with PDI value of 0.093, while
afterwards along with the polymerization PDI increased to 0.11. It should be noted that these
values are very close to the size and distribution parameters determined from scattering methods
(for SANS analysis experimental resolution was accounted).
Additionally, bilayer thickness is another parameter that can be gained from cryo-TEM images.
For unpolymerized vesicles, an average value of 2.82 nm and for polymerized ones 2.84 nm of
thickness were calculated. These values are all in very good agreement with results gained from
other methods above (see Table 6.8, Table 6.9, Table 6.11 and Table 6.12). Consequently,
results from cryo-TEM analyses are in very good agreement with those of the other methods
and demonstrate the morphological picture of the studied systems in a very remarkable way,
especially confirming the structural stabilization of vesicles by polymerization.
Figure 6.27. Number averaged vesicle size distribution histogram obtained from the
analyses of ~200 vesicles from cryo-TEM images of a) unpolymerized sample of 27.5 mM
TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS / 20 mM hexyl acrylate / 0.1 molar ratio of 1,
6-hexanediol diacrylate; b) cross-linked polymerized sample of 27.5 mM TDMAO / 0.275
mM L35 / 22.5 mM LiPFOS / 20 mM hexyl acrylate / 0.1 molar ratio of 1, 6-hexanediol
diacrylate. The solid lines show the Log-Normal (same function used in SANS analyses)
distribution of the data.
a)
b)
Chapter 6. The Effect of Acrylate Monomers
123
6.3.6 Neutron Spin Echo (NSE)
Neutron spin echo is an effective and versatile method to study the membrane dynamics of
vesicles which can focus on very small length scales. Since the vesicle bilayer enables the
loading of different moieties, hydrophobic agents, and in our case monomers, their stability and
viscoelastic properties are of crucial importance. The bilayer thicknesses obtained from small
angle neutron scattering measurements show the general behavior of the membrane along with
the monomer loading. However, in more detail, membrane fluctuations due to the monomer
loading and its subsequent polymerization play a key role to understand the membrane stability.
Therefore, we investigated these structures with NSE method complimentary to SANS and light
scattering measurements.
Basically, vesicle bilayers show undulation movements which are related to the bending rigidity
of the membrane and accordingly the mean bending modulus κ and the Gaussian modulus κ 57.
NSE makes it possible to determine the bending modulus κ via S(q,t) which is the intermediate
scattering function gained from the measurements. S(q,t) as described by 209:
𝑆(𝑞,𝑡)=exp (−𝐷(𝑞)𝑞2𝑡)((1−𝐴(𝑞))+𝐴(𝑞)𝑆𝑢𝑛𝑑(𝑞,𝑡))
(6.1)
where 𝑆𝑢𝑛𝑑(𝑞,𝑡) describes the undulation movement of membrane, D(q) is the translational
diffusion coefficient 𝐷(𝑞)=𝐷0/𝑆(𝑞) while 𝐷0is for infinite dilutions related with Stokes-
Einstein equation 3.5.
As known from the literature, membrane dynamics can be analyzed with different models.
Simply they all derive from Helfrich bending Hamiltonian 57. The difference arises from how
these models are considering the undulation. For small microemulsion spheres, Milner-Safran
model describe the motions by applying single exponentials to 𝑆𝑢𝑛𝑑(𝑞,𝑡) by counting in only
the longest undulation which is simply the sphere radius. Therefore this approach is only limited
to small spheres which has the form factor minima in the high q range 210,211.
On the other hand, Zilman-Granek model has been developed to determine the case of vesicles
predicting an integration over all undulation wave vectors between the length scale of the
vesicle and lower cut-off molecular length scale 212–214. Due to this model 𝑆𝑢𝑛𝑑(𝑞,𝑡)is described
as, 𝑆𝑢𝑛𝑑(𝑞,𝑡)=exp (−(Γ𝑍𝐺𝑞3𝑡)2
3) which decays with a stretched exponential. The Γ𝑍𝐺
(GammaZG) in the equation is described by =0.025γ√𝑘𝐵𝑇
κ𝑘𝐵𝑇
η. Here the parameters are γ≈
Chapter 6. The Effect of Acrylate Monomers
124
1− 3𝑘𝐵𝑇
(4𝜋𝜅)ln(𝑞𝜉)≈1 for 𝜅/ 𝑘𝐵𝑇≫1. In the end, the relation between the bending modulus 𝜅
and the Γ𝑍𝐺 for Zilman-Granek is given as Γ𝑍𝐺 ∝1/√𝜅 .
In this work we investigated 4 different samples with the neutron spin echo (NSE) method.
- Sample 1 was the bare vesicles (27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS)
- Sample 2 was the 20 mM hexyl acrylate loaded vesicles (unpolymerized mixture of 27.5 mM
TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS / 20 mM hexyl acrylate)
- Sample 3 was cross-linked polymerized vesicles of 15 mM monomer concentration (27.5 mM
TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS / 15 mM hexyl acrylate / 0.1 molar ratio of 1, 6-
hexanediol diacrylate (with respect to the total monomer amount))
- Sample 4 was cross-linked polymerized vesicles of 20 mM monomer concentration (27.5 mM
TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS / 20 mM hexyl acrylate / 0.1 molar ratio of 1, 6-
hexanediol diacrylate (with respect to the total monomer amount)).
We applied the Zilman-Granek method for data analysis to examine the membrane dynamic.
Figure 6.28 represents the measured NSE data applying a stretch exponential function to the
curves. Model derived from the equation 6.1 taking into account the 𝑆𝑢𝑛𝑑(𝑞,𝑡) and as seen
Figure 6.28. S(Q,t) of bare vesicles consisted from 27.5 mM TDMAO / 0.275 mM
L35 / 22.5 mM LiPFOS. Solid lines are the fits of Zilman-Granek model.
Chapter 6. The Effect of Acrylate Monomers
125
from the figure it agreed well with the curves. In Appendix 9.3.3.5 further curves are presented
for other samples, fitted with the same model and showing similar decays (Figure A25, A26
and A27).
In Figure 6.29 the apparent diffusion coefficient values, Dapp are presented and showing in
general similar trends for all samples. However, in detail the monomer insertion into the bilayer
has a different effect on dynamic behavior of the membrane. Sample 2 consisted of monomer
loaded vesicles has higher Dapp values than bare vesicles (sample 1) meaning by inserting
monomer, membrane became softened. On the other hand, cross-linking polymerization
(sample 3 and 4) leads to a noticeable toughening of the membrane which is inherently expected
and can be seen from the low Dapp data.
Additionally, with the Zilman-Granek model the bending rigidity of the membrane can be
determined via the relation Γ𝑍𝐺 ∝1/√𝜅 . However, it is known that obtaining absolute 𝜅
values with Zilman-Granek model poses some inherent difficulties 144,215,216. Different
assumptions were suggested in this regard and successfully deduced more realistic values of 𝜅
217,218. In a similar way as done in previous investigations, here we corrected the 𝜅 values simply
using an effective solvent viscosity ηeff = 3η 219–221. Similar to results above, monomer loaded
vesicles have the least rigid membrane and this may be explained such that here functions as a
cosurfactant that is incorporated within the bilayer (also leading to a slight thinning of it). In
contrast after polymerization significantly higher values of 𝜅 are observed and this stiffening
of the membrane can be directly associated with the polymerization of the contained cross-
Figure 6.29. Apparent diffusion coefficient Dapp of samples obtained from fitting the
equation 𝑆(𝑄,𝑡)=𝑒𝑥𝑝(−𝐷𝑎𝑝𝑝𝑄2𝑡) to the NSE data.
Chapter 6. The Effect of Acrylate Monomers
126
linking monomer. The formed polymer shell appararently leads to a stiffening of the membrane,
as one would also have expected.
6.3.7 Encapsulation Efficiency
Investigations on enhancing the stability of vesicles have crucial importance in regard to their
applications in controlled release of encapsulated drugs or any other agents. It is well-known
that these hollow spherical structures can widely be used for the aim of delivery depending on
their structural properties. Within this frame, different studies have been done to understand the
encapsulation capacity of liposomes or different vesicle types such as catanionic vesicles or
polymeric vesicles 222,223 and entrapment of fluorescence dyes 224,225, drugs 50,68,226 or glucose
227 has been studied intensively. For that purpose, different spectroscopic or microscopic
methods were applied for determining their entrapment and release ability, such as fluorescence
spectroscopy, confocal imaging or cross-correlation spectroscopy 182,225.
Vesicles
𝜿[𝑲𝑩𝑻]
Sample 1
6.63
Sample 2
4.65
Sample 3
8.48
Sample 4
8.37
Figure 6.30. Schematic presentation of calcein entrapment in vesicles and quenching
function of cobalt (II) ions.
Table 6.13. Bending rigidity 𝜿[KBT] of the bilayer as obtained from NSE measurements.
Sample descriptions are listed above (see page 124).
Vesicles
𝜿[𝑲𝑩𝑻]
Sample 1
0.14
Sample 2
0.14
Sample 3
Sample 4
0.14
Table 6.13.
Chapter 6. The Effect of Acrylate Monomers
127
In this part of the work, we studied the encapsulation properties and membrane permeability of
polymer stabilized vesicles, in particular cross-linked polymerized vesicles, and compared them
with bare vesicles of TDMAO/LiPFOS/L35. Calcein as a water-soluble fluorescence agent, was
used for determining the encapsulation capacity of the vesicles. It is known that calcein is
soluble in water and self-quenches at higher concentrations 228. Our strategy (see Figure 6.30)
was based on determining the encapsulation efficiency of vesicles by loading their hydrophilic
core with calcein dye below self-quenching concentrations. When the system reaches the
equilibrium state (fluorescence intensity was measured regularly until remaining constant),
cobalt (II) chloride was added to the solution for quenching the unentrapped calcein molecules
in the bulk media. Thereby, only the fluorescence intensity of entrapped dye can be detected
afterwards.
For all experiments, 1 µM concentration of calcein was added to each sample, and later the
same amount of cobalt (II) chloride. We tested the entrapment of calcein in two different ways
(see Figure 6.31), as a first attempt dissolving it into the stock solutions (TDMAO/L35 or
LiPFOS stocks) prior to polymerization and in the second way it was added after the end of the
polymerization. In the first way, calcein was added before the formation of vesicles, which also
means before polymerization. Therefore, it needs to be dissolved either in TDMAO/L35 or in
LiPFOS solutions. When we added 1 µM concentration of dye in TDMAO/L35 stock solution,
calcein’s fluorescence intensity remained unchanged as expected. However, interestingly when
it was dissolved in LiPFOS solution, intensity dropped down. To avoid this somehow
quenching effect of LiPFOS, we dissolved the dye in TDMAO/L35 stock and subsequently
Figure 6.31. Different routes for calcein encapsulation into the vesicles.
Chapter 6. The Effect of Acrylate Monomers
128
mixed this solution with LiPFOS. Right after that, polymerization was started. At the end of the
reaction, we still observed that fluorescence intensity of calcein decreased drastically in the
absence of cobalt ions. This can happen for different reasons, the dye can be quenched or react
with the radicals during the reaction. Therefore, the second method, where we added the dye
directly to the freshly polymerized vesicles, was chosen for further analyses.
At the end of polymerization, 1 µM concentration of calcein was added and equilibrated by
slowly mixing in the dark at room temperature until the fluorescence intensity remained
unchanged after ~1 hour (of 4 ml sample volume). This means while dye was transferring from
the outside medium through the polymerized bilayer into the vesicle core, its intensity was
fluctuating due to the different reasons such as collision or adsorption. Later on, 1 µM cobalt
(II) chloride was added to the solution and fluorescence was measured continuously. In Figure
6.32 we present the dynamics of Co+2 quenching calcein in polymerized vesicles. In the
presence of cobalt (II) chloride, free calcein in the outside media was quenched and intensity
decreased very fast in 4-5 minutes. Then intensity went down slowly until it remained constant
in 30 minutes. Since the calcein dye was in equilibrium in the solution, we assume Co+2 ions
diffused through the membrane in 30 minutes and some of the dye quenched during this time
period. However, after 30 minutes we observed only the fluorescence from the encapsulated
dye that was not quenched anymore with Co+2 ions.
Figure 6.32. Dynamics of Co+2 quenching calcein of polymerized vesicle,
measured at 25 °C.
Chapter 6. The Effect of Acrylate Monomers
129
The encapsulation efficiency of vesicles was calculated via the quenching ratio: =𝐹𝛼
𝐹𝛽 . 𝐹𝛼 is the
quenched fluorescence intensity of 1 µM pure calcein (at pH=9.9, which is measured for vesicle
solutions) after cobalt chloride was added and 𝐹𝛽 is the fluorescence intensity of 1 µM pure
calcein (at pH=9.9) itself. Fluorescence of vesicles (pure or polymerized) with calcein is
defined as 𝐹𝑐𝑎𝑙𝑐𝑒𝑖𝑛,𝑣𝑒𝑠 and after adding cobalt chloride to them the intensity is termed as
𝐹𝑞𝑢𝑒𝑛𝑐ℎ,𝑣𝑒𝑠. If all calcein molecules combined with cobalt ions, the fluorescence intensity of
vesicles would be decreased to a theoretical value of 𝐹𝑡ℎ𝑒𝑜,𝑣𝑒𝑠 and 𝐹𝑡ℎ𝑒𝑜,𝑣𝑒𝑠= 𝐹𝑐𝑎𝑙𝑐𝑒𝑖𝑛,𝑣𝑒𝑠∗𝛾.
Accordingly, encapsulation efficiency is: 𝐸𝐸=𝐹𝑞𝑢𝑒𝑛𝑐ℎ,𝑣𝑒𝑠−𝐹𝑡ℎ𝑒𝑜,𝑣𝑒𝑠
𝐹𝑐𝑎𝑙𝑐𝑒𝑖𝑛,𝑣𝑒𝑠−𝐹𝑡ℎ𝑒𝑜,𝑣𝑒𝑠∗100%.
In Figure 6.33 we present the measured fluorescence intensities of different sample types. The
parameters and conditions of the measurements are described in section 3.1.12. As expected,
pure vesicles (27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS) and polymerized
vesicles (27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS / 15 mM hexyl acrylate / 0.2
molar ratio of 1, 6-hexanediol diacrylate (with respect to hexyl acrylate concentration)) without
calcein were not fluorescent, as seen in the same figure. For comparison, fluorescence
intensities of pure and polymerized vesicles were measured after addition of 1 µM calcein to
each. Subsequently, 1 µM cobalt (II) added to both solutions and the intensities after quenching
Figure 6.33. Fluorescence intensities measured at 25 °C. t = 61 min for
samples without Co+2, and t = 30 min for samples with Co+2.
Chapter 6. The Effect of Acrylate Monomers
130
reduced to 200-380 (a.u). From the intensity and related equations above we calculated the
encapsulation capacities. As a consequence, it was found that while cross-linked polymerized
vesicles can entrap 35 % of calcein, pure vesicles can encapsulate 18 % of it. This means
basically polymerization stiffens the vesicle membrane, prevents the release of dye through the
outside media, therefore enhances the encapsulation efficiency of vesicles.
As a second attempt, we compared the effect of cross-linking ratio on entrapment capacity as
well. Total monomer concentration of 15 mM was varied for 0.1 and 0.2 molar ratio of cross-
linker (with respect to hexyl acrylate concentration). The results displayed in Figure 6.34
revealed that the polymerized vesicle with 0.1 molar ratio of cross-linker can encapsulate 25 %
of calcein dye while the polymerized vesicles with higher cross-linker amount of 0.2 molar ratio
can entrap 35 % of calcein. These results agree with previously presented encapsulation
efficiency values of surfactant vesicles 32,182,229.
Overall, investigation on encapsulation efficiency of TDMAO/L35/LiPFOS vesicles and their
polymer stabilized types have shown that, polymerization led to an effective increase on
encapsulation capacity. The encapsulation capacity of polymer-stabilized vesicles is at least
twice as high as pure vesicles. This can be explained by the fact that due to the polymerization,
membrane wall becomes more robust, limiting the transportation of dye in and out and
Figure 6.34. Fluorescence intensities measured at 25 °C. t = 61 min.for
samples without Co+2, and t = 30 min for samples with Co+2.
Chapter 6. The Effect of Acrylate Monomers
131
controllably encapsulates it into the nanocapsule. On the other hand, increasing the cross-linker
ratio from 0.1 to 0.2 strengthens the polymer network formed into the vesicle membrane and
therefore changes the encapsulation capacity proportionally. Additionally, it should also be
considered that dye can be adsorbed to the bilayer 182. In either case, dye is sequestered in such
a way efficiently.
Chapter 6. The Effect of Acrylate Monomers
132
6.4 Summary
Due to the flexibility of their alkyl chain and the low water solubility, acrylate monomers were
suggested to have better compatibility for dissolving into the hydrophobic vesicle membrane.
With this idea, acrylates with different chain lengths and associated with different water
solubility, were employed to prepare monomer loaded vesicles. Butyl-, hexyl-, isooctyl-, and
dodecyl acrylate monomers were dissolved in TDMAO/L35 solutions prior to the preparation
of vesicles and they were soluble in micellar solutions at characteristic ratios depending on their
structural properties. The monomer dissolved micellar aggregates were monitored by light and
small angle neutron scattering (SANS) measurements and resulted in the transition from the
cylindrical micelles to the spherical droplet with the increase of monomer concentration.
As second step, micellar solutions of TDMAO/L35/acrylate monomer were added to LiPFOS
solution with the aim of forming vesicles. Scattering experiments indicated that acrylates loaded
vesicles were formed successfully being smaller in size (radius ~ 26 nm) than bare vesicles and
retaining the low polydispersity of 0.09-0.1. The structural transition from micellar to vesicular
aggregates were remarkably observable from SANS analyses by means of the changes in
scattering patterns (Figure 6.35).
In the last part of this section, the effect of polymerization of hexyl acrylate loaded vesicles was
comprehensively investigated. It can be said that among other acrylates, hexyl acrylate was best
suited to become incorporated into the hydrophobic membrane as it showed the largest
concentration range for the vesicle phase. Results demonstrated that after polymerization,
vesicles were effectively stabilized around the radii of ~29 nm, and structural integrity could
be kept successfully. Especially the low PDI of the vesicles could be retained. With the help of
Figure 6.35. Schematic representation of structural transition from hexyl acrylate dissolved
TDMAO/L35 micelles to hexyl acrylate loaded TDMAO/L35/LiPFOS vesicles and their
cross-linked polymerized types.
Chapter 6. The Effect of Acrylate Monomers
133
neutron spin echo measurements, we monitored the membrane fluctuations and it was shown
that polymerization lead to a substantial increase of the membrane rigidity. Lastly, the
encapsulation efficiency (EE) of bare and polymer stabilized vesicles were compared via
loading them with a fluorescence dye, calcein. While the EE of polymerized vesicles was 35%,
bare vesicles had a value of 18%, and with increasing the cross-linking ratio from 0.1 to 0.2,
EE increases to 1.5 times more. In summary, polymerization not only stabilized the vesicles but
also improved their membrane permeability. This means that, with the association of
polymerization, vesicles can suitably be used in delivery systems or other potential future
applications.
Chapter 6. The Effect of Acrylate Monomers
134
7 Summary and Outlook
In this work, we aimed to provide an effective way for stabilizing a well-defined vesicle system
which could potentially be used as nanocarrier for different application areas from detergency
to electronics 32,184,185,230. Polymerization is a key method within this context and has been used
in many studies to improve the vesicle structure by inserting hydrophobic monomeric moieties
into their bilayer and subsequently fixating them by polymerization 97,196,231,232. This
strengthens the vesicle architecture providing a polymer shell interior of the hydrophobic
vesicle membrane and thereby enhances the entrapment capacity and reduces the permeability
of vesicle membrane.
Although it is a straightforward method, different problems have been encountered during the
studies. A very known issue reported by Jung is about phase separation 103,108. In
dioctadecyldimethylammonium bromide (DODAB) vesicles, a phase separation in the bilayer
occurred due to the styrene polymerization and the formed structures showed a so called
parachute-like shape 103. These structures lost their original shape of hollow spherical geometry
and deviates substantially in structure, thereby being of little use as templated structures. In
addition, polymerization often needs high temperatures or organic solvents which poses other
limits to the method.
This thesis offers an alternative way avoiding these problems. In particular, we were interested
in stabilizing a well-defined vesicle system via UV-initiated polymerization. Spontaneously
formed TDMAO/L35/LiPFOS monodisperse vesicles are interesting structures as templates for
further applications such as drug delivery 114,115,233 and were studied before and after their
fixation by polymerization For this purpose, we inserted monomers with different chemical
properties into the vesicle system and they were then reacted by UV-initiated polymerisation.
Choosing an appropriate monomer, was the most important point because of its vital effect on
the formation of vesicle template and its compatibility with the membrane. Therefore, common
monomers which can polymerise easily and accurately, such as styrene and alkyl acrylates with
different alkyl chain lengths were employed.
Monomers can be inserted into the vesicle bilayer via concurrent or diffusion loading ways 111.
In order to prevent aging problem of vesicles and having entirely homogenous system, we chose
concurrent loading where monomer was loaded simultaneously with the formation of the
136
bilayer. Thus, monomer was dissolved in 50 mM TMDAO / 0.5 mM L35 solution prior to the
formation of vesicles. We varied the monomer concentration in a systematic fashion in order to
obtain the loading capacity of hydrophobic micellar core or vesicle bilayer.
In the first part of the thesis (Chapter 4), TDMAO/L35 micelles were treated with styrene
monomer. Interestingly, we ended up with the finding that styrene had a dual function of being
both hydrophobic and having a cosurfactant character due to its relatively high polarity 234.
Investigations revealed a structural transition from rod-like micelles to vesicles only with the
presence of styrene monomer as was observed before for medium chain alcohols 51,166. Namely,
the generation of vesicles is linked to dissolving a certain amount of styrene into the amphiphilic
palisade layer, thereby acting as a cosurfactant. Polymerization transformed the polar styrene
to less polar polystyrene and this led to a corresponding change of the packing parameter again
and highly viscous rod-like structures were formed. For still higher styrene content in TDMAO
/L35 mixtures microemulsion droplets are formed as here the further added styrene can no
longer become incorporated into the amphiphilic monolayers and then acts as an oil that
becomes solubilized into the micellar cores. Polymerization led to the formation of nanolatices
of identical size, which means that here templating works perfectly well. The entire structural
progression was examined with turbidity, UV-vis, light and small angle neutron scattering,
NMR and rheology measurements
For our model vesicle system based on 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS
formulation, the effect of styrene on this template was addressed in the second part of the work
(Chapter 5). Monomer loaded vesicles were prepared by mixing the styrene dissolved
TDMAO/L35 solutions with 50 mM LiPFOS in the molar ratio of 55:45. Visual observation
and turbidity measurements demonstrated no significant phase change due to increasing the
amount of styrene. To gain a better insight scattering experiments were applied and we obtained
that vesicle morphology was retained after monomer loading, however their size and
polydispersity increased significantly. As our main goal was to stabilize the vesicles by
polymerization, we were successfully able to retain the monomer loaded vesicles with their
initial size and polydispersity 202.
In a similar way, acrylate monomers were studied intensively in the last part (Chapter 6). The
solubilisation behaviour of butyl-, dodecyl-, hexyl-, and isooctyl acrylate monomers were tested
firstly in TDMAO/L35 micelles, following that monomer loaded vesicles were prepared by
mixing with LiPFOS as identically described above. Possessing a short alkyl chain and
therefore having high water solubility, butyl acrylate was differing from the other acrylates.
137
Oppositely, isooctyl and dodecyl acrylates could hardly have solubilized in the micellar
TDMAO/L35/water solutions. Detailed structural information of this process has been studied
with mostly scattering experiments. On the other hand, hexyl acrylate monomer facilitated a
very good compliance either with micellar aggregates or vesicular bilayer. Its hydrophobicity,
and chain length made it a perfect prototype that can easily be located in the bilayer but retaining
the thickness of ~2.8 nm, which means that it has to act similar to a cosurfactant, becoming
incorporated within the amphiphilic palisade layer. Another important point to be considered
in the polymerization part, is a proper cross-linker possessing the same chemical structure as
monomer. 1, 6-hexanediol diacrylate was used for this aim. Hexyl acrylate dissolved micelles
and their further vesicular progression were monitored by turbidity, light and small angle
neutron scattering, cryo-TEM, neutron spin echo and NMR techniques. Particularly, the change
of the SANS patterns indicated a clear transition from micelles to vesicular structures. Our
results demonstrated a decrease in vesicle size from 35 nm to 29 nm with increasing the amount
of hexyl acrylate in the hydrophobic shell and the monodisperse size distribution remains.
Furthermore, cryo-TEM measurements confirmed that no morphological changes occurred
either before or after polymerization. This means hexyl acrylate monomer was a perfectly
suitable choice in regard to stabilize the former vesicular structure via polymerization in all
aspects. Finally, the encapsulation efficiencies of the pure, and polymerized vesicles were
compared by loading a fluorescence dye calcein into the core of the hollow spheres. These
experiments confirm nicely that polymerization leads to a much more robust shell that
correspondingly makes the transport of molecules out of the vesicles more difficult, i. e. by
polymerization one can control the release properties of these nanocapsules.
To summarize, our work in this thesis presents a new point of view for the preparation of
monodisperse polymeric hollow spherical nanoparticles in a straightforward way. Its ease for
application and advantages makes it a good alternative for the production of delivery agents.
Future work in this area can be directed through its development and application into the
biocompatible or biodegradable systems. In the similar manner, their functionalized analogues
might provide a very broad application area. Consequently, long term stability of polymer
stabilized vesicles is one of the open questions which might be addressed in future studies and
can raise their importance in the field of nanocarriers.
138
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151
9 Appendix
9.1 Appendix of Chapter 4
9.1.1 Refractive Index Increment
Styrene
amount
(mM)
dn/dc
(ml/g)
0
0.1490
10
0.1498
20
0.1505
30
0.1512
40
0.1517
50
0.1522
60
0.1526
70
0.1530
80
0.1534
90
0.1537
100
0.1540
120
0.1545
140
0.1549
150
0.1551
180
0.1556
200
0.1558
Table A1. Refractive index increments for different concentration of
styrene in 50 mM TDMAO / 0.5 mM L35 at 25 °C:
Table A1. Refractive index increments for different concentration of
styrene in 50 mM TDMAO / 0.5 mM L35 at 25 °C:
152
9.1.2 Determination of Aggregation Numbers
Aggregation numbers, Nagg, were calculated for surfactant by using the formula:
𝑁𝑎𝑔𝑔=𝑉.𝜌.𝑁𝐴
𝑀𝑤𝑖
where V is the volume, NA Avogadro’s number, 𝜌 is the density, and Mwi is a sum of the
molecular weight of components due to the molar fractions 𝑥, in each sample.
9.1.3 Density of the aggregates
The aggregate densities were derived from the dilution series at constant composition of
samples for 0-50 mM. For higher styrene concentrations, values were obtained via:
𝜌= 𝜒𝑇𝐷𝑀𝐴𝑂𝑀𝑇𝐷𝑀𝐴𝑂+𝜒𝐿35𝑀𝐿35+𝜒𝑠𝑡𝑦𝑟𝑒𝑛𝑒𝑀𝑠𝑡𝑦𝑟𝑒𝑛𝑒
𝜒𝑇𝐷𝑀𝐴𝑂𝑉𝑚𝑜𝑙𝑇𝐷𝑀𝐴𝑂+𝜒𝐿35𝑉𝑚𝑜𝑙𝐿35+𝜒𝑠𝑡𝑦𝑟𝑒𝑛𝑒𝑉𝑚𝑜𝑙𝑠𝑡𝑦𝑟𝑒𝑛𝑒
𝜒𝑖 is the mole fraction of the component i in the sample, 𝑀𝑇𝐷𝑀𝐴𝑂 is the molecular weight, and 𝑉𝑚𝑜𝑙
is the molar volume.
Styrene
amount
(mM)
d/(g/ml)
0
0.9131
10
0.9126
20
0.9122
30
0.9118
40
0.9114
50
0.9111
60
0.9109
70
0.9106
80
0.9104
90
0.9102
100
0.9100
120
0.9097
140
0.9094
150
0.9093
180
0.9090
200
0.9088
Table A2. The density of the aggregates for samples of 50 mM TDMAO / 0.5
mM L35 / Styrene.
Figure A1. a) Background subtracted Kratky–Porod plots of the scattering
curves for 50 mM TDMAO/0.5 mM L35/styrene (50-60-70-80-90-100 mM). b)
SANS curve of 50 mM TDMAO / 0.5 mM L35 / 70 mM styrene. Inset shows
the Kratky-Porod plot to the q region indicated by arrows. The fitted curves for
samples containing 50-60-70-80-90-100 mM styrene concentrations can be
seen below.Table A2. The density of the aggregates for samples of 50 mM
TDMAO / 0.5 mM L35 / Styrene.
153
9.1.4 Kratky-Porod Plots
Figure A1. a) Background subtracted Kratky–Porod plots of the scattering curves for 50 mM
TDMAO/0.5 mM L35/styrene (50-60-70-80-90-100 mM). b) SANS curve of 50 mM TDMAO
/ 0.5 mM L35 / 70 mM styrene. Inset shows the Kratky-Porod plot to the q region indicated
by arrows. The fitted curves for samples containing 50-60-70-80-90-100 mM styrene
concentrations can be seen below.
a)
b)
154
Sample: 50 mM TDMAO / 0.5
mM L35 / 50 mM styrene.
Sample: 50 mM TDMAO / 0.5
mM L35 / 60 mM styrene.
Sample: 50 mM TDMAO / 0.5 mM
L35 / 70 mM styrene
Sample: 50 mM TDMAO / 0.5
mM L35 / 80 mM styrene.
Sample: 50 mM TDMAO / 0.5 mM
L35 / 90 mM styrene.
Sample: 50 mM TDMAO / 0.5 mM
L35 / 100 mM styrene.
Figure A2. The fitted Kratky-Porod curves for samples containing 50-60-70-80-90-100
mM styrene concentrations.
155
9.1.5 Small Angle Neutron Scattering (SANS) of Polymerized Samples
Figure A3. SANS intensity patterns of polymerized 50 mM TDMAO
/ 0.5 mM L35 / styrene mixtures at 25 °C.
156
9.2 Appendix of Chapter 5
9.2.1 Refractive Index Increment
9.2.2 Kratky-Porod Plots
Styrene
amount
(mM)
dn/dc
(ml/g)
0
0.0598
10
0.0740
15
0.0795
20
0.0844
25
0.0886
30
0.0925
35
0.0959
38
0.0977
41
0.0995
44
0.1013
46
0.1024
50
0.1044
58
0.1081
Table A3. Refractive index increments for different ratio of styrene to 27.5 mM TDMAO /
0.275 mM L35 / 22.5 mM LiPFOS at 25°C.
Table A3. Refractive index increments for different ratio of styrene to 27.5 mM TDMAO /
0.275 mM L35 / 22.5 mM LiPFOS at 25°C.
Figure A4. Background subtracted Kratky–Porod plots of the scattering curves for 27.5
mM TDMAO/0.27 mM L35/ 22.5 mM LiPFOS/styrene (0-10-20-30-38-41-44-46 mM).
157
Figure A5. The fitted Kratky-Porod curves for samples containing 27.5 mM TDMAO /
0.275 mM L35 / 22.5 mM LiPFOS / different styrene amounts.
158
Figure A6. The fitted Kratky-Porod curves for samples of cross-linked polymerized
samples containing 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS / different
styrene amounts with 0.1 ratio of cross-linker.
159
9.2.3 Small Angle Neutron Scattering (SANS) of Samples after 1.5 Years
Figure A7. SANS curves of two samples after 1.5 years of preparation. Left: 41 mM styrene
loaded vesicle of 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS, Right: cross-
linked polymerized sample of 41 mM styrene / 27.5 mM TDMAO / 0.275 mM L35 / 22.5
mM LiPFOS at 25 °C
160
9.2.4 Small Angle Neutron Scattering (SANS) of samples before and after
polymerization
Figure A8. I(q)q2 vs q plot of the SANS data for sample 27.5 mM TDMAO /
0.275 mM L35 / 22.5 mM LiPFOS / 38 mM styrene black square: before
polymerization, red circle: after polymerization.
Figure A9. I(q)q2 vs q plot of the SANS data for sample 27.5 mM TDMAO /
0.275 mM L35 / 22.5 mM LiPFOS / 41 mM styrene black square: before
polymerization, red circle: after polymerization.
161
Figure A10. I(q)q2 vs q plot of the SANS data for sample 27.5 mM TDMAO /
0.275 mM L35 / 22.5 mM LiPFOS / 44 mM styrene black square: before
polymerization, red circle: after polymerization.
Figure A11. I(q)q2 vs q plot of the SANS data for sample 27.5 mM TDMAO /
0.275 mM L35 / 22.5 mM LiPFOS / 46 mM styrene black square: before
polymerization, red circle: after polymerization.
162
9.2.5 Calculation of the Polymer Shell Thickness
Assuming a full conversion of monomer, the volume of polystyrene was calculated via:
𝑽𝒑𝒐𝒍𝒚𝒔𝒕𝒚𝒓𝒆𝒏𝒆=𝑵𝒂𝒈𝒈 𝒎𝒐𝒏𝒐𝒎𝒆𝒓.𝑴𝒘𝒎𝒐𝒏𝒐𝒎𝒆𝒓
𝒅𝒆𝒏𝒔𝒊𝒕𝒚(𝒊𝒏 𝒃𝒖𝒍𝒌) 𝑵𝑨 , then dividing by the surface area for the vesicle
(radius= R0+D/2, from SANS), the thickness of the polymer shell was theoretically
deduced:
𝑡𝑝𝑜𝑙𝑦𝑠𝑡𝑦𝑟𝑒𝑛𝑒= 𝑉𝑝𝑜𝑙𝑦𝑠𝑡𝑦𝑟𝑒𝑛𝑒
4𝜋(𝑅0+𝐷/2)2
163
9.3 Appendix of Chapter 6
9.3.1 Appendix to Studies with Dodecyl Acrylate Monomer
Figure A12. The fitted Kratky-Porod curves for samples containing 27.5 mM TDMAO /
0.275 mM L35 / 22.5 mM LiPFOS / different dodecyl acrylate concentrations.
164
9.3.2 Appendix to Studies with Isooctyl Acrylate Monomer
Figure A13. The fitted Kratky-Porod curves for samples containing 27.5 mM TDMAO /
0.275 mM L35 / 22.5 mM LiPFOS / different isooctyl acrylate concentrations.
165
9.3.3 Appendix to Studies with Hexyl Acrylate Monomer
9.3.3.1 Refractive Index Increment
Hexyl
acrylate
amount
(mM)
dn/dc
(ml/g)
0
0.1490
20
0.1397
30
0.1361
50
0.1304
60
0.1281
70
0.1260
80
0.1242
90
0.1225
Hexyl
acrylate
amount
(mM)
dn/dc
(ml/g)
0
0.0598
7
0.0632
10
0.0644
20
0.0679
25
0.0694
30
0.0707
Table A4. Refractive index increments for different concentration of
hexyl acrylate in 50 mM TDMAO / 0.5 mM L35 at 25 °C:
Table A5. Refractive index increments for different ratio of hexyl
acrylate to 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS at
25°C.
Table A4. Refractive index increments for different concentration of
hexyl acrylate in 50 mM TDMAO / 0.5 mM L35 at 25 °C:
Table A5. Refractive index increments for different ratio of hexyl
acrylate to 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS at
25°C.
Table A5. Refractive index increments for different ratio of hexyl
acrylate to 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS at
25°C.
166
9.3.3.2 Kratky-Porod Plots
Figure A14. The fitted Kratky-Porod curves for samples containing 27.5 mM TDMAO /
0.275 mM L35 / 22.5 mM LiPFOS / different hexyl acrylate concentrations.
167
Figure A15. The fitted Kratky-Porod curves for polymerized samples containing 27.5
mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS / different hexyl acrylate
concentrations.
168
Figure A16. The fitted Kratky-Porod curves for cross-linked polymerized samples
containing 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS / different hexyl
acrylate concentrations for different cross-linker ratios.
169
Figure A16 (continue). The fitted Kratky-Porod curves for cross-linked polymerized
samples containing 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS / different hexyl
acrylate concentrations for different cross-linker ratios.
170
9.3.3.3 Porod-Debye Plots
Figure A17. a) I(q)q4 vs q4 plots for the samples of 50 mM TDMAO / 0.5 mM
L35 / different hexyl acrylate concentrations. b) For clarity the data sets were
multiplied each with a scale factor of 3. The intercept of the straight line is the
specific surface S/V.
a)
b)
171
Figure A19. Specific surface of the polymerized sample of 27.5 mM TDMAO
/ 0.275 mM L35 / 22.5 mM LiPFOS / different hexyl acrylate concentrations
deduced by Porod analysis as function of monomer concentration.
Figure A18. Specific surface of the system of 50 mM TDMAO / 0.5 mM L35
/ different hexyl acrylate concentrations deduced by Porod analysis as function
of monomer volume fraction.
172
9.3.3.4 Small Angle Neutron Scattering (SANS) of samples before and after
polymerization
Figure A20. I(q)q2 vs q plot of the SANS data for sample 27.5 mM TDMAO /
0.275 mM L35 / 22.5 mM LiPFOS / 10 mM hexyl acrylate black square:
before polymerization, red circle: after polymerization.
Figure A21. I(q)q2 vs q plot of the SANS data for sample 27.5 mM TDMAO /
0.275 mM L35 / 22.5 mM LiPFOS / 15 mM hexyl acrylate black square:
before polymerization, red circle: after polymerization.
173
Figure A22. I(q)q2 vs q plot of the SANS data for sample 27.5 mM TDMAO /
0.275 mM L35 / 22.5 mM LiPFOS / 20 mM hexyl acrylate black square:
before polymerization, red circle: after polymerization.
Figure A23. I(q)q2 vs q plot of the SANS data for sample 27.5 mM TDMAO /
0.275 mM L35 / 22.5 mM LiPFOS / 25 mM hexyl acrylate black square:
before polymerization, red circle: after polymerization.
174
Figure A24. I(q)q2 vs q plot of the SANS data for sample 27.5 mM TDMAO /
0.275 mM L35 / 22.5 mM LiPFOS / 40 mM hexyl acrylate black square:
before polymerization, red circle: after polymerization.
175
9.3.3.5 Neutron spin echo measurements
Figure A25. S(q,t) of unpolymerized vesicles consisted from mixture of 27.5 mM
TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS / 20 mM hexyl acrylate (Sample 2).
Solid lines are the fits of Zilman-Granek model.
Figure A26. S(q,t) of cross-linked polymerized vesicles consisted from
mixture of 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS / 15 mM
hexyl acrylate / / 0.1 molar ratio of 1, 6-hexanediol diacrylate (with respect
to the total monomer amount) (Sample 3). Solid lines are the fits of Zilman-
Granek model.
176
Figure A27. S(q,t) of cross-linked polymerized vesicles consisted from
mixture of 27.5 mM TDMAO / 0.275 mM L35 / 22.5 mM LiPFOS / 20 mM
hexyl acrylate / / 0.1 molar ratio of 1, 6-hexanediol diacrylate (with respect
to the total monomer amount) (Sample 4). Solid lines are the fits of Zilman-
Granek model.
