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Macro-, Micro-, and Nanospheres from
Cellulose – Their Preparation,
Characterization and Utilization
Christopher Carrick
Doctoral Thesis
Kungliga Tekniska Högskolan, Stockholm 2014
AKADEMISK AVHANDLING
Som med tillstånd av Kungliga Tekniska Högskolan i Stockholm
framlägges till offentlig granskning för avläggande av teknisk
doktorsexamen fredagen den 26 september 2014, kl. 10.00 i sal F3,
Lindstedtsvägen 26, KTH, Stockholm. Avhandlingen försvaras på
engelska. Fakultetsopponent: Professor Derek Gray, McGill
University.
Supervisor
Professor Lars Wågberg
Copyright © 2014 Christopher Carrick
All rights reserved
Paper I © 2013 RSC Advances
Paper II © 2014 Langmuir
Paper III 2014 Manuscript
Paper IV © 2014 RSC Advances
Paper V 2014 Manuscript
TRITA-CHE Report 2014:32
ISSN 1654-1081
ISBN 978-91-7595-231-4
Abstract
The structure of a polymeric material has a great influence in many
fundamental scientific areas as well as in more applied science, since it
affects the diffusion, permeability, mechanical strength, elasticity, and
colloidal properties of the materials. The results in this thesis demonstrate
that it is possible to fabricate solid and hollow cellulose spheres with a
cellulose shell and encapsulated gas, liquid or solid particles and with a
sphere size ranging from a few hundreds of nanometres to several
millimetres, all with a tailored design and purpose.
The sizes of the different spheres have been controlled by three
different preparation methods: large cellulose macrospheres by a solution
solidification procedure, hollow micrometre-sized cellulose spheres by a
liquid flow-focusing technique in microchannels, and nanometre-sized
cellulose spheres by a membrane emulsification technique.
The spheres were then modified in different ways in order to
functionalize them into more advanced materials. This thesis demonstrates
how to control the cellulose sphere dimensions and the wall-to-void volume
ratio, the elasticity and the functionality of the spheres as such, where they
were prepared to be pH-responsive, surface specific and X-ray active. These
modifications are interesting in several different types of final materials
such as packaging materials, drug release devices or advanced in vivo
diagnostic applications.
In the more fundamental science approach, surface-smooth solid
cellulose spheres were prepared for characterization of the macroscopic
work of adhesion when a cellulose surface is separated from another
material. Using these ultra-smooth macroscopic cellulose probes, it is
possible to measure the compatibility and the surface interactions between
cellulose and other materials which provide an important tool for
incorporating cellulose into different composite materials.
Sammanfattning
Strukturen av polymera material har en stor inverkan i många
grundvetenskapliga och mera applicerade vetenskapsområden eftersom den
påverkar materialets diffusionsegenskaper, permeabilitet, mekaniska
egenskaper, elasticitet och deras kolloidala egenskaper. Denna avhandling
visar att det är möjligt att tillverka solida samt ihåliga cellulosasfärer som
utgörs av en cellulosavägg med inkapslad gas, vätska eller solida partiklar
inom storleksintervallet från några få hundratals nanometer till flera
millimeter med skräddarsydd design för olika slutapplikationer.
Resultaten visar övergripande att sfärernas storlek kunde kontrolleras
med hjälp av tre olika tillverkningsmetoder. De millimeterstora
cellulosakapslarna preparerades med hjälp av en solidifiering av en upplöst
cellulosablandning. De ihåliga mikrometerstora cellulosakapslarna
tillverkades med flödesfokusering av vätskor i mikrometerstora kanaler och
de nanometerstora kapslarna tillverkades med en
mebranemulgeringsteknik.
De tillverkade cellulosakapslarna skräddarsyddes sedan i flera
efterföljande modifieringssteg för att funktionalisera cellulosakapslarna till
mer avancerade slutmaterial. Resultaten visar att det är möjligt att
kontrollera kapselns dimensioner såsom volymsförhållandet mellan
kapselväggen och kapselhåligheten, kapselns elasticitet samt kapselytans
specificitet och responsivitet. De sistnämnda egenskaperna visades genom
att tillverka kapslar som svarade mot ändringar i pH, specifik växelverkan
med olika biomolekyler och att var röntgeninteraktiva. Dessa olika
modifieringar gör materialet intressant för olika slutanvändningsområden
som förpackningsmaterial, kontrollerad frisläppning av läkemedel eller för
avancerad medicinsk diagnostik.
För mera fundamentala studier tillverkades mycket ytsläta
cellulosasfärer för att karaktärisera det makroskopiska adhesionsarbetet
som krävs för att separera ett cellulosamaterial från ett annat material.
Genom att använda dessa makroskopiska och släta cellulosaprober är det
möjligt att mäta kompatibiliteten och ytinteraktionerna mellan cellulosa och
andra material vilket är ett omistligt verktyg då cellulosafibrer/fibriller
inkorporeras i olika kompositmaterial.
List of Papers
This thesis is a summary of the following papers:
I “Hollow cellulose capsules from CO2 saturated cellulose solutions —
their preparation and characterization”, Christopher Carrick, Marcus
Ruda, Bert Pettersson, P. Tomas Larsson and Lars Wågberg, RSC
advances 2013, 3, 2462-2469
II “Lightweight, Highly Compressible, Noncrystalline Cellulose
Capsules”, Christopher Carrick, Stefan B. Lindström, P. Tomas
Larsson and Lars Wågberg, Langmuir, 2014, 30 (26), 7635–7644
III “Macroscopic Spherical Cellulose Probes with low surface roughness
– their preparation and applications as adhesion probes for
interaction measurements”, Christopher Carrick, Sam Pendergraph
and Lars Wågberg, Manuscript
IV “Native and functionalized micrometre-sized cellulose capsules
prepared by microfluidic flow focusing”, Christopher Carrick, Per A.
Larsson, Hjalmar Brismar, Cyrus Aidun and Lars Wågberg, RSC
Advances, 2014, 4, 19061-19067
V “Immunoselective cellulose nanospheres – a versatile platform for
nanotheranostics”, Christopher Carrick, Lars Wågberg and Per A.
Larsson, Manuscript
The contributions of the author of this thesis to these papers are:
I Principal author. Planned and performed most of the experimental
work
II Principal author. Active in planning the experiments and performed
most of the experimental work.
III Principal author. Planned and performed most of the experimental
work
IV Principal author. Planned and performed all the experimental work
V Principal author. Active in planning the experiments and performed
most of the experimental work.
Abbreviations
AFM Atomic force microscopy
BSA Bovine serum albumin
Ca Capillary number
CED Cupriethylenediamine
CNS Cellulose nanosphere
CSLM Confocal scanning light microscopy
DMAc Dimethylacetamide
EGFR Epidermal growth fractor receptor
FITC Fluorescein isothiocyanate
ELISA Enzyme-linked immunosorbent assays
GNP Gold nanoparticles
JKR Johnson-Kendall-Roberts
MCAM Macroscopic contact adhesion measurement
MFFD Microfluidic flow focusing device
NMMO N-Methylmorpholine N-oxide
PBS Phosphate buffered saline
PDMS polydimethylsiloxane
QCM Quartz crystal microbalance
Re Reynolds number
RH Relative humidity
SEM Scanning electron microscopy
THF Tetrahydrofuran
W/O Water-in-oil
W/O/W Water-in-oil-in-water
We Weber number
XRD X-ray diffraction
Contents
1. Introduction ......................................................................................... 1
1.1 Setting the scene ...................................................................... 1
1.2 Methods for the preparation of spherical objects ..............2
1.2.1 General emulsification techniques ................................2
1.2.2 Microfluidic emulsification techniques ..........................4
1.3 Cellulose ..................................................................................... 7
1.4 Stability, solubility and regeneration of cellulose
structures ................................................................................... 8
1.5 Cellulose spheres .....................................................................9
1.6 Swelling of a cellulose gel network ..................................... 10
1.7 Cellulose functionalization .................................................... 13
1.8 Cellulose bulk functionalization techniques ...................... 14
1.9 Smooth cellulose surface – controlling the topography . 15
2. Experimental ..................................................................................... 16
2.1 Materials ................................................................................... 16
2.2 Cellulose fibres ........................................................................ 16
2.3 Experimental procedures ...................................................... 16
2.3.1 Preparation of charged cellulose fibres ...................... 16
2.3.2 Preparation of cellulose solutions ............................... 17
2.3.3 Preparation of cellulose capsules ............................... 17
2.4 Characterization techniques ................................................. 20
2.4.1 Mechanical compression response ........................... 20
2.4.2 Field-Emission Scanning Electron microscopy
(FE-SEM) ...................................................................... 21
2.4.3 Atomic force measurement (AFM) .............................. 21
2.4.4 Confocal scanning light microscopy (CSLM) ............. 21
2.4.5 X-ray diffraction (XRD) ................................................ 22
2.4.6 Macroscopic contact adhesion measurement
(MCAM) ........................................................................ 22
2.4.7 Monitoring the adsorption of CNSs ............................ 22
3. Results and Discussion ............................................................. 24
3.1 Cellulose capsule preparation: controlling the shape,
geometry and size (Papers I to V) ..................................... 24
3.2 Mechanical properties of the cellulose macrospheres
and capsules (Papers I & II) ................................................ 32
3.2.1 Mechanical response of porous cellulose
macrocapsules (Paper I) ............................................ 33
3.2.2 Mechanical response of non-porous cellulose
macrocapsules (Paper II) ........................................... 34
3.3 Adhesion measurements using solid cellulose
macrospheres (Paper III) ..................................................... 39
3.4 Swelling of the cellulose capsule gel structure
(Papers I, II, IV & V) .............................................................. 43
3.5 Surface modifications using antibody conjugation
(Paper V) .................................................................................. 47
3.6 Encapsulation of gold nanoparticles (Paper V) ............... 51
3.7 Micro- and nanocapsules as an extended release
device or as a diagnostic tool (Paper IV & V) .................. 53
4. Conclusions ..................................................................................... 56
5. Future Work ...................................................................................... 58
6. Acknowledgments ........................................................................ 60
7. References ........................................................................................ 62
1
1. Introduction
1.1 Setting the scene
The study, understanding and shaping of different geometries of
materials has for several millennia been of fundamental interest and has
fascinated many people. Examples are the fantastic architecture of the
pyramids in Egypt or the spectacular observations by Galileo and Galilei
regarding the shape of the earth.
In recent years, there has been an increasing interest in nanotechnology,
which provides a totally new way of optimizing the final material
properties by a nano-scale ordering of well-defined nano-components.
Due to the small size of these entities they have properties totally
different from those of the corresponding macroscopic materials since
they can adopt discrete energy states.1 Nanoscience has also created a
huge interest in the cellulosic research field due to the preparation of the
nanofibrils, liberated from larger cellulose fibres by homogenization
procedures creating smaller fibrils with a much higher aspect ratio, i.e.
diameter-to-length ratio, than the original fibre.2 In this work, a different
route was pursued to fabricate solid and hollow spherical cellulose
materials. This relatively unexplored cellulose field further enabled us to
study the behaviour of a cellulose polymer matrix in a more typical
colloidal application, where the swelling of a hollow cellulose sphere or
the diffusion of encapsulated substances through a cellulose shell
structure has been characterized as well as the interfacial cellulose
adhesion behaviour when interfaced with other polymers. The use of
2
these hollow cellulose spheres, which can also be considered as a
capsules, in in vivo applications is of specific interest since cellulose
possesses features such as excellent biocompatibility with human tissue,3
absence of immunostimulatory reactions and lack of enzymatic in vivo
degradation.4 This means that cellulose has an excellent potential for use
in pharmaceuticals, e.g. as a drug delivery matrix.
1.2 Methods for the preparation of spherical objects
There are several methods for the preparation of spherical drops
including emulsification techniques,5, 6 microfluidic flow techniques,7, 8
phase separation techniques,9, 10 dripping11 and spraying techniques.12 All
these techniques generate spherical liquid drops due to the minimization
of surface energy, since a spherical geometry will always have a lower
surface-to-volume ratio than an irregular geometry.
1.2.1 General emulsification techniques
When forming an emulsion, two immiscible fluids are distributed within
each other, for example as when vinegar and oil are mixed together into a
salad dressing. Neglecting the influence of the type of surfactant used, the
mixing shear forces will in most cases break the fluid with the lower
volume into small drops. When preparing a typical salad dressing, less
vinegar than oil is used and hence the vinegar breaks into drops, forming
a water-in-oil (W/O) emulsion. When the system is sheared, small water
volumes are formed which immediately start to rearrange into perfectly
spherical drops because of the surface energy minimization and a
thermodynamically unstable macroemulsion is created. The
thermodynamically stable state is a layered structure with the lowest
3
density fluid sitting on top of the denser fluid. However, in the emulsion,
the size of the drops formed is related to the applied shear force. The
greater the shear force that is transferred to the emulsion, the smaller is
the drop size.13, 14 When a single W/O emulsion has been prepared, it is
possible to generate a double emulsion by adding the emulsion to a larger
volume of a third liquid and again introducing shear forces. In this case, a
polar water phase is added which is immiscible with the previous
continuous oil phase. To avoid breaking the initial emulsion it is
important to apply a lower shear force in the second mixing step than was
applied in the first emulsification step.15 If the initial emulsion is broken,
the two aqueous phases may come in contact and mix to create an oil-in-
water (O/W) emulsion since a larger volume of the aqueous phase is now
present. If a lower shear force was successfully applied to the initial W/O
phase, a water-in-oil-in-water (W/O/W) double emulsion is generated.
This process can be continued to generate triple/quadruple emulsions
etc..16
To increase the stability of the prepared emulsion, surfactants or surface
active particles (pickering emulsion)17 can be used. Surfactants which are
amphiphilic molecules have a polar head-group and a non-polar tail
which make them surface active. The molecules will be dissolved in the
solution and will be located between the immiscible fluids at the liquid–
liquid interfaces.18, 19 The adsorption at the interface lowers the surface
energy of the drops and therefore decreases the interfacial tension
between the two liquids.15 These surfactant molecules will also ultimately
lead to greater stability by introducing repulsion between the drops of the
same liquid, as illustrated in Figure 1. The type of surfactant also affects
the emulsion according to the Bancroft rule which states that the
surfactant solubility in the different phases is the determining factor for
which fluid will be emulsified and form drops. 20, 21 If, for example, the
4
surfactant dissolves better in the water phase, an O/W emulsion is
formed. This is the case for mayonnaise where the dispersed phase
consists of approximately 75% oil and the 25% continuous phase is water
stabilized by natural surfactants emanating from the egg yolk.
Figure 1. Schematic image of an emulsion stabilized by a surfactant.
1.2.2 Microfluidic emulsification techniques
In the last ten years, microfluidic techniques have been of great interest
for use in many scientific fields such as fluid mechanics,7 molecular
biology,22, 23 colloidal chemistry,24, 25 electro-chemistry26 and medicine25.
This is because it has proven to be very efficient for the preparation of
uniform and monodisperse drops, since the shear forces acting on the
different fluids can be finely tuned. This microfluidic technique enables
the continuous preparation of single droplets, at one single location
inside a capillary tube (Figure 2). The general strategy for forming an
emulsion inside a microfluidic device is to allow immiscible liquids to
flow inside micrometre-sized capillary channels. When the immiscible
5
liquids meet in the micro-channels, drop formation occurs due to
Rayleigh-Plateau instabilities.27, 28 A thin thread of the injected liquid is
created and this ultimately breaks to generate a drop, thus lowering the
surface energy after a certain distance from where the two liquids meet.
The same phenomenon can be seen when turning on the faucet when
washing your hands; at a high water flow a continuous thread/stream of
water hits the sink, but if the distance between the faucet and the sink
were increased to an infinite distance, water drops would inevitable be
generated for the same reason as before, assuming that the thread of
water will become continuously thinner due to acceleration, so that at
some point the inertial forces of the fluid will overcome the viscous forces
and will thus minimize the surface-to-volume ratio by forming drops.
Another way of demonstrating this is to reduce the flow of water from the
faucet and soon after you have reduced the flow rate sufficiently water
drops will be generated before the water phase reaches the sink. In this
case, the emulsion is created by the continuous gas phase (air), which can
be considered as a fluid with low density, and the liquid water coming
from the faucet.
There are two main ways of forming microdrops using this technique: the
T-junction29-31 and the microfluidic flow focusing devices (MFFD)32-34.
The most frequently used system is the T-junction system (Figure 2a)
which is based on micro-channels molded, in most of the cases, using
polydimethylsiloxane (PDMS). This system has, in the simplest approach,
two inlets that at an angle of 90 ᵒ (T-junction) where a droplet is pinched
off, creating a single emulsion after the intersection where the two
immiscible fluids come into contact. The main advantage using the T-
junction concept is that it can easily be built and it is possible to create a
large number of different geometries and flow patterns through the
device. In the MFFD system, the device is constructed from capillary glass
6
tubes placed inside square glass tubes that are aligned to create a
symmetric junction where three different fluids can be injected into a
collection tube (Figure 2b). This system can therefore generate a double
emulsion in a single junction. Since the main objective of our work was to
prepare a double emulsion, the MFFD was the only technique
investigated.
Figure 2. Illustration of the T-junction (a) and the microfluidic flow-focusing device (b).
In practice, the shear forces are controlled by the size of the capillaries,
the flow rates pushing the fluids inside the device and the solvent
properties.34 The dispersity in the microfluidic techniques is a function of
how far from the junction the thread breaks into drops. The closer to the
junction the jet pinches off into drops, the more monodisperse will the
drops become.34 Depending on how far from the junction the droplet is
being pinched off, the system can be defined as being in the dripping or in
the jetting regime. If the droplet is pinched off before three times the
diameter of the collection tube, it is considered to be in the dripping
regime and if the drop is pinched off after that distance it is considered to
be in the jetting regime (where the polydispersity of the drops formed is
greater). The distance where the drop is pinched off is controlled by e.g.
the device geometry, the viscosity of the liquids, the flow rates, the surface
tensions and the density of the fluids.34, 35 Even though the drop is
pinched off in the jetting regime, the droplet formation is still less
polydisperse than can be achieved with normal emulsification
7
techniques.35 There are however limitations with this technique, the most
severe limitation being the production rate. In a normal microfluidic
device, a typical volume of drops generated is of the order of microlitre to
millilitre per hour. Yet, there are several interesting materials that have
been developed using microfluidics, for example thermo-responsive
poly(N-isopropylacrylamide) (PNIPAAm) spheres,36 and capsules for the
controlled release of encapsulated drugs,37 cosmetics38 and pesticides.39
1.3 Cellulose
Cellulose is the most abundant renewable polymer on earth. It is the main
constituent of wood and plants and it is estimated that 7.5 · 1010 tonnes
are produced per year.40 The cellulose polymer is hydrophilic,
biodegradable and biocompatible,3, 41 a stiff linear homoploymer linked
together with β (1 4) glucosidic bonds forming a rod-like polymer with a
degree of polymerization of 500-4000 cellobiose repeating units when
extracted from wood,42 with an elastic modulus of approximately 130 GPa
for the cellulose crystal.43, 44 The main procedure for extracting and
purifying cellulose is from wood raw materials, consisting of
approximately 40% cellulose together with lignin and hemicelluloses,45
using e.g. the kraft46 or sulphite pulping methods47. These pulping
methods liberate the cellulose-rich fibres from the wood raw material
primarily by dissolving the lignin-rich adhesive lamellae joining the fibres
together. The pulping process can be performed so that the
hemicelluloses are retained or dissolved from the fibres depending on the
process conditions. Due to the abundance and chemical properties of
cellulose, it is nowadays refined into materials such as paper, textiles,
hygiene products and drug dispersants. However, the excellent properties
of cellulose are definitely also suitable for use in more advanced materials
8
and applications, and this has recently achieved considerable interest in
the forest-rich countries due to the rapid down-turn in the use of
newsprint and other printing and writing papers.
1.4 Stability, solubility and regeneration of cellulose
structures
Cellulose has several different crystalline structures. The natural form of
cellulose is called cellulose I and it exists in two crystalline forms;
cellulose Iα and cellulose Iβ. Cellulose Iα is dominant in bacteria and algae
whereas cellulose Iβ is present in plant structures such as wood.48 The
crystal structure can however be changed to other packing structures.
When textiles are produced using the viscose process, cellulose is
dissolved by generating a cellulose xanthogenate derivative
(Cross et al.)49 which increases its solubility in alkali, since cellulose itself
is not dissolved in conventional solvents such as water, ethanol, acetone
or toluene. The dissolved cellulose derivative can then be precipitated in
acidic water to remove the xanthate, and the cellulose chemical structure
is regained, i.e. the cellulose is regenerated.50 This process changes the
cellulose crystalline structure from the parallel chain stacking in
cellulose I to an antiparallel chain stacking in cellulose II. The
regeneration is an irreversible process with regard to the crystalline
structure, which means that the cellulose II structure is
thermodynamically more stable than cellulose I, which is considered to be
meta-stable.48 This transformation of the cellulose structure can however
be achieved using solvents other than carbon disulphide, such as
N-Methylmorpholine N-oxide (NMMO).50 This cellulose solvent is not
however considered to be a true solvent, since the cellulose is not
completely dissolved.51 The solution contains cellulose crystals that are
9
approximately 5 nm in size, consisting of 50–100 cellulose chains.52, 53 In
order to completely dissolve cellulose, an ionic liquid is commonly
used,54 cupriethylenediamine (CED)55 or lithium chloride in
N,N-dimethylacetamide (LiCl-DMAc)56. By dissolving the cellulose in e.g.
LiCl-DMAc solution and subsequently solidifying the cellulose in a non-
solvent such as water or ethanol, the degree of crystallization in the dry
state is reduced from about 60 % to below 1 %, creating a non-crystalline
cellulose structure.57
1.5 Cellulose spheres
Dyed solid cellulose spheres with a diameter of 1–2 mm were first
prepared by Pettersson and Eriksson (2000)11 by dissolving cellulose in
LiCl-DMAc and then solidifying the dissolved cellulose solution by
dripping it into a non-solvent water reservoir forming solid cellulose
spheres. The spheres were then used to measure the degradation kinetics
and the activity of endoglucanase while enzymatically degrading the
cellulose. Depending on the preparation conditions, it was possible to
control the amount of cellulose and the dye content in the prepared
cellulose spheres, and this provided a facile and precise way of measuring
the kinetics of amorphous cellulose degradation in water due to the
continuous release of dye as the amorphous and solid cellulose was
degraded. At this point, the preparation of cellulose spheres was limited
to solid spheres. From a material point of view, preparing a hollow
cellulose capsule would be interesting to enable more advanced
applications of regenerated cellulose. A cellulose capsule, i.e. a hollow
cellulose sphere, with a wall structure of cellulose could for example lower
the material consumption for different end-use applications and can for
example be achieved by encapsulating a gas such as air. Furthermore, the
10
cellulose capsule could protect and encapsulate liquid or solid drugs for
extended release applications, or contrast agents for pharmaceutical
applications. The cellulose could then be further functionalized to be
compatible with other materials or to be, for example, pH responsive58
which could be an interesting property for controlled-drug-release
applications where the release of a drug is triggered by swelling or
delayed by shrinkage of the cellulose wall structure. A pH change is a
naturally occurring process when digesting food and extracting energy
and other vital components in our human body where the pH is
approximately 1.5–3.5 in the stomach and 7–9 in the small intestine.59
Many different drug formulations use this effect by coating the drugs with
an acid-insoluble enteric coating which is stable at pHs below 5–6 and
dissolves at a higher pH.60
1.6 Swelling of a cellulose gel network
The swelling of a wet cellulose network is dependent on the free energy of
swelling caused by the charges of the cellulose ( ), the free energy of
restraining properties of the cellulose polymer network ( ) and
the free energy emanating from the interaction between the cellulose and
the solvent ( ) present in the cellulose gel/network.
(1)
The charges of the gel, i.e. in the case of cellulose fibres the carboxyl
groups from the hemicellulose, create an osmotic pressure inside the gel
due to the imbalance of concentration of low molecular mass ions outside
and inside the gel. Together with a difference in activity coefficient of
water in the proximity of the cellulose inside the gel and of the bulk water,
11
this will lead to a combined swelling pressure inside the cellulose
gel/network. This term can in turn be related to the quality of the solvent
which is usually described by the -parameter. For good solvents, this
parameter is less than 0.5, whereas for bad solvents the value is greater
than 0.5.61 In equilibrium, this combined swelling pressure is
counteracted by the restraining network pressure caused by the fibrillar
network in the gel and this is mainly of entropic origin.62 By describing
the swelling of a cellulose network in this way, it is possible to design a
swelling cellulosic gel. In practice, the most common way of preparing a
swelling and pH- or salt-concentration-responsive cellulose gel is to
increase the ionic contribution, i.e. the charge density, of the cellulose
backbone by for example carboxymethylation, creating a weak
polyelectrolyte.63 This weak cellulose polyelectrolyte has carboxyl groups
which are protonated or deprotonated at low and high pH respectively or
screened at high salt concentrations. This swelling effect on a cellulose
polymer gel was demonstrated by the early modelling work of Grignon
and Scallan, where the swelling, denoted E in Figure 3, was a function of
the salt concentration and the solution pH.58 The greatest swelling
occurred when no salt was added to the continuous phase in the pH-
interval of 9–12. Swelling was induced by deprotonation of the carboxyl
group at pH 4–5 at moderate salt concentration. When the salt
concentration in the continuous phase was increased, the swelling started
at a lower pH but the maximum swelling was lower than in the situation
without added salt. This effect was attributed to the ions available in the
bulk solution, which enable the transport of protons from the carboxyl
groups attached to the cellulose gel while preserving charge neutrality in
the cellulose gel by the attraction of sodium ions. When the salt
concentration was increased even further, the difference in osmotic
pressure between the cellulose polymer gel and the bulk phase became
smaller and this ultimately lead to less swelling. When the pH was
12
increased to above 13, the swelling was drastically reduced due to the high
ion concentration, reducing the osmotic pressure and hence the swelling
pressure.58
Figure 3. Theoretical plots of charge density (which is intimately linked to the degree of
swelling, E) as a function of pH and salt concentration. The charges consist of weak acid
groups.
This swelling effect is interesting in the preparation of stimuli-responsive
devices for e.g. drug release applications since the rate of diffusion of
molecules is dependent on the polymer network density. A swollen and
porous gel structure will allow significantly more rapid diffusion through
a capsule wall than a densely packed structure.64
E,
eq
uiv
ale
nts
/litre
Solution pH
0.02
0.04
0.06
0.08
0.10
2 4 6 8 10 12 14
Pure water
High [salt]
Moderate [salt]
Low [salt]
13
1.7 Cellulose functionalization
The chemical structure of cellulose is shown in Figure 4, where it is clear
that the structure contains many hydroxyl groups.
Figure 4. Chemical structure of a repeating unit of cellulose.
When cellulose is functionalized by covalently binding substances,
particles or chemical monomers, they are mostly attached to the hydroxyl
groups. Common cellulose functionalizations are esterification and
carboxymethylation.65 Esterification reactions are performed to increase
the hydrophobicity of the cellulose polymer or to increase the
compatibility with other materials66 whereas carboxymethylation is
performed to increase the charge density and therefore induce e.g. higher
electrostatic interactions with polyelectrolytes, to be pH sensitive or water
soluble, or to produce cellulose nanofibrils since the charges increase the
inter-molecular repulsion between the cellulose polymers and this leads
to swelling and an easier defibrillation.67 The cellulose can furthermore be
oxidized with, for example, sodium periodate to generate aldehydes that
can easily bind to primary amines. The oxidized cellulose can also be
reduced by adding for example a borohydrate solution to reduce the
aldehydes to alcohols. This oxidation however affects the material
properties of the initial cellulose fibres and the structure becomes
amorphous and ductile.68 It is possible to partially oxidize the cellulose
polymer by reducing the reaction time, sodium periodate concentration
14
or reaction temperature to preserve a high elastic modulus and
mechanical strength.68 The partially oxidized cellulose can then be
functionalized to be e.g. surface-specific by antibody conjugation onto a
cellulose surface using the available primary amines, from the antibodies,
or to increase the strength by adding the cross-linker
butanetetracarboxylic acid,69 or mussel-inspired by attaching dopamine
to the cellulose surface70.
1.8 Cellulose bulk functionalization techniques
Apart from chemical bulk functionalization, it is also possible to
incorporate functional particles by physically entrapping the particles
inside a cellulose matrix. One interesting route for bulk functionalization
of cellulose is to bind magnetic ferrite nanoparticles in a cellulose
network.71 This attachment of well dispersed magnetic nanoparticles was
demonstrated by the fact that the bulk magnetic functionalized cellulose
material could be used as a loudspeaker with a good spectrum of audible
frequencies.72 In medical science, diagnostics encapsulation of gold
nanoparticles has been of increasing interest in recent years, since the
gold nanoparticles can provide in vivo image contrast by using surface-
enhanced Raman spectroscopy,73 light microscopy74, fluorescent
imaging74 and enhanced X-ray scatter imaging75. These techniques
provide a faster and more effective route as a diagnostic tool than
conventional enzyme-linked immunosorbent assays (ELISA) where the
samples cannot be analysed in vivo. ELISA is therefore in most cases
limited to liquid samples such as blood samples, and this makes it much
harder to analyse in vivo tissue or organs.
15
1.9 Smooth cellulose surface – controlling the
topography
Controlling the topography of cellulose materials, such as preparing
cellulose smooth films, is of fundamental interest.76, 77 This is especially
important in the preparation of composite materials such as liquid
containers which are built up of several layers of different films where the
functions of each individual layer are combined in a laminate film. If the
different layers delaminate due to e.g. having a rough surface or having
poor compatibilities between the individual layers, the entire structure
and its functionalities can be lost. In model studies, cellulose thin-films
have been prepared to increase the understanding of the adhesive
properties of cellulose and other materials.76, 77 Thin films have been used
because the topography or the surface roughness is greatly affected when
the film thickness is greater than approximately 50 nm. To achieve good
contact adhesion between different surfaces, the material surfaces have to
enable molecular interactions over the entire contact area, and this
requires smooth surfaces.78 Using thin-films of cellulose has however the
limitation that only a few cellulose raw materials can be studied and that
the substrate on which the cellulose film is cast may interact with the
other surface, creating incorrect contributions to the adhesive
properties.77 To overcome these shortcomings, the cellulose surface can
be modified with other polymers to decrease the surface roughness.79
However, the true adhesion between cellulose and other materials is then
lost, since the adhesion between the functionalized surfaces is the only
data that can be seen from these types of material.
16
2. Experimental
2.1 Materials
More detailed information about the chemicals and materials employed
can be found in the attached papers and only the most important details
are presented in this summary.
2.2 Cellulose fibres
The cellulose fibres used were from a dissolving grade pulp, mainly
spruce, and were provided by Domsjö Aditya Birla AB, Sweden
(Dissolving Plus grade). The pulp contained 93% cellulose with a degree
of polymerization of about 780, determined by the CED viscometry
method80 and the rest (7%) was hemicelluloses with small traces of lignin
and extractives.
2.3 Experimental procedures
2.3.1 Preparation of charged cellulose fibres
The charge density of the cellulose fibres was modified by
carboxymethylation according to the method of Wågberg et al.63 utilizing
1-chloro-acetic acid for cellulose modification to four different
substitution levels; one batch of non-modified fibres and three batches
with different charges. The charge density was measured by
conductometric titration according to an earlier described procedure,81
giving values of 73, 114 and 350 µeqv./g for the modified fibres, while the
17
non-modified fibres had a total charge density of 29 µeqv./g emanating
from residual charged hemicellulose in the fibres.63
2.3.2 Preparation of cellulose solutions
Modified and non-modified cellulose fibres were dissolved using lithium
chloride in N,N-dimethylacetamide (LiCl-DMAc) solution according to
Berthold et al.82 If water is present in the solvent, it impairs the
dissolution of cellulose83 and promotes the formation of polymer
aggregates.84 The solvent was therefore heated to 105 °C for 30 min to
remove traces of water before adding the cellulose fibres to the highly
hygroscopic solvent mixture. The cellulose concentration was controlled
by adding different amounts of pre-swollen fibres in pure DMAc to reach
different concentrations. The solution was then re-heated to
approximately 80 °C to further remove traces of water and promote the
dissolution of the cellulose. The cellulose dissolution was regarded as
complete when clear solutions were achieved by ocular inspection with
support from Röder et al.85
2.3.3 Preparation of cellulose capsules
Millimetre-sized cellulose capsules were formed by a solution
solidification method (Paper I, II and III).57, 86 It includes, in most of the
experiments, saturating the LiCl-DMAc cellulose solution with a suitable
gas such as carbon dioxide, nitrogen or propane. When preparing solid
spheres, the gas dissolution step was excluded. The cellulose solution
with or without dissolved gas was then added drop-wise into a non-
solvent such as water, ethanol or methanol to solidify the cellulose-
containing droplet into a hollow gel particle (Figure 5). (Details about the
creation of the hollow centre of the capsules will be given on page 25)
18
Figure 5. Schematic presentation of the solution solidification method. Dissolved cellulose
is saturated with a gas (left); a sample of the solution is then taken (middle) and added
drop-wise into a non-solvent (for example pure water where it immediately solidifies (right).
Micrometre-sized cellulose spheres were prepared using a MFFD
technique which involves allowing immiscible fluids to flow in
micrometre-sized glass tubes. The centrally aligned junction made by
placing cylindrical glass tubes with matching outer diameters inside
square tubes with a matching inner diameter creates voids in the corners
of the square tube where the middle and the continuous fluids are
injected (Figure 6).16 The inner octane was injected into the centred
cylinder coming from the left in Figure 6. This MFFD set-up enabled the
preparation of a double emulsion in one junction where the three fluids
(octane, cellulose solution and silicone oil) came into contact, also
illustrated in Figure 6.
19
Figure 6. Schematic description of the MFFD, showing inlet locations, fluids and flow
directions of the three fluids. The capillary tube inner diameter, outer diameter and total
inner width of the microfluidic flow focusing device were Di = 580 µm, Do = 1000 µm and
Dtot = 1050 µm respectively.
To prepare cellulose nanospheres (CNSs), an emulsion of cellulose
solution in silicone oil was first prepared by mixing (vortex mixer) the
fluids at a volume ratio of 1:4. This mixing step creates spheres of
cellulose solution with a size of a few tens to a few hundreds of
micrometers. The emulsion was then further processed by flowing the
emulsion through a 2 µm membrane into a non-solvent where the
cellulose emulsion was broken into even smaller spheres and
subsequently solidified, creating spheres with a diameter of
approximately 160 nm. This process is schematically illustrated in Figure
7.
Dto
t
Do Di
Cellulose solution
Octane
Silicone oil
20
Figure 7. Schematic illustration of how dissolved cellulose droplets in silicone oil are
pushed through a 2 µm membrane into ethanol where the dissolved cellulose solidifies as
cellulose nanospheres in ethanol.
2.4 Characterization techniques
Detailed information about the different characterization and
instrumental techniques can be found in the individual papers.
2.4.1 Mechanical compression response
In order to determine the dry mechanical properties of the capsules, such
as compressibility, elastic modulus and gas permeability, a Deben micro-
tensile tester with a 50 N load cell was used. The tests were performed on
conditioned cellulose capsules at 50% RH and 23 oC where a single
capsule was compressed between two flat steel plates to different
compressive strains or loads where the change in load was continuously
Large drops
of dissolved
cellulose in oil
Filter with
2 µm pore-
size
Solidified
cellulose
nanospheres
in non-solvent
21
monitored as a function of compressive strain or as a function of time in
comparison with an initial compressive load.
2.4.2 Field-Emission Scanning Electron microscopy
(FE-SEM)
The capsule’s morphology was imaged using SEM, equipped with a cold
field emission electron source. The capsules were either freeze-dried to
preserve the porous wet structure or air dried at different temperatures to
provide information about the different structures of the capsules
prepared.
2.4.3 Atomic force measurement (AFM)
The topography and surface roughness of the dried cellulose spheres were
characterized with AFM, operating in the Scanasyst mode with a
cantilever having a tip radius of approximately 8 nm and a spring
constant of 5N/m.
2.4.4 Confocal scanning light microscopy (CSLM)
The diffusion of an encapsulated 4 kDa dextran model drug with
covalently attached fluorescein isothiocyanate, FITC, in cellulose
microspheres was studied by CSLM. The FITC was excited using a laser
and the emitted light was detected. The experiment was conducted by
injecting wet dextran-FITC-containing capsules in FITC-free water
solution where the decreasing amount of encapsulated fluorescein in the
cellulose capsules into the continuous water phase was continuously
monitored over approximately 2 hours.
22
2.4.5 X-ray diffraction (XRD)
The degree of crystallinity of the prepared capsules as well as the signal
from the encapsulated gold nanoparticles were analysed by XRD where
the intensity was measured as a function of the 2 scattering angle. The
detection of encapsulated gold nanoparticles were determined at
scattering angles of 30–110o.
2.4.6 Macroscopic contact adhesion measurement
(MCAM)
The dry work of adhesion was measured using the MCAM apparatus in a
controlled environment (50% RH and 23 oC) between functionalized or
non-functionalized solid cellulose spheres with a diameter of about 1 mm
and a flat 3 mm thick PDMS film.76 The contact radius and the load acting
on the two surfaces were continuously monitored and stored using a
microscope equipped with a camera and a microbalance and a specially
prepared control program. The two surfaces were brought into or out of
contact by a high resolution step motor operating at a constant speed of
10 µm/min.
2.4.7 Monitoring the adsorption of CNSs
The specific adsorption of antibody-conjugated CNSs and the
corresponding protein was investigated using quartz crystal microbalance
(QCM).87 A protein solution was first pumped into the QCM chamber at
0.1 ml/min and allowed to be adsorbed onto an oxidized silica quartz
crystal surface. Subsequently, antibody-conjugated cellulose nanospheres
were injected using the same injection flux. The change in the third
resonance frequency overtone of the crystal was used to estimate the
adsorbed mass according to the Sauerbrey model.88 This procedure
23
determines both the solid amount of adsorbed CNSs and the amount of
immobilised water.
24
3. Results and Discussion
3.1 Cellulose capsule preparation: controlling the
shape, geometry and size (Papers I to V)
The largest cellulose macrocapsules, approximately 3 mm in diameter,
were prepared using the solution solidification method where a dissolved
cellulose solution with dissolved gas was dripped into a non-solvent.57
When in contact with the non-solvent, the cellulose droplet is solidified
into a sphere where the dissolved gas is nucleated in the centre of the
sphere creating a gas-filled cellulose capsule. This process is triggered by
a decrease in solubility of the gas when the cellulose is solidified. The
cellulose wall thickness and the wall porosity of the prepared capsules
could be tuned by adjustment of the concentration of the dissolved
cellulose and the amount of dissolved gas, controlled by the applied gas
pressure and type of gas (carbon dioxide, nitrogen or propane) prior to
capsule formation. A higher concentration of the dissolved cellulose
resulted in a thicker cellulose wall structure, as shown in Table 1. The
table also shows that the type of gas had a large impact on the cellulose
wall structure. When carbon dioxide was used as gas and the cellulose
concentration was increased from 1 to 1.5 and 2 wt%, the wall thickness
increased from 130 to 240 and 300 µm. With nitrogen gas, the wall
thickness was almost unaffected, having an approximate thickness of
350 µm. However, with pentane, the wall thickness decreased
dramatically to approximately 4 µm and 8 µm with cellulose
concentrations of 1 and 1.5 wt%, respectively. This large difference in wall
thickness can be explained by the amount of dissolved gas in the cellulose
solution. The solubilities of the nitrogen, carbon dioxide and
25
propane were therefore determined, and it was found that 0.039, 1.37 and
1.89 g gas/kg of the respective gases were dissolved in the
cellulose/LiCl-DMAc solution. The low amount of dissolved nitrogen
resulted in a small encapsulated gas volume and a thick cellulose wall of
350 µm. When the more soluble carbon dioxide was used instead, the
encapsulated gas volume increased and the wall thickness decreased to
130 µm. When the gas solubility was further increased using propane, the
encapsulated gas volume increased further and the wall thickness
decreased to 4 µm. The relatively large difference in capsule wall
thickness between carbon dioxide and propane (with only a small
difference in gas solubility) can be explained by the fact that the solubility
of CO2 in the water non-solvent is 1.5 g/l,89 while the solubility of propane
in water is only 0.040 g/l.90 This means that CO2 can easily escape into
the non-solvent during regeneration, while propane becomes entrapped
inside the cellulose capsule. Consequently, the relative gas solubility in
the cellulose solution and in the non-solvent is very important when
designing the dimensions of the capsule.
The influence of carbon dioxide pressure on the wall thickness was
further investigated. When the gas pressure and thereby the gas solubility
were increased in the cellulose solvent, according to Henry’s law, the wall
thickness of the prepared capsules decreased and the hollow void created
by the dissolved gas increased in volume linearly with increasing gas
pressure (Figure 8). The cellulose wall densities of the CO2- and the
propane-prepared capsules were gravimetrically determined and
calculated to be 30 kg/m3 and 1200 kg/m3, which dramatically influenced
the capsule stability in solvents, porosity and mechanical behaviour, as
will be reported in more detail later.
26
Table 1. Properties of the differently prepared cellulose capsules as a function of the
cellulose concentration in solution.
Cellulose concentration (wt%) 1 % 1.5 % 2 %
Capsule size CO2 (mm)
Wall thickness CO2 (µm)
2.7
130
2.8
240
2.9
300
Wall thickness N2 (µm) 350 360 360
Wall thickness C3H8 (µm) 4.0 7.5 -
Wall density CO2 (kg/m3) 15.2 18.9 20.8
Wall density C3H8 (kg/m3) 1202 1206 -
Capsule density CO2 (kg/m3) 28.4 29.7 30.0
Capsule Density C3H8 (kg/m3)
Average BET pore diameter CO2 (nm)*
7.6
14.7
14.2
15.9
-
24.1
Cellulose solution viscosity (mPa·s) 70 301 1095
*Pore size of pores in the capsule wall
27
Figure 8. Total volume, wall volume and void volume of the capsules as a function of gas
pressure. The lines are merely a guide to the eye.
As mentioned above, the type of non-solvent also played an important
role controlling the amount of gas encapsulated in the cellulose sphere. If
a less polar solvent than water was used, such as ethanol, the solubility of
the hydrophobic gases (propane, carbon dioxide and nitrogen) increased,
and this resulted in a smaller volume of encapsulated gas. Thus, when
preparing solid cellulose spheres without any encapsulated gas, a less
polar solvent was used. In this study (Paper II), the goal was to minimize
the surface roughness and create a macroscopic cellulose probe for e.g.
adhesion measurements. It was found that the optimum concentration of
dissolved cellulose prior to sphere fabrication was 1.5 wt% (Figure 9a–c).
When a lower cellulose concentration of 1 wt% was used, the surfaces of
the spheres started to buckle upon drying, producing a surface-rough
cellulose probe (Figure 9d), and when a 2 wt% cellulose solution was
used, semi-spherical particles with a characteristic tail were formed
28
(Figure 9e). This is presumably due to a higher viscosity (1095 mPa s
compared to 301 mPa s for the 1.5 wt% cellulose concentrations) of the
cellulose solution, which could not be counterbalanced by the surface
tension forces before solidification in the non-solvent when dripping from
approximately 1 cm height. When the dripping height was increased, to
provide more time for reorientation into a perfectly spherical shape, the
drop encountered a larger impact force when penetrating the surface of
the non-solvent, and this induced a larger surface roughness (detailed
results not shown). The forces acting on the cellulose droplets when they
fall into the non-solvent are naturally dependent on the surface tension of
the non-solvent. Several different non-solvents were studied and it was
found that when water was used, the dripping height had to be
approximately 10 cm to allow penetration through the surface. When
exchanging the non-solvent to ethanol, the dripping height could be
reduced to less than 1 cm, which greatly reduced the surface roughness,
according to AFM measurements, to approximately 6 nm of the dried
cellulose spheres at a cellulose concentration of 1.5 wt%. The surface
roughness could however be further reduced to approximately 2 nm for
cellulose spheres having a diameter of 0.9 mm (Figure 9c) by reswelling
the spheres in tetrahydrofuran (THF) and redrying the spheres. This
roughness is similar to that of the cellulose model surfaces used for
adhesion measurements,77 but the thickness of these films is only 10–50
nm.77
29
Figure 9. Cellulose probes for macroscopic contact adhesion measurements. a) and b) SEM
micrograph and c) an AFM measurement (3 µm x 3 µm) displaying the cellulose probe
topography for spheres prepared using 1.5 wt% cellulose concentration and solidified in
ethanol, d) and e) capsules prepared from 1 and 2 wt% cellulose solutions.
When microcapsules were prepared using a MFFD, a double emulsion of
an octane droplet inside a cellulose solution dispersed in a continuous
silicone oil phase was prepared (Figure 10 a, b). The cellulose drops were
subsequently solidified in the MFFD using a non-solvent in the silicone
oil, as schematically shown in Figure 10 c, d. The average outer and inner
diameters of the cellulose capsules were estimated to be 44 and 29 µm
a b
30 nm
0 nm
c
d e
30
respectively. A photomicrograph and a SEM micrograph of the solidified
cellulose capsules are shown in Figure 10 e, d.
Figure 10. Photomicrographs from a MFFD experiment with octane, cellulose and silicone
oil. (a) The contact zone where the three fluids meet. (b) The inner liquid (octane) is injected
from the tapered cylinder on the left (diameter 50 µm) and is focused by the middle fluid (a
0.7 wt% cellulose solution) entering from the left in the outer square tube. These two fluids
are focused into the inner tapered tube on the right (diameter of 150 µm), i.e. the collection
tube, by the continuous fluid (silicone oil) entering from the right in the square outer tube.
(b) Magnification of a part of the collection tube. (c) Schematic illustration of the
solidification of a cellulose capsule from the double emulsion. In (e) and (d) a
photomicrograph and a SEM micrograph of the MFFD-prepared cellulose capsules.
Cellulose solutionSilicone oil
Octane
100 µm50 µm
Continiuous silicone phase Dissolved cellulose phase Inner octane phase
Precipitated celluloseNon-solvent in silicone oil
100 µm 100 µm
a b
c d
e d
31
The formation of an emulsion is controlled by three dimensionless
numbers; the Reynolds number (Re),91 the Capillary number (Ca)92 and
the Weber number (We)93.
[2]
[3]
[4]
where ρ, v, η, l, and σ are the density, mean velocity, viscosity,
characteristic length of the fluid (the jet diameter) and interfacial tension
respectively. To pinch off a droplet, the Reynolds number, i.e. relation
between the inertial forces to viscous forces on the droplet, must be
greater than the capillary number, i.e. the relation between the viscous
forces and the surface tension forces. At low capillary numbers, i.e. at low
viscous drag, the Weber number (Equation 4) which describes the
balance between forces created by inertia and surface tension, becomes
increasingly important in describing the droplet formation. To enable the
preparation of cellulose capsules, a double emulsion of an encapsulated
octane droplet inside a cellulose droplet dispersed in a continuous
silicone oil phase was used. The Re and Ca numbers for the preparation of
this double emulsion were calculated to be 1.3·10-2 and 0.6 respectively
for the cellulose solution phase and 4.5·10-2 and 3.2 respectively for the
continuous silicone oil phase. Furthermore, the Weber number of the
cellulose solution, describing the relation between inertial forces and
surface tension forces, was 8.0 · 10-3 for droplet formation which
indicates the upper limit for drop formation.
32
When cellulose nanospheres, CNSs, were prepared, higher shear forces
had to be introduced to the cellulose emulsion. Here a membrane
emulsification method5 was used involving two steps: first the
preparation of a cellulose emulsion by mixing a cellulose/LiCl-DMAc
solution in oil at a volume ratio of 1:4 followed by pushing this emulsion
through a 2 µm pore size membrane into a non-solvent according to
Figure 7. Employing this method, nanospheres of cellulose could be
prepared as shown in Figure 11.
Figure 11. SEM micrograph CNSs prepared from a 1 wt% cellulose solution pushed through
a 2 µm filter into an ethanol non-solvent where they solidified. The spheres were then
solvent-exchanged to water and freeze-dried.
3.2 Mechanical properties of the cellulose
macrospheres and capsules (Papers I & II)
The mechanical properties of two different types of capsules were
determined; porous cellulose macrocapsules prepared using CO2 as the
33
dissolved gas and non-porous cellulose capsules prepared using propane.
Two characterization techniques were used: the Hertz approach assuming
pure elastic interactions between the macrospheres, and neglecting
adhesive interactions (Paper I) and Von Mises approach for thin elastic
shells (Paper II).
3.2.1 Mechanical response of porous cellulose
macrocapsules (Paper I)
In order to determine the dry elastic properties of the capsules, the elastic
modulus of solid freeze-dried solid cellulose spheres solidified from LiCL-
DMAc solutions was determined by compressing a cellulose sphere
between two rigid metal plates and applying the Hertz equation94:
( )
[5]
where E, ν, F, D and d are the Young’s modulus, Poisson’s ratio,
compression load, diameter of the sphere and contact spot diameter
respectively. Since Equation 5 is valid for solid spheres, solid cellulose
spheres without encapsulated gas were prepared with the same cellulose
concentration as the hollow capsules and it was assumed that the
mechanical response of the wall of the hollow capsules would be similar
to that of the solid spheres. Here, the growth of spot diameter, d, was
measured as a function of the applied compressive load seen in Figure 12.
The elastic modulus of the porous cellulose wall was then calculated using
Equation 5, and was found to be 1.8 MPa and 7.3 MPa respectively for
cellulose capsules prepared from 1 and 2 wt% cellulose solutions.
34
Figure 12. Contact spot diameter versus compression load for solid cellulose macrospheres
prepared from solutions with cellulose concentrations of 1% and 2% by weight. The solid
lines represent Equation 5 assuming a Poisson’s ratio of 0.4.
3.2.2 Mechanical response of non-porous cellulose
macrocapsules (Paper II)
The porous and non-porous cellulose capsules behave completely
differently in terms of mechanical response to deformation due to the
different material structure of the capsules as listed in Table 1. The dry
non-porous, thin-walled capsules could be compressed to over 98%
compression without catastrophic failure (which is very different, for
example, from normal paper made of fibres with a high cellulose content
which typically shows values of 3–5% elongation at break). To be able to
establish the fundamental mechanism behind this mechanical response,
an extended mechanical model was applied using the Von Mises
approach (Equation 6) where the plastic and elastic responses of the
cellulose capsule were characterized, taking into account the increase in
internal gas pressure upon compression according to:
35
( )
(
)
[6]
where are respectively the critical compressive yield
strain, the capsule wall thickness, the radius of the sphere, the yield
stress, the atmospheric pressure and the ratio of specific heats for air.
Using this model, an estimated value of the critical compression, i.e. the
compression needed for the capsule to be plastically deformed, was
calculated to be 85%. This critical compression was also determined
experimentally by cyclic compression measurements of single cellulose
capsules (Figure 13). The data from the consecutive compression cycles
almost overlapped each other when the peak deformation was increased
from ɛ = 0.1 (10% compression) to ɛ = 0.7, indicating that the capsule
recovered its shape when it was unloaded. After extended compression
cycles to ɛ = 0.9, plastic deformations could be observed as large,
irreversible wrinkles on the surface of the capsules (Figure 13 and
image 3). Furthermore, when the capsules were compressed a second
time to ɛ = 0.9 (not shown) the load versus compressive strain-curves no
longer overlapped, which supports the conclusion that a plastic
deformation regime starts at ɛ = 0.7–0.9. It should be noted that no
failure of the capsules was detected even at these high compressive
strains.
36
Figure 13. Compression cycling of a cellulose capsule prepared from 1 wt% cellulose
solution with propane as the dissolved gas. The capsule was compressed to a peak strain of
ɛ = 0.1 (green), 0.3 (purple), 0.5 (blue), 0.7 (red) and 0.9 (black) in consecutive cycles.
Micrographs show 1) a never-deformed capsule, 2) a capsule deformed to ɛ = 0.9 and 3) a
capsule after release of a compressive load to ɛ = 0.9.
The reason for the extraordinary elastic compressibility was found to be
related to the function of the packed cellulose wall structure as a gas
barrier. When the capsules were compressed, the encapsulated gas was
retained in the sphere and compressed, providing mechanical
compressibility to the capsule. Subsequently, when the compressive
forces were released, the capsules with pressurized encapsulated gas
could relax back to their initial volume. To quantify this, long-time
compressive experiments were performed for single capsules where the
compressive strain was kept constant at three different initial
compressive loads and the relaxation was continuously measured during
approximately three days (Figure 14a). This load decay was then related
1 2
3
37
to the amount of gas which escaped from the interior of the capsule. The
compressive resistance was assumed to be largely controlled by the gas
“trapped” inside the strong and densely packed cellulose shell structure.
It was decided to test this assumption by quantifying the gas permeability
through the cellulose wall membrane. The relationship for the gas
permeability, P, can be found by solving Equation 7 for P.
[7]
where are respectively the volume of the penetrating
gas, the duration of the experiment, the exposed area for gas removal, the
membrane thickness and the relative pressure pushing the gas through
the membrane. In the case of cellulose macrocapsules kept under a
constant compressive strain, the amount of gas escaping is related to the
amount of gas in the interior of the capsule through the conservation of
mass and, employing the ideal gas law for the interior,
( ) ( ) ( ) the equation can be rewritten as:
( ) ( )[ ( )
( ) ] ( ) [8]
where ( ) ( ) ( ) ( ) are respectively the transmural pressure
(the pressure difference between the both sides of the cellulose capsule
wall) induced by compressing the capsule, the exposed surface area, the
force at time t, and the initial force when the capsule is compressed to a
constant compressive strain. Plotting the left-hand side of Equation 8
against time for different cellulose capsules compressed under different
38
initial compressive forces yields overlapping, straight lines with the same
slope, as demonstrated in Figure 14b.
Figure 14. Force relaxation between 24 and 72 hours for dry cellulose macrocapsules
prepared from a 1 wt% cellulose solution, using propane as the dissolved gas, compressed at
23 oC and 50% RH to 1 N (solid squares and dash-dotted trend line), 6 N (open circles, solid
trend line) and 10 N (solid triangles, dashed trend line). In a) the force is plotted as a
function of time and in b) the left-hand-side of Equation 8 is plotted as a function of time
where the dash-dotted, dashed and solid lines represent the linear trend lines for 1 N, 6 N
and 10 N, respectively. The slopes of these trend lines represent the gas permeability.
The gas permeability through the cellulose wall membrane was calculated
through a least-squares fit in Figure 14, to be 0.40, 0.36 and
0.36 (ml µm/(m2 days kPa) for the 1 wt% cellulose capsules compressed
to 1 N, 6 N and 10 N, respectively. These values are very similar to values
reported earlier for regenerated cellulose films.95 The fit in Figure 14
supports the underlying assumptions in Equation 8, i.e. that the gas
permeation rate depends on the transmural pressure induced by
compressing the capsules to different initial loads, and on the exposed
surface area through which the gas can penetrate the capsule wall.
39
3.3 Adhesion measurements using solid cellulose
macrospheres (Paper III)
Adhesion measurements on cellulose surfaces have previously been
performed using thin films (10–40 nm) of cellulose77, 96, 97 since there are
no known methods of preserving a smooth film surface profile when the
film thickness is increased. For larger samples, adhesion measurements
using colloidal probe AFM with cellulose spheres having diameters
between 10 and 15 µm have also been performed.79 It is however evident
that these spheres had a rather rough surface, typically with an rms value
of 32 nm.98 In this work, the use of bulk cellulose macrospheres as probes
for macroscopic adhesion testing is reported for the first time. Cellulose
probes having a diameter of around 0.9 mm were used (Figure 9), almost
two orders of magnitude larger in diameter than already existing
probes.79 The rms roughness was measured to be more than one order of
magnitude lower, 2.0 ± 0.2 nm, for pure cellulose probes, and
1.0 ± 0.2 nm for alkylated cellulose probes. The low surface roughness
and the spherical shape enable a smooth and uniform contact zone that
can easily be imaged with an optical microscope through a transparent
material such as a PDMS film (Figure 15).
Figure 15. Typical image of the contact zoon between a cellulose probe and a flat 4 mm
thick PDMS model surface.
50 µm
40
By determining the contact radius as a function of the applied load and
applying the Johnson-Kendall-Roberts, JKR, contact mechanics theory99
it is possible to determine the thermodynamic work of adhesion, W,
between the materials by plotting the cube of the contact radius, a, as a
function of the applied load and fitting the results to Equation 9. The
loading and unloading data are tabulated in Table 2. The experimental
results and the fitting to Equation 9 are also shown in Figure 16.
[ √ ( ) ] [9]
where R is the radius of the sphere, K is an elastic constant representative
of the experimental system and F is the applied load acting on the PDMS
film and cellulose probe.
Figure 16. Adhesion experiment using non-modified cellulose spheres (left) and alkylated
cellulose spheres (right), where the probe is forced into contact with a flat PDMS surface.
The experiment was performed at 23 oC and 50 % RH at a strain rate of 10 µm/min where
the elastic constants, K, were calculated to be 4.00 ± 0.06 and 4.10 ± 0.10 MPa for cellulose
and alkylated cellulose, respectively.
41
The work of adhesion, W12, was calculated from contact angle
measurements using Equation 10, which is based on the dispersive and
acid/base interactions between materials.100
√
(√
√
) [10]
Prior to the work of adhesion calculations, the specific surface energies of
the dispersive and polar contributions were determined by measuring the
contact angles, , on the different materials of water, ethylene glycol and
methylene iodide solutions and using the equation:
( )
√
√
√
[11]
where 1 and 2 are the surface energies of PDMS (taken from the work of
M. Owen101) and cellulose, and the subscripts s and l represent the solid
and liquid respectively in the contact angle measurements. The surface
energies thus obtained can be used to calculate the thermodynamic work
of adhesion between the solid cellulosic probes and the PDMS surface,
using Eq. 10, and these results are summarized in Table 2.
The adhesion energy at the critical pull off force, Wmin was calculated
from Equation 12:99
[12]
Where Fs is the force when the surfaces spontaneously separate
42
Table 2. Work of adhesion for a cellulose probe and a PDMS surface in loading and
unloading was determined by MCAM, Eq. 9. The work of adhesion, W12tot, was also
calculated from dispersive and polar interactions as determined from contact angle
measurements using Eq. 10 and Eq. 11. Wmin represents the work of adhesion at the
minimum force from the contact mechanics measurements (Eq. 12).
Probe Wload
(mJm-2)
Wunload
(mJm-2)
W12tot
(mJm-2)
Wmin
(mJm-2)
Cellulose 41 ± 5 104 ± 5 65 107 ± 10
Alkylated 40 ± 4 142 ± 13 59 143 ± 12
The cellulose and alkylated cellulose both have similar work of adhesion
in the loading sections although their total surface energies were different
from the static contact angle measurements, the dispersive component of
the surface energies were similar. Previous work on the adhesion of
PDMS has suggested that the dispersive component of the surface energy
was the predominant factor in the loading work of adhesion for JKR
studies compared to contact angle measurements.102 When the probes
were retracted and subsequently detached, the unloading work of
adhesion was 36% higher in the alkylated cellulose opposed to the
unmodified cellulose. The hysteresis, detected as the difference between
the loading and unloading is typically seen with PDMS on cellulose but it
is much less then has been reported in previous literature.76, 97 This
reduced hysteresis can be explained by the system used. When large
cellulose probes with a diameter of 0.9 mm are used, the underlying
substrate has almost no influence compared with when a thin cellulose
film with a width of 40 nm is used. Furthermore, when model films of
cellulose are prepared an anchoring cationic polymer has to be used to
prevent delamination of the cellulose film and the silica wafer. When two
surfaces are forced together under compression, the cationic polymer
may enter the PDMS and contribute to the adhesion.
43
It should also be remembered that the calculated work of adhesion as
determined using the acid/base theory, i.e. Equation 10, is heavily
debated since the liquids used are known to affect the properties of the
surfaces. It is furthermore difficult to obtain an accurate value of the
acid/base properties of water, which must be used to quantify the
acid/base properties of the solids using Equations 10 and 11. All this
means that the MCAM gives a more accurate evaluation of the adhesive
interactions between the surfaces used in the experiments. Further
experiments are needed to establish the molecular mechanisms behind
the hysteresis for the different systems. However, the close similarity
between the work of adhesion from the unloading values and the pull-off
experiments is very encouraging and shows that the MCAM experiments
with the new cellulose probes open up new possibilities for quantifying
and understanding the role of different surface modifications of cellulose.
3.4 Swelling of the cellulose capsule gel structure
(Papers I, II, IV & V)
Salt- and pH-responsive cellulose capsules were prepared by introducing
charges through carboxymethylation on the cellulose fibres prior to
dissolution of the cellulose and subsequent capsule formation. For the
larger macrocapsules, the effect of pH and salt concentration for four
differently charged cellulose capsules were studied. The determined
charge densities were 29, 73, 114 and 350 µmol charges per gram
cellulose (µeqv./g). As can be seen in Figure 17a–b, the capsules exhibited
swelling and shrinkage when exposed to a change in pH, and shrunk
when the salt concentration was increased. This swelling was theoretically
expected since an increase in pH increases the degree of dissociation of
44
the carboxyl groups within the capsule wall and this increases the osmotic
pressure in the wall, which in turn leads to an expansion of the capsule
wall.58, 103 An increase in pH from 10 to 12 also increases the ionic
strength in the solution, and this counteracts the swelling due to a
decrease in the difference in concentration between ions inside and
outside the capsule wall and thus a decrease in osmotic pressure, since all
the carboxyl groups should be fully dissociated at pH 10.63
45
Figure 17. Wall volume of cellulose macrocapsules prepared from cellulose with charge
densities: 29, 73, 114 and 350 µeqv./g using 1 wt% cellulose concentration and CO2 as
encapsulated gas. All the solutions had a background salt (NaCl) concentration of 10 mM in
order to avoid large changes in ionic strength when the pH was changed. The expansion in
capsule wall volume is shown for (top) a change in pH, and (bottom) a change in salt
concentration.
The effect of pH was further investigated for the microcapsules prepared
by the MFFD where the relative swelling is shown in Figure 18. Here, two
46
different cellulose microcapsules with different charge densities
(prepared from cellulose fibres having charge densities of 29 and
350 µeqv./g) were used. The same trend as for the macrocapsules can be
seen, but in relative numbers the microcapsules swelled less. This can be
explained by the cellulose concentration in the capsules, where the
microcapsules were prepared from 0.7 wt% cellulose and the
macrocapsules from 1.5 wt% cellulose. It can also be due to the structure
of the capsule wall, and it should be kept in mind that the swelling
pressure is counteracted by the restraining properties of the cellulose
polymer network in the capsule wall. These restraining properties will
naturally be different depending on how the capsule wall has been
solidified.
Figure 18. Relative capsule volume as a function of solution pH for microcapsules
prepared from carboxymethylated (black squares) and unmodified pulp (black circles).
The swelling behaviour of the capsules also means that the release
properties of an encapsulated drug can be tuned by the preparation
47
conditions to suit a particular condition where the encapsulated materials
in the internal cavity are to be released. A possible application is for the
capsules to be filled with an active therapeutic or diagnostic substance
that is to be released in the human duodenum. When the capsule enters
the stomach where the pH is approximately 3, the capsule wall will shrink
and there will be little release of active substances. However, when the
capsules reach the duodenum where the pH is approximately 8, the
capsules will swell and more easily release the encapsulated active
substance.
3.5 Surface modifications using antibody conjugation
(Paper V)
Surface modifications on the CNSs were performed by covalently binding
antibodies selective for bovine serum albumin (BSA) and epidermal
growth factor receptor (EGFR) protein to the CNSs. The conjugation was
performed according to a classical reductive amination procedure by first
partially oxidizing the CNSs to form dialdehyde cellulose.104 The antibody
was then attached by reacting the dialdehyde cellulose with the primary
amines of the antibody. The real-time specificity of the antibody-CNS
conjugates was then measured using QCM. As can be seen in Figure 19,
both the BSA (Figure 19a) and the EGFR (Figure 19b) were adsorbed on
the SiO2 surface. When the non-conjugated CNSs (RefCNS) were injected,
no significant and permanent adsorption to either the BSA- or the EGFR-
coated surface was detected. Almost all the detected interaction was of a
non-specific type since all the immobilized mass, i.e. the entire frequency
shift, was removed after phosphate buffer (PBS) washing. The CNSs
conjugated with the “wrong” antibody also showed the same almost
complete absence of interaction as the RefCNS. The spheres with the
48
“correct” conjugated antibody, however, showed significant binding to the
corresponding protein layer (seen as a higher frequency decrease in
Figure 19) and the immobilization continued over a longer time-period
compared to that of the spheres without specific interaction. Equally
important, the spheres did not completely detach during the washing
step, strongly indicating that they were permanently attached to the
protein surface and that the antibody maintained its specificity after
conjugation with the CNS.
49
Figure 19. QCM graph displaying the frequency shift (i.e. mass adsorption) as a function of
time. When (a) the protein BSA and (b) the protein EGFR with subsequent washing buffer
(PBS), non-conjugated CNSs (RefCNS), (a) anti-EGFRCNS or (b) anti-BSACNS and then the
“correct” antibody conjugated CNSs were added. The addition points are indicated by
arrows.
0 20 40 60 80 100 120 140 160−100
−80
−60
−40
−20
0
20EGFR
Time (min)
Fre
qu
en
cy s
hift
(Hz)
PBS
RefCNS
PBS
anti−BSACNS
PBS
anti−EGFRCNS
PBS
0 20 40 60 80 100 120 140 160−100
−80
−60
−40
−20
0
20BSA
Time (min)
Fre
qu
en
cy s
hift
(Hz) PBS
RefCNS
PBS
anti−EGFRCNS
PBS
anti−BSACNS
PBS
(a)
(b)
50
The anti-BSACNS conjugates were adsorbed more effectively than the
anti-EGFRCNS conjugates. The greater adsorption of anti-BSACNS can
presumably be explained by lower conjugation efficiency, quantified as a
conjugation of about 40% for the anti-BSA and about 20% for the anti-
EGFR.
To support the QCM results and to further show the absence of non-
specific interactions and the presence of highly specific interactions, silica
wafers with a pre-adsorbed protein layer of BSA or EGFR were
submerged for 45 min in test tubes containing each CNS solution (i.e.
non-conjugated and conjugated with anti-BSA or anti-EGFR) to enable
adsorption onto the different surfaces and then washed with PBS for
45 min (Figure 20). The experiment was performed with each CNS in
parallel rather than in the sequential series shown in Figure 19. As clearly
can be seen in Figure 20, a higher density of immobilised CNS was
detected where antibody-antigen interactions were present, fully
supporting the QCM observations (Figure 19).
51
Figure 20. SEM micrograph of an EGFR- or BSA protein layer adsorbed onto silica wafers
followed by dipping in a solution of RefCNS, anti-BSACNS and anti-EGFRCNS. Immediately
after the adsorption of the different CNSs, the silica plates were washed and freeze-dried to
preserve the CNS structure.
3.6 Encapsulation of gold nanoparticles (Paper V)
As mentioned previously, the nanospheres might be an interesting
substrate for use in medical diagnostics or treatments. To prepare a
diagnostic device, the CNSs were functionalized by incorporation of an
anti-BSACNS
RefCNS
anti-EGFRCNS
EGFR
surface
BSA
surface
52
image contrast agent, gold nanoparticles (GNPs), in the bulk of the
cellulose matrix. This was achieved by dispersing GNPs in the cellulose
solvent prior to CNS preparation. The typical XRD pattern of the different
crystalline gold planes of GNPs immobilized in CNSs can be seen in
Figure 21, corresponding to the peaks at scattering angles of 38, 44, 65,
78 and 82 degrees (the peaks at 36, 42 and 62 degrees are due to the
substrate). This means that it would presumably be possible to trace the
antibody-conjugated cellulose spheres in a human blood system and
image their progress by X-ray techniques.
Figure 21. X-ray diffraction of dry, gold-containing CNSs. The labelled peaks at scattering
angles of 38, 44, 65, 78 and 82 degrees correspond to the different crystalline planes of gold.
53
3.7 Micro- and nanocapsules as an extended release
device or as a diagnostic tool (Paper IV & V)
The diffusion of encapsulated substances in cellulose microcapsules
prepared from microfluidics was investigated by loading the capsules
with a FITC-labeled 4 kDa dextran. Figure 22 shows the release of
encapsulated dextran as a function of time when the capsules were placed
in a 0.01 M pH 7.4 phosphate buffer. It can clearly be seen that it took
approximately two hours for the dextran to be fully released from the
cellulose capsules and to reach equilibrium with the surrounding
solution. The diffusion constant, D, over the cellulose wall can be
calculated from Fick’s 2nd law:
[13]
The analytical solution, describing the integral amount diffusing from a
spherical object was derived by Crank105 under the assumption that the
system has a uniform initial concentration, C1, and that the surface
concentration is maintained constant at C0 throughout the experiments:
∑
(
) [14]
where Mt is the amount that has diffused after time t, is the amount
that has diffused after equilibrium has been reached and a is the radius of
the sphere. Equation 10 assumes a solid sphere, but Kräger et al.106 have
shown that the equation can be used for a capsule by rescaling the capsule
to a solid sphere with the same volume-to-surface-area ratio. The results
54
of the diffusion through the cellulose wall can be seen in Figure 22, where
the dashed line represents the calculated intensity using experimental
data as applied to Equation 14. From the best least squares fit, the
diffusion constant was calculated to be 6.5·10-14 m2/s. This value can be
compared to the diffusion of dextran through a cellulose fibre wall,
8.4·10-12 m2/s for a 10 kDa polymer, earlier reported by Hovarth et al.107.
The diffusion of the slightly shorter dextran molecule used in our system
is thus approximately two orders of magnitude slower than that in a
swollen wood fibre. This indicates that the capsule wall structure is less
porous and consequently a better encapsulation material. It also indicates
that the diffusion can be controlled over a rather wide range by changing
the structure of the capsule wall by tuning the cellulose solidification
process and the thickness of the cellulose shell being dependent on the
cellulose concentration.
Figure 22. Normalized intensity of a FITC-labelled 4 kDa dextran, in the centre of a
microcapsule, as a function of time when the microcapsule is placed in 0.01 M 4 kDa
phosphate buffer. Closed circles show experimental data and the dashed line represents the
intensity calculated according to Eq. 14 using a diffusion constant of, D=6.5·10-14 m2/s, that
gave the best fit to the experimental data.
55
In medical science, it can be hypothesized that the functionalized
nanospheres may act as an interesting theranostic in vivo tool, where the
extended release of encapsulated drugs could be used for therapeutic
purposes. Furthermore, in a diagnostic application, attaching antibodies
to the surface of the cellulose spheres can enable surface-specific
interactions and, together with embedded GNPs, it would be possible to
image diseased areas as illustrated in Figure 23.
Figure 23. Schematic illustration of how antibody-conjugated CNSs with embedded GNPs
might be used as an in-vivo contrast agent to image the specific interactions from antibody
conjugation on CNS with antigen present on the surface. In the first image (from the left) a
solution of surface-functionalized CNSs is injected into an infected location. Subsequently,
the functionalized CNSs bind to the antigen surface, where the encapsulated GNPs provide
contrast of the affected area.
56
4. Conclusions
This thesis describes three new methods for preparing model cellulose
spheres, resulting in spheres with diameters ranging from 160 nm to 3
mm. The solution solidification procedure used a dripping technique with
or without a dissolved gas where the cellulose solution solidified in
contact with a non-solvent generating a 3 mm cellulose sphere, with or
without encapsulated gas. The effect of using different preparation
conditions was investigated and it was found that the wall thickness and
the void diameter were controlled by the gas dissolution pressure (i.e. the
amount of dissolved gas), the type of gas, the cellulose concentration, the
type of non-solvent used and the solubility of gas in the non-solvent. If
the gas dissolution step was excluded, a solid cellulose sphere was
generated. The solid cellulose spheres were further optimized to achieve a
cellulose surface topography to enable a quantification of the macroscopic
compatibility of cellulose and other solid interfaces using a specially
developed adhesion measurement technique.
Two different emulsification methods were used to reduce the size of the
cellulose capsule. Micrometre capsules were prepared using a new type of
microfluidic flow-focusing device where immiscible fluids were injected
into micro-channels of glass where cellulose drops with octane droplets
were generated. The diffusion constant of dextran through this cellulose
shell structure was determined to be 6.5·10-14 m2/s which is lower than
the diffusivity of dextran in a cellulose fibre, indicating that a prepared
cellulose capsule could extend the release of encapsulated substances. To
prepare nanospheres of cellulose, a new membrane emulsification
procedure was used. This method involves the preparation of a cellulose
solution emulsion in silicone oil which is subsequently further broken
57
into smaller drops by injecting the emulsion through a 2 µm membrane
into a non-solvent, to yield cellulose spheres with an approximate size of
160 nm. These spheres were then surface functionalized to be specific by
antibody conjugation on the pre-oxidized cellulose sphere surface. The
antibody conjugation enabled specific interactions with corresponding
proteins with low non-specific interactions. For the nano-spheres, a bulk
functionalization was also developed by incorporating smaller gold
nanoparticles which could be detected by X-ray diffraction and UV-vis
spectroscopy, and these might be useful for theranostic applications.
58
5. Future Work
Given the findings in this thesis, there is no doubt that several different
cellulose capsules can be prepared, and that these spheres can be used as
a platform for both applied and fundamental scientific studies. For the
applied scientific part, it would be interesting to “glue” large numbers of
cellulose capsules together to form macroscopic structures such as an
elastic and lightweight foam material. This would potentially be a strong
replacement material in application areas where the plastic industry is
now dominant. For the fundamental scientific continuation, it would be
interesting to characterize the interactions of cellulose with materials
different from those used in this thesis. The swelling effect of non-
functionalized cellulose spheres in different solvents is relatively large, as
was indeed observed in Paper III, Figure 1. For a thorough investigation
of the solvent interactions, a number of different fibres could possibly be
investigated where the solvent effect of using differently charged cellulose
polymers or compositions of hemicelluloses and lignin could also be used.
Since the solid cellulose spheres can be prepared with an rms surface
roughness of only a few nanometres, the macroscopic adhesive properties
could be characterized when investigating the compatibility between
cellulose and other soft surfaces. Here it would also be interesting to
investigate the macroscopic adhesive behaviour of other types of
functionalized cellulose for improving and understanding how to tailor
the cellulose polymer for various composite materials. To even broaden
fundamental surface science, it would be interesting to investigate
whether these solid smooth spheres could be prepared in smaller sizes,
where it would be interesting to study the microscopic adhesive
behaviour using AFM. The nanospheres are also interesting for
biomedical in vivo applications where the non-toxic chemical properties
59
of cellulose are truly used. Here, the cellulose spheres could act as a drug
delivery device for extended release applications or for advanced
diagnostics where the bulk functionalized spheres with antibodies
conjugated on the surface of the spheres could target and locate infected
cells.
60
6. Acknowledgments
In this chapter I would like, from a personal point of view, to describe the
journey that resulted in this thesis, and acknowledge those who have
helped me on the way.
It all started with a master thesis work performed at KTH where I later
continued towards a PhD degree. For my master thesis work, I was
assigned to Prof. Lars Wågberg who has my most sincere appreciation,
and he is the main contributing person for why I decided to pursue a
PhD. I do not know for how many hours we have discussed relevant and
non-relevant things during these years, but they are many. From these
many hours together, I owe Lars a lot regarding how to be a good scientist
and person and for which I truly want to thank you. During my early time
as a PhD student, I met Bert Pettersson, with his truly inventive mind,
who is always curious and full of new ideas. Thanks a lot for the guidance,
and for the inspiration and motivation to continue the PhD journey.
As a PhD student, I have had some tough first years since it was not easy
to understand the basic fundamentals regarding capsule formation as I
hope this thesis describes them. Here, I would like to express my
appreciation to Stefan Lindström, Sam Pendergraph, Cyrus Aidun, Tomas
Larsson and my friend Marcus Ruda for many fruitful discussions.
As the fundamental knowledge of capsule formation was being developed,
I started to work more closely with Per Larsson whom I consider to be not
just one of the most important persons regarding the scientific outcome
of this thesis, but also a person whom I consider to be a good friend –
Thank you!
61
From all my years being a PhD student, I also wish to thank all the people
in the Department of Fibre and Polymer Technology especially my
colleagues for a good working atmosphere. An extra thanks to Maria and
Mona for administrative support, and to my room-mates; Erdem and
Pontus, for scientific and other small chats, and also Gunnar, Monika,
Emil, Nicholas, Andreas, Caroline, Erik, Christian, Anna, Rebecca,
Lousie, Oruc, Petri, Andrew, Maryam, Veronica, Linn, Carl and Dimitri
for all the support throughout the years.
Anthony Bristow is acknowledged for the linguistic revision of all the
scientific papers that I have published, and also Lars Ödberg for
reviewing this thesis.
Finally, I would like to express my deepest gratitude to my family; my
parents Elisabeth and Dick – thanks for all the care and support such as
feeding my endless appetite for any kind of food, for pushing and
encouraging me to educate myself and always respecting my own sincere
choices (even though I did not become an archbishop as my mother
wanted!); my sister, Catherine, for the endless love and all the adventures
we have shared. I am also looking forward to being a part of future
adventures together with the rest of your fantastic family, Johan, Mira
and Albert!; my parents in law, Bo and Karin, for letting me be a part of,
and really feeling welcome, in your family. And last, but not least, Linda –
you are the woman of my life and you are simply the best wife a man can
wish for!
62
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