Harnessing Mesoporous Spheres - transport studies and biotechnological applications

Harnessing Mesoporous Spheres
- transport studies and biotechnological
applications
Jovice Boon Sing Ng
黄雯沁
Department of Physical, Inorganic and Structural Chemistry
Stockholm University
Doctoral Thesis 2009
Department of Physical, Inorganic and Structural Chemistry
Stockholm University
10691 Stockholm University Sweden
Cover:
The front figure is a scanning electron microscopy micrograph of a small heart
formed by colloidal mesoporous silica spheres. The image has been touched up
using Photoshop to give it a red colour. Huge specific surface area and pore large
volume for a small heart.
Faculty Opponent:
Prof. Frank Caruso
Department of Chemical and Biomolecular Engineering
The University of Melbourne, Australia
Evaluation Committee:
Associate Prof. Ulla Elofsson, Institute for Surface Chemistry, YKI
Prof. Lars Wågberg, School of Chemical Science and Engineering, KTH Royal
Institute of Technology
Prof. Anders Palmqvist, Chemical and Biological Engineering, Chalmers University
of Technology
Substitute:
Professor Arnold Maliniak, Department of Physical, Inorganic and Structural
Chemistry, Stockholm University
©Jovice Boon Sing Ng, Stockholm 2009
ISBN 978-91-7155-898-5
Printed in Sweden by US-AB, Stockholm 2009
Distributor:
Department of Physical, Inorganic and Structural Chemistry, Stockholm University
To my parents,
and
my brother and sister,
Boon Hong and Boon Hiong,
知之为知之，不知为不知，是知也
《论语·为政》
Science is organized knowledge, wisdom is organized life.
Immauel Kant
Abstract
Abstract
Applications in controlled release and delivery calls for a good
understanding of molecular transport within the carrier material and the
dominating release mechanisms. It is clear that a better understanding of
hindered transport and diffusion of guest molecules is important when
developing new porous materials, e.g., surfactant templated silica spheres,
for biotechnological applications. Confocal laser scanning microscopy was
used to quantify the bulk release and intraparticle transport of small charged
fluorescent dyes, and fluorescently-tagged neutral dextran, from mesoporous
silica spheres. The time dependent release and the concentration profiles
within the spheres have been used to analyze the release mechanisms using
appropriate models. While the small, non-adsorbing anionic dye is released
following a simple diffusion driven process, the concentration of the cationic
dye varies radially within the spheres after loading. The release of the
cationic dye is controlled by diffusion after an initial period of rapid release,
which could be due to a significant fraction of the cationic dye that remains
permanently attached to the negatively charged walls of the mesoporous
silica spheres. The diffusion of dextran and the resulting flat concentration
profiles could be related to the complex structural feature of the cylindrical
pores close to the surface, and a possible conformational change of the
dextran with the concentration. The stability and leaching of a catalytic
enzyme, lipase, immobilized in hydrophobilized mesoporous support has
also been quantified. Colloidal monodisperse mesoporous silica spheres
were synthesized and transmission electron microscopy showed that the
inner pore structure display a radially extending pores. The mesoporous
spheres were used as solid supports for a lipid membrane incorporated with a
multi-subunit redox-driven proton pump, which was shown to remain
functional.
Key words: Mesoporous, spheres, particles, CLSM, TEM, controlled-release,
molecular transport, lipid membrane, enzyme, intraparticle, lipase, solid
support.
List of Publications
List of Publications
This thesis is based on the following papers:
1.
Jovice Boon Sing Ng, Padideh Kamali-Zare, Hjalmar Brismar and Lennart
Bergström, Release and molecular transport of cationic and anionic
fluorescent molecules in mesoporous silica spheres, Langmuir, 24, pp
2.
11096–11102, 2008.
Jovice Boon Sing Ng, Padideh Kamali-Zare, Malin Sörensen, Hjalmar Brismar,
Peter Alberius, Niklas Hedin and Lennart Bergström, Molecular transport
and release of dextran in silica spheres with cylindrical mesopores,
3.
manuscript.
Malin Sörensen, Jovice Boon Sing Ng, Lennart Bergström and Peter Alberius,
Improved enzymatic activity of Thermomyces lanuginosus lipase
immobilized in a hydrophobic particulate mesoporous carrier, submitted.
4. Jovice Boon Sing Ng, Petr O. Vasiliev and Lennart Bergström, The radial
dependence of the spatial mesostructure of monodisperse mesoporous
silica spheres, Microporous and Mesoporous Materials, 112, pp. 589-596,
5.
2008.
Gustav Nordlund, Jovice Boon Sing Ng, Lennart Bergström and Peter
Brzezinski, A Membrane-reconstituted functional proton pump on
mesoporous silica particles, submitted.
Papers not included in the thesis:
1.
Petr O Vasiliev; Bertrand Faure; Jovice Boon Sing Ng and Lennart Bergström,
Colloidal aspects relating to direct incorporation of TiO2 nanoparticles
into mesoporous spheres by an aerosol-assisted process, Journal of
2.
Colloid and Interface Science., 319, pp. 144-151, 2008.
Niklas Hedin, Jovice Boon Sing Ng, Peter Stilbs, Spectral deconvolution of
NMR cross polarization data sets, Solid State Nuclear Magnetic Resonance,
3.
accepted.
Ranjith Krishna Pai, Jovice Boon Sing Ng, Saju Pillai, Lennart Bergström, and
Niklas Hedin, Temperature induced formation of strong gels of
acrylamide-based polyelectrolytes, Journal of Colloid and Interface Science,
accepted.
List of Publications
4.
Boon Sing Ng, Kjell Jansson and Lennart Bergström, Using differential
scanning calorimetry to follow how gelcasting proceeds, Journal of
American Ceramic Society, 90, pp. 999-1001, 2007.
Patent
Patent
Inventor/ co-inventor (PCT/SE2006001488): Process for production of dental
component and arrangement of such component.
Table of Contents
Table of Contents
Abstract
List of Publications
Patent
Table of Contents
1. INTRODUCTION ................................................................................................1
1.1 POROUS MATERIALS .....................................................................................................3
1.2 SURFACTANT-TEMPLATED MESOPOROUS MATERIALS..................................................4
1.2.1 Synthesis of surfactant templated mesoporous spheres ......................................5
1.2.2 Functionalization of pore walls ..........................................................................7
1.3 MOLECULAR TRANSPORT IN POROUS SOLIDS ...............................................................8
1.3.1 Fick’s Law ..........................................................................................................8
1.3.2 Models for adsorption and desorption..............................................................11
1.3.3 Molecular transport in liquid-filled pores ........................................................12
1.3.4 Transport studies of the mesoporous solids......................................................13
1.4 MESOPOROUS SILICA AS POTENTIAL HOST FOR DRUGS AND ENZYMES .......................14
1.4.1 Membrane encapsulated mesoporous particles................................................14
1.4.2 Mesoporous solid supports for the enzymes .....................................................15
2. AIMS AND SUMMARY OF PAPERS .............................................................19
3. MATERIALS ......................................................................................................21
3.1 SURFACTANTS, SILICA SOURCES AND BUFFERS ...........................................................21
3.2 FLUOROPHORES..........................................................................................................21
4. METHODS AND EXPERIMENTAL TECHNIQUES....................................23
4.1 SYNTHESIS OF MESOPOROUS SPHERES ........................................................................23
4.1.1 Monodisperse aerosol-produced mesoporous spheres .....................................23
4.1.2 Large-pore mesoporous silica spheres by slow evaporation from emulsion
droplets......................................................................................................................25
4.1.3 Mesoporous colloids by modified Stöber Method.............................................26
4.2 IMMOBILIZATION OF MESOPOROUS SPHERES IN GELATINE ........................................27
4.3 CONFOCAL LASER SCANNING MICROSCOPY (CLSM).................................................28
4.3.1 Principle ...........................................................................................................28
4.3.2 Correcting and evaluating for possible fluorescent microscopy artefacts........31
5. SUMMARY OF KEY RESULTS AND DISCUSSIONS.................................37
Table of Contents
5.1 THE EFFECT OF GUEST MOLECULE-HOST PORE WALL ELECTROSTATIC INTERACTIONS
ON MOLECULAR TRANSPORT ............................................................................................37
5.2 INTRAPARTICLE TRANSPORT AND RELEASE OF DEXTRAN IN CYLINDRICAL MESOPORES
..........................................................................................................................................41
5.3 CATALYTIC ACTIVITY AND STABILITY OF TLL LIPASE IMMOBILIZED IN
FUNCTIONALIZED MESOPOROUS SILICA
...........................................................................46
5.4 COLLOIDAL SPHERES WITH ORDERED MESOPORES’ SYNTHESIS, CHARACTERIZATION,
AND THEIR USE AS A SOLID SUPPORTS FOR A BILAYER LIPID MEMBRANE .........................49
5.4.1 Mesoporous structure within the colloidal mesoporous spheres ......................49
5.4.2 Lipid bilayer membranes coating .....................................................................52
6. CONCLUSIONS .................................................................................................57
6.1 INTRAPARTICLE MOLECULAR TRANSPORT STUDIES USING CLSM ............................57
6.2 MESOPOROUS SILICA SPHERES AS SUPPORTS FOR LIPID BILAYERS AND TLL ENZYME
..........................................................................................................................................58
7. OUTLOOKS........................................................................................................59
ACKNOWLEDGEMENTS ...................................................................................61
REFERENCES........................................................................................................63
Introduction
1. Introduction
Materials with tailorable porosity and pore size distribution are of great
interest for applications where the uptake and/or release of active
components need to be controlled. Soft chemical derived inorganic or hybrid
structure, templated by surfactant or other structure directing agents, resulted
in a new family of mesoporous materials1, 2 characterized by a narrow pore
size distribution in the mesoscopic range (2-50nm), tuneable pore
connectivity, and well defined morphology.3-8 Mesoporous silica is
particularly attractive as support for adsorption/ separation of
macromolecules,9, 10 in biocatalysis,11, 12 and biosensing;13 and as host
reservoir materials for controlled release applications, e.g., in drug release,1418
gene delivery,19 and chemical release.20
Applications in controlled release and delivery rely on the ability of the
carrier materials to dispense the guest molecules at the desired rate. This
calls for a good understanding of the molecular transport within the carrier
material and the dominating release mechanisms. It is clear that a better
understanding of the hindered mass transport and diffusion of guest
molecules, in particularly macromolecules, in liquid-filled mesopores is
important when developing new porous materials for biotechnological and
controlled release applications.
Transport of solute molecules in liquid-filled pores is hindered when the
characteristic dimension of the solute molecules is no longer small compared
to the pore it traverse in.21-24 The geometrical confinement clearly reduces
the mobility and diffusion rates of solutes and polymer in various types of
porous media, e.g., porous glasses.25 Several theoretical descriptions of
liquid-phase mass transfer in fine pores have been developed based on
hydrodynamic models of diffusion.23, 24, 26 Hindered diffusion of
macromolecules within confined space has been extensively studied, e.g., by
Deen et al. who compared the diffusion of macromolecules of different
structural flexibility using track-etched membranes,27 and by Nicholson et al.
who quantified the diffusion of large flexible random coils of dextrans in
extracellular space.28, 29
Attempts to estimate the effects of confinement and tortuosity on the
release kinetics of drug molecules from mesoporous materials by bulk
release studies17, 30 have recently been reported. Studies have also shown that
the affinity between the guest molecules and the surfaces of the mesoporous
1
Introduction
material can have a significant effect on both the loading and release
kinetics.30-32 It is interesting to note that, in contrast to the numerous
investigations on bulk uptake and release, there are only a limited number of
studies where the molecular distribution and hindered transport within the
material have been analyzed. The poorly defined morphology and wide
particle size distribution of many of the investigated mesoporous materials
also limit the possibility to analyze the data quantitatively.
Various experimental techniques can be used to study molecular transport
dynamics within a mesoscopic space, e.g., nuclear magnetic resonance,33, 34
neutron scattering,35 holographic laser interferometery,36 and singlemolecule fluorescence microscopy.37 The ability of confocal laser scanning
microscopy (CLSM) to optically section the studied object38 and to directly
visualize and quantify the spatial (both 2-D and 3-D) fluorescence molecules
distribution within the porous system in real time makes this technique
highly suited for studies of the uptake and relatively slow molecular
transport in mesoporous materials39-41 and chromatographic media.42 Cheng
and Landry41 showed how confocal laser scanning microscopy (CLSM) and
an amine-binding dye can be used to probe the rate and the extent of amine
functionalization of the mesoporous silica spheres. Schröder et al.42
quantified intraparticle protein diffusion in chromatographic media by
measuring the intraparticle protein concentration profiles in a microcolumn
using CLSM, and determined the diffusion coefficient using a spherical
diffusion model.
Mesoporous silica materials, with their regular arrays of pores and narrow
pore size distribution, have biocompatible and easily functionalized surfaces.
These combined properties have motivated their use in biomedical (e.g.,
controlled drug release14, 15, 17 and gene delivery19) and biotechnological (e.g.,
enzymes immobilization43 and biochemical recognition and protein
function18) applications. The encapsulation or attachment of a
pharmaceutical agent within or on a solid support greatly improves drug
safety and stability. New therapies are made possible and have inspired
active research on the design of tailored materials and intelligent delivery
systems.44 Recent works have shown how, e.g., coumarin, quantum dots and
other molecules can open and close the mesopores triggering release of
specific molecules.45, 46 Particle-supported bilayer lipid membranes have
been studied extensively as models of the cell membrane.47-51 However, their
2
Introduction
applications as a durable encapsulation of the active molecules in
mesoporous particles have only been recently pursued.18, 40
This thesis has been written in 7 chapters, which includes this
introduction (Chapter 1) covering some backgrounds on mesoporous
materials, molecular transport in porous solids, and application of
mesoporous silica spheres as host materials for drugs and lipases. The aims
of this PhD study are listed and the summary of the five papers, which this
thesis is based on, can be found in Chapter 2
Details of the materials used during the study for this thesis are described
in Chapter 3. Chapter 4 briefly describes the synthesis of two types of
mesoporous silica studied, 1) monodisperse aerosol-produced, and 2)
monodisperse modified Stöber method produced mesostructured silica
spheres. This chapter also covers some principles of the confocal laser
scanning microscopy, with special emphasis on the procedures used for
removing the fluorescent microscopy artefacts (photobleaching and light
attenuation).
Chapter 5 is a collected summary of key results and discussions of the
five papers included in this thesis. The first two sections are devoted to the
studies of molecular transport within the mesoporous spheres using CLSM.
These sections discussed the important effects of guest molecule-host pore
wall electrostatic interactions on the spatial distribution and the transport
kinetics of two small charged dyes (Paper I), and the hindered transport of a
neutral and flexible macromolecule (Paper II). The next two sections include
demonstrations of the use of mesoporous spheres in biotechnological
applications: a solid support for immobilization of TLL lipase (Paper III);
and a bilayer lipid membrane encapsulated solid support with inner porous
structure mimicking a cytoskeleton (Paper IV and V).
Finally, after drawing the conclusions (Chapter 6), a list of possible
further studies inspired by this work is included in Chapter 7.
1.1 Porous materials
A porous material is a solid frame or matrix with an interconnected
network of pores permeable to gases or fluids. Many substances ranging
from natural rocks, soils, biological tissue (e.g., bone), to man-made
materials like cements, foams and ceramics are porous. Porous materials
have been intensively studied due to their scientific interest and
technological importance as they interact with atoms, ions and molecules not
3
Introduction
only at the exterior surfaces but also throughout the bulk of the material.
Conventional applications of porous materials involve ion exchange,
adsorption (for separation) and catalysis, and many of these applications
benefit from the size selectivity and enormous surface area of the well
defined pores present in solids such as zeolites.52, 53 According to the IUPAC
definitions, porous materials can be divided into 3 classes; microporous
materials where the pore size is equal to or below 2 nm, mesoporous
materials where the pore size is between 2 nm to 50 nm, and macroporous
materials characterized by a pore size above 50 nm. The distribution of pore
sizes, pore volumes and pore connectivity directly affect the capability of the
porous material to perform the desired function in a particular application.
Ordered pore structure with narrow pore size distribution has become
increasingly important during recent years as it can lead to superior
properties. For example, a material with uniform micropores, such as zeolite
molecular sieves, can discriminate between molecules particularly on the
basis of size. It selectively adsorbs small molecules from a mixture
containing molecules too large to enter its pores. However, microporous
materials like zeolites present severe limitations when large reactant
molecules are involved, especially in liquid-phase systems. Most small
biomolecules are too big to enter the micropore cavities. This is unfortunate
as the high surface areas, the mechanical and chemical resistance, and the
well controlled pore wall chemistry of zeolite materials are attractive for e.g
enzyme catalyzed fine chemical synthesis.54 Hence, an important line of
research has focused on the enlargement of the pore sizes into the mesopore
range. This would allow larger molecules to enter the pore system, where
they can be immobilized, separated from a mixture, and also released at a
controlled rate.
1.2 Surfactant-templated mesoporous materials
The soft chemistry derived inorganic or hybrid materials templated by
structuring directing agents feature unique textural properties. Like the
microcrystalline zeolite, this class of materials is characterized by very large
specific surface area and pore volume, narrow pore size distributions,
tuneable ordered pore structure that my display a long-range order,1-3 and in
particular for mesoporous silica, easily functionalized surfaces.55 Unlike the
zeolites, the surfactant-templated mesoporous silica exhibit amorphous pore
walls.
4
Introduction
The morphology of mesoporous silica based materials can also be
controlled to yield, e.g., rods,56 spheres,4, 57, 58 and fibres.7 The well-known
representatives of this class of materials are siliceous MCM-41, MCM-48
and MCM-50 (Scheme 1). Recent work has investigated the potential use of
mesoporous materials in applications such as chromatography,58-60
catalysis,61, 62 as chemical and biochemical sensors,63 and as platforms for
molecular screening.40
a
b
c
Scheme 1 Structure of mesoporous materials a) MCM-41 with 2D
hexagonal structure and p6mm plane group, b) MCM-48 with cubic structure
and Ia 3 d space group, and c) MCM-50 with lamellar structure. Drawings
kindly contributed by Dr. Keiichi Miyasaka.
1.2.1 Synthesis of surfactant templated mesoporous spheres
Porous silica spheres are of interest in, e.g., chromatography,58 catalysis62
and chemical and biochemical sensor63 applications and as platforms for
molecular screening.48 Monodisperse spherical mesoporous particles in the
colloidal size range with well defined pore sizes and pore structure are
attractive in applications where the uptake and/or release of active
components needs to be controlled, e.g., in controlled drug or gene
delivery.18, 19
Evaporation-induced self-assembly (EISA) utilizes the evaporation of the
solvent to increase the surfactant concentration and eventually induce the
formation of liquid crystalline phases.64 By changing the original
alcohol/water/surfactant ratio it is possible to control the resulting
mesostructure. EISA combines the simplicity of the sol-gel process with the
efficiency of surfactant self-assembly, allowing rapid preparation of
mesostructured colloidal particles, fibres, thin films with controlled
mesostructure.65 EISA also provides a general and flexible approach for
5
Introduction
nanocomposite fabrication by incorporation of non-volatile components, e.g.,
nanoparticles66 into the silica mesoporous matrix.
Aerosol-based techniques that utilize the EISA process have been used to
produce both hollow and dense mesoporous particles where the relatively
large interfacial tension between the droplet surface and the air yields
spherical powders.4, 5, 67-69 In 1997 Bruinsma et.al67 pioneered the continuous
production of hollow mesoporous particle using the aerosol process. The
aerosol-assisted process was further modified to enhance it robustness and
yield by Andersson et. al.70 The mesoporous particles produced by these
groups are all polydispersed. The production of monodisperse mesoporous
particles by an aerosol-assisted method was demonstrated by Rao et al.,5, 69
who utilized a vibrating orifice aerosol generator to form the monodisperse
primary droplets of the precursor solution.
Mesoporous silica spheres can be synthesized in solution at both acidic71,
72
and basic conditions.57, 58 Unger and co-workers73, 74 showed that the
classical Stöber method, which is able to produce silica colloids from
tetraalkoxysilanes-ammonia-water-alcohol mixture,75 can be modified and
yield mesoporous spheres by the addition of n-alkyl amine. The simplicity of
this modified Stöber route for mesoporous spheres has generated a
substantial interest for various applications.19, 76, 77 However, the modified
Stöber route has been plagued by problems related to polydispersity,
agglomeration and/or absence of a long-range mesoscopic order. Recently,
Yano and co-workers demonstrated how monodisperse submicron silica
spheres with a long-range ordered 2D hexagonal mesostructure can be
synthesized from solutions containing tetramethoxysilane and nalkyltrimethylammonium surfactants by using a high surfactant to silicon
ratio in a dilute system.78, 79 Studies of the internal pore structure of the
spheres by electron diffraction80-82 and direct imaging by TEM of particles
partially infiltrated by platinum79 suggest radially aligned pores. Recent
work by Nakamura et al83 showed that a radial alignment of the mesopores is
retained even after seeded growth. The pore structure in the core of the
mesoporous spheres is less well understood and there is only a few studies
analyzing how the pore structure varies with the distance from the centre of
the sphere.80-82
6
Introduction
1.2.2 Functionalization of pore walls
An important feature of mesoporous silica is the multitude of possibilities
(Scheme 2) to functionalize the pore surface. Hence, surface functionality
can be modified and the textural properties can be changed independently by
various in situ and/or post synthesis procedures. The microenvironment
inside the mesopores can be drastically changed by surface coating with
organic or inorganic species, or direct incorporation of organic species into
the inorganic matrix to form a hybrid framework. The mesoporous support
material itself can also be functionalized by incorporation of different
components into the pore walls. Many of the modification steps make use of,
e.g., the silanol groups present on the silica surfaces as anchor sites for metal
species or silane coupling agents.
In situ functionalization or post synthesis
Substitution
grafting
Immobilization
Surface
coating
L
M
L
M+
Ion
exchange
Silylation
Nanoparticle
R
R
Enzyme
encapsulation
Non-silica
material
Organic-inorganic
hybrid framework
Scheme 2 The sketch illustrates the different functionalization of the
mesopore walls. Sketch is modified from Taguchi et al.52
Several reports have indicated a lower density of silanols in ordered
mesoporous materials, especially in MCM-41, than in hydroxylated silica
surfaces of macroscopic dimensions where the density of silanol groups is
about 4–6 Si–OH per nm2.84 The density of silanol groups for ordered
mesoporous materials has been estimated to vary between 1.4 to 3 groups
per nm2, for MCM-41 and MCM-48,85, 86 depending on factors such as
surface curvature, template removing method and rehydration process. There
is also evidence of a non-homogeneous distribution of the silanols, with
patches of more hydrophobic (e.g., isolated silanols and siloxane bridges)
7
Introduction
and more hydrophilic (adjacent silanols), over the surface of MCM-41.87, 88
This may lead to inhomogeneous distribution of groups anchored to the
surface.
1.3 Molecular transport in porous solids
The study and the control of guest molecule–host pore-wall interactions
in porous materials call for a good understanding of the molecular transport
within the carrier material and the dominating release mechanisms. This is
especially important and essential when developing new porous materials
with tailored properties for specific applications.
1.3.1 Fick’s Law
The Fick’s equation,
J = −D
∂C
∂x
(1)
where J is the diffusion flux (mol/cm2/s1) and C is the concentration
(mol/cm3), provides a convenient starting point for the quantitative analysis
of intraparticle transport in porous materials. The diffusivity (D) may be
called the transport diffusivity since this quantity measures the ratio of the
solute flux to the concentration gradient.
To describe the rate of adsorption or release generally requires
information on the change of concentration as a function of time describable
by a continuity equation,
∂J
∂C
=−
∂x
∂t
(2)
which becomes
∂C
∂ 2C
=D 2
∂t
∂x
(3)
This gives us the Fick’s Second Law in one dimension which describes
how diffusion causes the concentration to change with time, if D is
independent of C.
It should be noted that for diffusion in a binary fluid phase the flux (J) is
defined relative to the plane of no net volumetric flow and the coefficient D
is called the mutual diffusivity. The same expression can be used to
characterize migration within a porous (or microporous) solid, but in that
8
Introduction
case the flux is defined relative to the fixed frame of reference provided by
the pore walls. The diffusivity is then more correctly termed the transport
diffusivity. Note that the existence of a gradient of concentration (or
chemical potential) is implicit in this definition89 (Scheme 3a). In the case of
self-diffusion the physical situation is different since there is now no
gradient of species concentration, describing an uncorrelated random
movement of molecules (Scheme 3b). This process is described by following
the molecular trajectories of a large number of molecules and determining
their mean square displacement. This difference in the microphysical
behaviours between the transport diffusion and self diffusion implies that D,
the transport diffusion coefficient, and D*, the self diffusion coefficient, are
generally different.
b
a
Scheme 3 Schematic representation of molecular movements in the case of a)
transport diffusion and b) self diffusion. Sketch adopted from Ruthven.89
As per mentioned earlier, the flux, as well as the diffusion coefficient, has
to be chosen relative to a frame of reference. The solid framework of the
pore provides a convenient and unambiguous frame of reference for
measuring the diffusive flux. Considering diffusion in a porous solid as a
special case of binary diffusion where the diffusivity of the solid atoms (of
the pore frame) is zero, the ‘interdiffusion’ can thus be described by a single
diffusivity i.e. the interdiffusivity is simply the diffusivity of the mobile
species
Diffusion in a sphere
For cases in which the diffusion is radial, the diffusion equation with a
constant diffusion coefficient takes the form
2
∂C
⎛ ∂ C 2 ∂C ⎞
= D⎜ 2 +
⎟
∂t
r ∂x ⎠
⎝ ∂x
(4)
9
Introduction
where r is the radius and x is the distance from the centre of the sphere.
Letting u = Cr
Eq. 4 becomes
∂u
∂ 2u
=D 2
∂t
∂r
(5)
Since this is the equation for linear flow in one dimension, the solutions
of many problems in radial flow in a sphere can be deduced immediately
from those of the corresponding linear problems.
For non-steady state diffusion, an isothermal spherical particle subjected
to a step change in adsorbate concentration at the external surface at time
zero, the total amount of diffusing substance entering or leaving the sphere is
given by90, 91
Mt
6 ∞ 1
t ⎞
⎛
= 1 − 2 ∑ 2 exp⎜ − Dn 2π 2 2 ⎟
M∞
π n=1 n
r ⎠
⎝
The short and long time asymptotes are given by
Short time: M t = 6
M∞
r
Dt
(6)
π
Long time: M t = 1 − 6 exp⎛⎜ − Dπ 2 t ⎞⎟
2
2
M∞
π
r ⎠
⎝
(7)
where Mt and M∞ are the amount of adsorbate at time t and time infinity,
respectively.
Surface resistance model
For particles where the mass transfer resistance is much higher at the
surface than in the interior of the adsorbent particle due to, e.g., partial
closure of the pore entrance/exits, the concentration profile typically show
step-like form with a sharp change in concentration at the surface, and a flat
intraparticle concentration profile (even under partial loading conditions). A
particle with surface resistance control follows a simple exponential
approach to equilibrium92:
Mt
⎛ − 3kt ⎞
= 1 − exp⎜
⎟
M∞
⎝ r ⎠
(8)
where the r/3k is the time constant and k is the rate constant.
10
Introduction
1.3.2 Models for adsorption and desorption
Nanoporous materials are widely used in adsorption-related applications.
Adsorption phenomena are found in many areas including catalysis and
medicine. Adsorption is also used to characterize porous solids in terms of
surface area, pore volume and pore size; and to understand the behaviour of
the molecules in the porous materials investigated.93 Adsorption from
solutions depends upon the properties of the organic and inorganic solutes
(molecular mass, molecular size, geometry, polarity, solubility and etc.), and
the structure and nature of the adsorbent’s surface (surface area, porosity,
chemical structure etc.). In addition, the experimental conditions such as the
pH and the ionic strength of the solution and the temperature of adsorption
also determine the adsorption capacity and the nature of the adsorption
process.
A number of adsorption isotherm equations such as the Langmuir
equation, the Freundlich equation, the Brunauer-Emmett-Teller (BET)
equation, and Temkin equation have been developed to represent the gasadsorption data.94 Several of the isotherm equations for gas-adsorption can
be extended to correlate the liquid-phase adsorption equilibrium of a single
component system by simple replacement of adsorbate pressure by its
concentration. In this thesis only the Langmuir equation and the Temkin
equation are described.
Langmuir adsorption isotherm
The Langmuir adsorption isotherm, originally for gas adsorption, is the
first theoretically developed adsorption isotherm. It has a defined adsorption
maximum and assumes linear adsorption at concentrations far below this
maximum. The Langmuir equation may be described by,
⎛ KP ⎞
⎟
⎝ 1 − KP ⎠
(9)
θ =⎜
where θ is the fraction of surface coverage, K is a constant assuming the
enthalpy of adsorption is independent of θ, and P is the applied pressure or
concentration. However this Langmuir model is seldom obeyed and the heat
of adsorption, q, normally falls with increasing surface coverage.
11
Introduction
Temkin adsorption model
The Temkin adsorption is derived from the Langmuir adsorption isotherm
by inserting the condition that the heat of adsorption decreases linearly with
surface coverage. Such an effect can arise from repulsive forces on a
uniform surface, or from an inherent surface heterogeneity of the surface.
This isotherm is characterized by linear variation of ln p with θ evaluated by
ln p = ln A0 +
q 0αθ
RT
(10)
where A0 = Kexp(qo/RT) is independent of θ, α is a constant, q is the heat
of adsorption and qo is the heat of adsorption when θ = 0.
1.3.3 Molecular transport in liquid-filled pores
When the dimensions of solute molecules are at least several times larger
than those of the solvent, the resistance to the Brownian motion of the solute
may be equated to the hydrodynamic drag on a particle of equivalent size
and shape. For spherical solutes this leads to the Stokes-Einstein equation
D∞ =
kT
(11)
6πηrs
where D∞ is the diffusivity in dilute bulk solution, k is the Boltzmann’s
constant, T is the temperature, η is the solvent viscosity, and rs is the radius
of the solute. For non-spherical solutes of known D∞ the value of rs inferred
from eq. 1 is termed as the Stokes-Einstein radius, the radius of a sphere of
equal diffusivity. For solutes large enough to behave as hydrodynamic
particles, hindered diffusion can be explained in part by the fact that the
constrained space of a pore causes the molecular friction coefficient
(denominator of eq. 12) to exceed its value for an unbounded solution i.e.
Deff/D∞ < 1, where Deff is the effective diffusion coefficient.
Several theoretical descriptions of liquid-phase mass transfer in fine pores
have been developed based on hydrodynamic models of diffusion.23, 24, 26 The
principle objective has often been to understand the permeability properties
of biological structures of, e.g., capillary walls and cellulose membranes, by
applying the hydrodynamic diffusion models with the concept of hindered
transport in equivalent pores as a description of the path taken by lipidinsoluble molecules.95
12
Introduction
1.3.4 Transport studies of the mesoporous solids
Several methods for measuring transport diffusion are ‘macroscopic’ as
they depend on measuring the flux under a well-defined gradient of
concentration. Uptake rate measurements using evaluation of
chromatographic,96 zero-length column97 and temporal analysis of
products;12, 16 and traditional bulk release studies followed the change in the
solutes concentration of the liquid media as a function of time14, 17, 19, 30 are
just some examples.
The diffusion in mesoporous materials may be determined using as
equilibrium techniques in which the self-diffusivity is measured, and by nonequilibrium techniques in which the transport diffusivity is measured.89 True
equilibrium measurements are made on a length scale smaller than the
dimensions of an individual crystal by following the mean square
displacement of the molecules in a known time interval by using, e.g.,
nuclear magnetic resonance,33, 34 or neutron scattering.35
The optical interference microscopy and IR sorption techniques98
(holographic laser interferometery,36 and single-molecule fluorescence
microscopy37) may be classified as mesoscopic methods that allow a direct
measurement of sorption/desorption rates on the single-crystal scale. The
form of the transient concentration profiles obtained from these mesoscopic
methods provides direct experimental evidence relating the nature of the
rate-controlling resistances to mass transfer.99 Confocal laser scanning
microscopy (CLSM), also a mesoscopic method, has the possibility to
optically section the studied object38 to obtain the transient intraparticle
profile of the adsorbing/desorbing fluorescent molecules in real time. This
technique is therefore well suited for comparing the transport of molecules in
mesoporous particles of different connectivity and pore size. CLSM has been
employed together with an amine-binding dye to provide an elegant and
facile way to probe the rate and the extent of amine functionalization of the
mesoporous silica spheres.41 We have recently used CLSM to follow the
release of an anionic and a cationic fluorescent dyes, and have related the
time and spatially dependent variations in the concentration of dyes (within
the mesoporous silica spheres) to the specific interactions with the
negatively charged walls.100
13
Introduction
1.4 Mesoporous silica as potential host for drugs and enzymes
1.4.1 Membrane encapsulated mesoporous particles
There is a significant interest in developing novel approaches to control
and trigger the release of molecules, say drugs, which have been
immobilized in the pores of mesoporous silica. Recent works have shown
how, e.g., coumarin, quantum dots and other molecules can open and close
the pores to trigger the release of specific molecules.45, 46
Cu
Ca
Mg
Lipid
Heme
Cu
Figure 1 Three-dimensional structure (side view) of cytochrome c oxidase
from R. sphaeroides enzyme. Subunit I, light gray; subunit II, green; subunit
III, pink; subunit IV, yellow; heme (see indication); Cu dark red; magnesium,
dark green; calcium, dark gray; lipids, bright green and red.101 Images
generated from the coordinates in the pdf files 1m56 available in the Protein
Data Bank (http://www.rcsb.org/pdb/home/) using the Jena3D Viewer.
Particle-supported bilayer lipid membranes have been studied extensively
as models of the cell membrane.47-51 Deposition of lipid membranes as
durable encapsulations covering entire surface of the mesoporous particles
(with active molecules immobilized within) has recently been recently
pursued.18, 40 18 The release of these molecules could be mediated by specific
channels or transporters incorporated into the membrane. This requires
development of methodology and techniques for quick and facile
incorporations of integral membrane proteins into the particle-supported
14
Introduction
lipid layers. Such protein-membrane particle systems are also of significant
interest for functional studies of membrane proteins as a robust system for
mechanistic investigations of transport mechanisms. Indeed, recent reports
describe successful incorporation of a number of membrane protein model
systems into membranes supported on solid51 and porous102 silica particles.
Encapsulating a support such as the silica mesoporous spheres with bilayer
lipid membrane incorporated with membrane-bound transport proteins
produced a supramolecular architecture with an inner pore structure
mimicking a cytoskeleton. This solid-supported biofunctional cellular
surface can serve to provide basis for functional studies of membrane-bound
transport proteins such as. cytochrome c oxidase (CytcO) from Rhodobacter
(R.) sphaeroides (Fig. 1), and also for applications within pharmaceutical
drug delivery.
1.4.2 Mesoporous solid supports for the enzymes
Lipases are used as versatile biocatalysts for ester synthesis, hydrolysis,
alcoholysis, acidolysis, aminolysis and interesterfication; reactions of great
importance in, e.g., the food, detergent, leather, textile, cosmetic, paper and
pharmaceutical industries.103 The number of commercially available lipases
has steadily increased since the first lipase developed for detergent industry,
Lipolase®, was launched in 1988.
Lipases are enzymes classified as glycerol ester hydrolases (EC 3.1.1.3)
and can be found in the human blood plasma, panaceas, in different bacterial
and mold. The lipase primarily catalyzes the hydrolysis of triglycerides and
partial glycerides (the substrate or reactant), the main constituents of
vegetable oil and animal fats, to free fatty acids and glycerol. A majority of
lipases undergo ‘interfacial-activation’ which relates to the dramatic increase
in catalytic turnover of lipases at a (hydrophobic) substrate interface.104 This
phenomenon can be explained by a conformational change of the protein at
the interface.105 Most lipases has an oligopeptide chain referred to as the ‘lid’
that covers the active site in aqueous solution, this is called the ‘closed form’
(Fig. 2a). This lid folds when associated with an appropriate surface, e.g., the
substrate, to expose the active site and is called the ‘open form’ (Fig. 2b).
Heterogeneous catalysis when the enzyme is attached onto solid supports,
which can be polymers, ceramics, or zeolites,106, 107 have several advantages
over homogeneous catalysis with respect to, e.g., the ease of recovery and
reusability.107-109 The stability of the enzyme can also be increased by the
15
Introduction
immobilization. The enzyme stability has also been shown to improved
when immobilized in pores having pore size matching closely to the size of
the enzyme.11, 110 The development of mesoporous materials with pore sizes
of 5 nm and above111 opened the possible range of enzymes, such as
proteases and lipases,112 for immobilization on the internal surfaces of the
solids. Immobilization of biomolecules into the mesoporous carrier may be
carried out either by chemical binding or physical adsorption, e.g., through
cross-linking of the enzyme onto the pore-walls or by encapsulation of the
enzyme into the pore channels (Fig. 3) of the support.43, 107, 113 Physical
adsorption is also the simplest method and thus the most commercially
viable alternative.
Lid
a
Active site
Se
Lid
b
Active site
Oleic acid
Ser
Figure 2 Three-dimensional structure of the a) closed and b) open form of
Thermomyces lanuginosus. The Ser is in light red and the oleic acid is in
bright green around the active site. lipase. Images generated from the
coordinates in the pdf files 1tib and 1gt6 respectively available in the Protein
Data Bank (http://www.rcsb.org/pdb/home/) using the Jean 3D Viewer.
16
Introduction
The major drawback with physical adsorption is enzyme leakage due to
weak interactions between the support and the enzyme. Attempts to reduce
leakage by cross-linking of the enzyme inside the mesoporous carrier, or by
silylation of the pore openings to reduce the size of entrance/exit of the pore
channels, have only been partially successful and frequently resulted in a
reduction of the enzymatic activity.12, 112, 114, 115 On the other hand,
hydrophobic treatments of the supports show a reduced leakage and studies
also indicate an increase in the enzymatic activity.12, 112, 114, 116
Figure 3 Strategies for enzyme immobilization.43
17
18
Aims and Summary of Papers
2. Aims and Summary of Papers
To tailor the properties of porous materials requires learning about the
control of host-guest interactions. One important aim of this thesis is to
develop a facile and direct method to investigate the effects of guest
molecules-host pore walls interactions, on (relatively slow) molecular
transport within mesoporous silica materials. An experimental protocol was
developed to monitor the spatial distribution of fluorescently-tagged
molecules within the same spheres over an extended period of time using
Confocal Laser Scanning Microscope (CLSM). Procedures were established
to check and correct the intensity data obtained for photobleaching,
refractive index mismatch, and light attenuation. In Paper I a positively
charged dye, Oregon Green 488, and a negatively charged dye, Rhodamine
6G were used to probe the effect of guest-host electrostatic interactions on
the spatial evolution and molecular transport within mesoporous silica
spheres produced by an aerosol-assisted route. In Paper II, the hindered
transport of macromolecules in liquid-filled mesopores was investigated by
following the transport of a fluorescently tagged-dextran, from the bacterial
strain Leuconostoc mesenteroides, within mesoporous spheres produced by
an emulsion and evaporation (ESE) method. Various diffusion and
adsorption models were employed to analyze the intensity data corrected for
both photobleaching and light attenuation. The guest-host interactions in the
nanoscale pore system exert a profound influence on both the spatial
arrangement of the molecules and its transport kinetics.
This thesis also aimed to extend the potential biotechnology applications
of the mesoporous silica spheres. Two different applications were
investigated, as carrier material to enhance the stability of the immobilized
enzyme lipase, and as a solid support for lipid membranes where
transmembrane proteins have been incorporated. In Paper III the spatial
distribution and stability of a lipase were quantified using CLSM. The timedependent leaching of lipase from the ESE spheres was evaluated for
materials with a hydrophobic and a hydrophilic pore surface. The higher
specific enzyme activity of the lipase immobilized onto the hydrophobilized
spheres was related to reduced lipase-denaturating and lower lipase leakage
rate. In Paper IV colloidal mesoporous silica spheres were synthesized via a
modified Stöber method. Analysis of the thin microtome slices taken at
different height of the sphere using TEM revealed bundles of hexagonally
19
Aims and Summary of Papers
ordered cylindrical channels extending radially to the particle surface. In
Paper V these spheres were successfully used as solid support for formation
of lipid bilayers incorporated with a transmembrane complex ‘molecular
machine’, cytochrome c oxidase (CytcO, cytochrome aa3).
20
Materials
3. Materials
3.1 Surfactants, silica sources and buffers
Polyethylene oxide hexadecyl ether, Brij 56 (Sigma-Aldrich) is an nonionic amphiphilic templating surfactant used for the synthesis of micron size
small pore mesoporous silica (SMS) spheres (details found in Paper I). A
cationic surfactant, hexadecyltrimethylammonium bromide, C16TAB
(Aldrich, 95%) was used as template for the synthesis of colloidal small-pore
mesoporous silica (CMS) spheres (details found in Paper IV and V).
Tetramethoxysilane, TMOS (Aldrich, 98 %) and tetraethoxysilane, TEOS
(Sigma, Purum >98 %) are alkoxysilanes used as the inorganic silica sources.
The Dulbecco’s phosphate buffered saline, PBS (135 mM, pH 7.2, Sigma)
and 3-morpholinopropanesulfonic acid, MPOS pH 7.5 (0.2 M, MOPS+NaCl)
were used as buffers. Milli-Q grade water was used in all experiments.
3.2 Fluorophores
Small charged dyes, cationic Rhodamine 6G, Rh6G (Sigma Aldrich), and
anionic Oregon green 488 5-isomers, OG (Molecular Probes Europe BV,
Leiden, The Netherlands) were both diluted to 0.1 mmol/dm3 with
MilliporeTM-grade water. These dyes were used to study the effects of guest
molecule-host pore wall electrostatic interactions (Paper I, section 5.1 in
thesis).
Dextrans from the bacterial strain Leuconostoc mesenteroides,
characterized by their high molecular weight, low toxicity, relative inertness,
and good water solubility are often used to model, e.g., large biological
molecules.117 Texas Red®-tagged dextran with molecular weight of 3000
g/mol (Dex3k), a diameter of gyration, dg = 5.2 nm,118 a diameter of
hydration, dh = 2.9 nm 118 and a bulk diffusion coefficient of D = 23 x 10-7
cm2/s28was obtained from Molecular Probes Europe BV (Leiden, The
Netherlands). The Dex3k was diluted to 0.1 mmol/dm3 with MilliporeTMgrade water, and filtered with a 0.2 µm filter to remove any large
agglomerates. This solution was used for the study of the hindered transport
of macromolecules in mesoporous materials (section 5.2, Paper II).
Alexa Fluor 488-labelled lipases were prepared in YKI following a
procedure described in Paper III. The lipase originated from the fungus
21
Materials
Thermomyces (formerly Humicola) lanuginosus, with the commercial name,
Lipolase®, kindly supplied by Novozymes A/S (Bagsvaerd, Denmark, ∅ =
46 Å, MW = 31000 g/mol, c = 20 mg/ ml). The concentration of the Alexa
Fluor 488-labelled lipases was measured to be 27.1 µmol/dm3, and the
fluorophore concentration was measured to be 0.27 Alexa Fluor 488 per
enzyme. The tagged lipase was used for the study of enzyme immobilization
within the mesoporous supports (section 5.3, Paper III).
22
Methods and Experimental Techniques
4. Methods and Experimental Techniques
4.1 Synthesis of mesoporous spheres
The following sections describe the different methods used to produce the
three types of mesoporous particles (Fig. 4) used in this thesis.
a
b
100 μm
10 μm
c
1 μm
Figure 4 Scanning electron microscopy images of calcined mesoporous
spheres. a) The aerosol-assisted produced monodisperse spheres (Paper I), b)
the polydisperse spheres produced by the emulsion and evaporation method
(Paper II and III), and c) the monodisperse colloids by modified Stöber
method (Paper IV and V).
4.1.1 Monodisperse aerosol-produced mesoporous spheres
Using a vibrating orifice aerosol generator (VOAG Model 3450, TSI Inc.,
USA), monodisperse-mesostructured silica spheres have been produced
based on a modified recipe from previously established procedures.5, 66
23
Methods and Experimental Techniques
Schematic views of the aerosol generator setup (Scheme 4 and 5),
demonstrating the principle of the mono-droplets formation from the
precursor solution using a vibrating orifice. A solution containing the nonionic templating surfactant Brij 56 and ethanol was mixed with a solution of
HCl, ethanol and TEOS to form the precursor solution before being
introduced into the aerosol generator. The operation of the aerosol generator
is based on the instability and breaking up of a cylindrical liquid jet via a
vibrating orifice (20 μm) into uniform droplets. The generated droplets were
carried by a great volume of laminar airflow through the drying-chamber
where evaporation of solvents (essentially ethanol and water) takes place.
HEATING ZONE
DRYING ZONE
Ethanol evaporation
Vibrating
orifice
Water evaporation and
polymerization of silica
Filter
PARTICLE
COLLECTION
Carrier and
dispersion Air
To exhaust fan
Precursor Solution
Scheme 4 The transformation of monodisperse droplets of precursor solution
(gray circles) to solid mesostructured spheres (black circles) in a vibrating
orifice-aerosol generator (VOAG).
As the surfactant concentration reaches or goes above its critical
micellization concentration (CMC) self-assembly of the surfactant molecules
occurs forming the template for the hydrolyzed silica source. Further silica
condensation occurred as the particles pass through the heating zone before
the mesostructured particles were collected at the filter and calcined to
remove the surfactant template. The particles collected were calcined to
remove the surfactant. The calcined particles are mesoporous spheres with
average sphere diameter ∅ = 6.0 ± 0.2 μm (Fig. 4a). These particles have a
Type IV nitrogen isotherm and the pore size, surface area and pore volume
24
Methods and Experimental Techniques
determined are 2.7 nm, 800 m2/g and 0.44 cm3/g, respectively. The TEM and
XRD analyses indicate that the spheres have disordered mesopores (details
available in the supporting information of Paper I). The mesoporous spheres
were used for transport studies of two small, oppositely charged dyes in
Paper I.
Monodisperse
droplets
~40μm
20μ
Vibrating dispersion orifice
(40-80kHz)
Piezoelectric ceramic
Scheme 5 Breaking-up of a cylindrical liquid jet (precursor solution) by the
vibrating orifice to produce monodisperse droplets.
4.1.2 Large-pore mesoporous silica spheres by slow evaporation from
emulsion droplets
Spherical mesostructured particles, templated by Pluronic F127 together
with a swelling agent poly(propylene glycol), were produced via the
emulsion and solvent evaporation (ESE) method119, 120 This method consists
of five distinct steps: (1) preparation of a precursor solution, (2)
emulsification of the precursor solution, (3) evaporation of ethanol and water
by applying vacuum, (4) separation of particles and (5) surfactant removal
by calcination. The final mesoporous spheres are polydispersed with particle
diameter of 5 - 40 μm (Fig. 4b). The calcined spheres were silylated to
produce particles with hydrophilic pore surfaces. Both the as-calcined (with
hydrophilic pore surfaces) and the silylated (with hydrophobic pore surfaces)
have a Type IV nitrogen isotherm with an adsorption-desorption hysteresis
loops of Type H2. The specific surface area, the pore volume and the
average pore diameter of the spheres determined from the nitrogen sorption
isotherm are summarized in Table 1. The silylation treatment slightly
25
Methods and Experimental Techniques
reduced the pore diameter (Table 1) but the internal mesostructure has been
preserved.
Table 1 Nitrogen sorption data of the calcined and the silylated mesoporous
particles.
1
Particle
Pore size1
(Å)
Pore volume
(cm3/g)
Surface Area
(m2/g)
Calcined
67/39
0.42
417
Silylated
65/36
0.34
314
The first value corresponds to the pore size calculated from the adsorption isotherm
and the second value to the pore size calculated by the desorption isotherm.
The internal mesoporous structure determined using TEM and XRD
suggested that the spheres consist of domains of a two-dimensional
hexagonal structure with p6mm symmetry (see details of particles synthesis,
functionalization and characterizations in Paper III). The ESE spheres with
hydrophilic surfaces were used for investigating dextran diffusion (Paper II),
and the ESE spheres with hydrophilic and hydrophobic surfaces were used
as solid supports for the enzyme lipase (Paper III).
4.1.3 Mesoporous colloids by modified Stöber Method
Mesostructured colloidal silica particles were synthesized from a dilute
solution
of
alkali/methanol/water
that
contains
hydrolyzed
tetramethylorthosilicate
(TMOS)
as
the
silica
source,
and
hexadecyltrimethylammonium bromide (C16TAB) as the cationic templating
surfactant. Water was removed from the precipitate by evaporation in
ambient conditions overnight followed by drying at 80 ºC for 24 hours. The
dried mesostructured particles were calcined to remove the templating
surfactant. The calcined particles are monodisperse mesoporous spheres with
average particle diameter of 0.5 μm (Fig. 4c). These particles have a Type
IV nitrogen isotherm and the pore size, surface area and pore volume
calculated are 3.0 nm, 1027 m2/g and 0.72 cm3/g, respectively. The internal
mesoporous structure determined using XRD suggested that the spheres
consist of a two-dimensional (2D) hexagonal structure with p6mm symmetry
(see details of synthesis and characterization in Paper IV). TEM observation
of microtome slices of these spheres revealed bundles of hexagonally
26
Methods and Experimental Techniques
arranged pore channels extending radially towards the surface of the
particles. The study of the structure within these spheres will be discussed in
details in the next chapter. The colloidal small-pore mesoporous silica
particles were sealed in a glass container and kept in desiccators until use.
These spheres were used as a solid support for the formation of a bilayer
lipid membranes incorporated with transmembrane proteins in Paper V.
4.2 Immobilization of mesoporous spheres in gelatine
To facilitate the monitoring of the same spheres over extended period of
time, the aerosol-produced mesoporous particles (Paper I) were immobilized
by adding a small amount of gelatine (5 mg of gelatine in 100 ml of water)
and allowing the polymer to form a percolating gel (Scheme 6). The use of
gelatine to immobilize the particles enabled the monitoring of individual
spheres over an extended period of time, thus allowing a facile yet wellcontrolled experiment. The gel used contained more than 99.99 wt % water
and was kept hydrated at all times. The weak interaction between the dyes
and the gelatine was confirmed by checking the fluorescent-free background
after the saline solution exchange to remove the dye-containing solution. The
high molecular weight and large architecture of the gelatine (a helix polymer,
Mw ~300000 g/mol and length ~208 nm, which folds when cooled down)121
prohibits gel penetration into the pores of the mesoporous spheres.
VOAG generated
mesoporous spheres
Glass-bottom Petri dish
Buffer = PBS, PH 7.2, 130 mmol/dm3
Scheme 6 Sketch of how monodisperse mesoporous spheres are
immobilized in a diluted gel. The right figure is an enlarged view of the gel
that sets in the glass-bottom Petri dish.
27
Methods and Experimental Techniques
4.3 Confocal laser scanning microscopy (CLSM)
4.3.1 Principle
Confocal laser scanning microscopy (CLSM) allows imaging of a
fluorescent sample with high resolution and depth discrimination.
Fluorescence is the result of an excitation-relaxation process that occurs in
certain molecules, generally polyaromatic hydrocarbons or heterocycles,
called fluorophore. The excitation and emission wavelength is dependent on
the molecular structure of the fluorophore (Fig. 5b). In CLSM only one point
in the specimen is lit at a time instead of a broad, extended region of the
sample as in wide-field fluorescence microscope. A focused laser beam is
used to create a point source illumination, where the width of the beam in the
order of 0.5 μm. Reflected or fluorescent light from the illuminated spot is
collected by the objective and separated by a dichroic mirror so that the
emitted fluorescence can be focused onto the detector, which is typically a
photomultiplier tube (PMT).
A small pinhole aperture in the range of 10-100 μm is placed in front of
the detector and effectively blocks light from all out-of-focus parts of the
sample (Fig. 5a). Hence, a confocal microscope essentially consists of a
point light source and a pinhole at the image detecting plane that are
optically aligned to suppress out-of-focal plane information. Indeed, a
confocal microscope may be compared to the microtome method used to
obtain the desired slice mechanically. The principle of a confocal
microscope is seen in Fig. 5a and c. The dichroic beam splitter and emission
filter must match the excitation and emission spectra of the fluorophore (Fig.
5b and c). Rotating mirrors that scan the laser spot in x-, y-, and z-direction
over the specimen are used to create an image (a micrograph). The beam
scanning setup enables a higher temporal resolution compared to stage
scanning microscopes. Due to the depth-discriminating properties of the
instrument, optical sectioning and 3D-reconstructuring of a sample can be
performed.
28
Methods and Experimental Techniques
Fluorescence emission
a
Plane not in focus
Specimen
Plane in focus
Adsorption
b
Objective lens
Wavelength (nm)
Dicroic mirror
Specimen
Laser
c
Condenser
lens
Laser
NFT
LP
Pinhole
aperature
Detector
Detector
Pinhole
aperature
Figure 5 Principles of a confocal microscope. a) The pinhole aperture in
front of the detector effectively blocks reflections from out-of-focus planes
of the specimen. b) Adsorption/emission spectra and molecular structure of
Oregon Green 488 (www.invitrongen.com). c) Filter settings to match the
absorption and emission spectra of fluorescein; NFT = dichroic mirror, LP =
long pass filter.
The confocal microscope is diffraction limited, i.e. the size of the focused
laser spot is only limited by diffraction by the lenses in the microscope. The
resolution is dependent on the wavelength of the light and the numerical
aperture (NA) of the focusing objective. The lateral Rayleigh resolution of a
fluorescent sample, assuming the excitation and emission wavelengths are
equal, reads:
Rlateral ∝
λ
(12)
N .A.
When imaging an infinitely thin layer, the confocal microscope produces
an axial intensity function with a full-width-half-maximum (FWHM) that
can be approximated with eq. 5:
FWHM ∝
λ
(13)
(N .A.)2
29
Methods and Experimental Techniques
The depth resolution, or the optical slice thickness, is defined as the
FWHM and roughly taken as doubled of the lateral resolution. Thus, using a
wavelength λ = 500 nm, an objective with N.A. = 1.4 and pinhole size
corresponding to Airy Unit (AU) of one, the theoretically best resolution is ~
0.18 μm laterally and ~ 0.36 μm in depth. The dye-containing spheres were
optically sectioned into 5-20 equidistant slices separated by approximately 1
μm. By simple spherical geometrical consideration, the slice with the largest
diameter (nearly identical to the diameter of the sphere) is taken as the
equatorial slice. The position of the equatorial slice was verified every time
the spheres were imaged by the procedure described above.
a
a
b
c
d
Figure 6 CLSM optical section of spheres loaded with a) an anionic dye,
Oregon Green 488, (Fig. 1 Paper I) b) an cationic dye, Rhodamine 6G, c)
Texas Red®-tagged Dex3k (Fig. 1 Paper II, scale bar only valid for this
image) and d) Alexa Fluor 488-tagged enzyme (Fig. 3 Paper III). Images
were obtained after buffer exchange, the variation in slice size resulted from
height or size variations of the spheres at the optical plane where the image
was obtained.
30
Methods and Experimental Techniques
An inverted Axiovert 100 M microscope with a Zeiss LSM 5 Pascal
scanner was used together with a 488 nm argon laser (for Oregon Green 488,
Fig. 6a) or a 543 nm HeNe laser (for Rhodamine 6G, Fig. 6b), and a 40× /1.3
NA oil immersion objective lens to image the dye-containing mesoporous
spheres in Paper I. An Axiovert 200 inverted microscope with a Zeiss LSM
510 scanner was used together with a 543 nm HeNe laser and a 60 x /1.4 NA
oil immersion objective lens to image the Texas Red®-tagged dextrancontaining mesoporous spheres (Fig. 6c) in Paper II; and together with a 488
nm argon laser and a 60 x /1.4 NA oil immersion objective lens to image the
Alexa Fluor 488-tagged enzyme containing spheres (Fig. 6d) in Paper III.
4.3.2 Correcting and evaluating for possible fluorescent microscopy
artefacts
Quantitative fluorescent measurements require that the effects of light
attenuation, refractive index mismatch and dye quenching are insignificant
or corrected, and that the effect of photobleaching is quantified.
Photobleaching correction
The dye-loaded spheres are typically subjected to a number of scans at
the same position over the entire course of the release study. To quantify the
photobleaching of the dye-loaded spheres, they can be subjected to an
equivalent number of scans, without any time delay, as during the release
study. The photobleaching effect is evaluated by fitting the measured
intensity (as a function of the number of scans intensity) to a simple
exponential function,
I N = I 0 exp( − k * n)
(14)
where IN = corrected intensity, I0 = initial intensity (after the first scan), k
is the bleaching constant determined from the logarithm fit, and n = number
of scans. Figure 7 shows the change in fluorescence intensity as a function of
number of scans for Oregon green 488, Texas Red®-tagged dextrans and
Alex Fluor 488-tagged enzyme-containing mesoporous spheres. Rhodamine
6G is a highly photostable dye122 that displays minimal photobleaching
within the typical number of scans performed in this study and is therefore
not compensated for photobleaching.
31
Methods and Experimental Techniques
Light attenuation and refractive index mismatch
Normalized
intensity
Intensity (a.u)
Light extinction by the adsorbent matrix and photon re-adsorption by, e.g.,
dye or dye-conjugated molecules can result in variations in the detected light
intensity. Since the attenuation of light depends on the location of the
focused point, this may result in, e.g., lower intensity values in the inner
particle region.123
1
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
a
0
b
0.0
10
20
30
40
50
Number of scans
Number
of scans
1
0
50
100 150 200 250
Number
of of
scans
Nnumber
scans
300
0.8
0.6
0.4
0.2
c
0
0
20
40
60
Numberof
of scans
scans
Number
80
100
Figure 7 Bleaching profile mesoporous spheres containing a) Oregon Green
488 (Paper I), and Texas-Red tagged dextran b) with Mw = 3000 g/mol
(Paper II), and c) Alexa Fluor 488-tagged enzyme (Paper III). The
fluorescence intensities have been normalized.
The effect of light extinction was investigated by analyzing the
fluorescent intensity profiles of different optical sections of the dye
containing spheres, where the light path has to travel different distances
before it reaches the detector. We do not find any significant difference
between the top and bottom sections for Oregon Green 488 and Rhodamine
6G containing spheres (see details in supporting information of Paper I),
showing that the effect of light extinction is insignificant. The absence of
any significant axial aberration of the spherical sections is also a strong
32
Methods and Experimental Techniques
indication that any possible effects of the small refractive index mismatch
between the spheres and the medium on the loss of the fluorescent signal
should be insignificant. For the Texas Red®-tagged dextran-containing
spheres, however, the fluorescence intensity of the optical sections obtained
from the top section are higher than those from the bottom section (e.g., Fig.
8 for Dex3k), showing the effect of light attenuation. No observable axial
aberration of the spherical sections indicates that there is a negligible
refractive index mismatch between the spheres and the medium.
1
2
4
5
3
6
1
4
7
7
Figure 8 CLSM images of the optical sections of the Dex3k-containing
spheres (slices 1-3 are from above the centre slice and slices 5-7 are below
the centre slice) with a section thickness of 1 µm. A schematic representation
of the positions (represented by lines) where the sectioning was performed is
also included. (Supporting Information from Paper II)
33
Methods and Experimental Techniques
Susanto et al.124 introduced a short cut method based on the Lambert-Beer
equation for correcting light attenuation inside a spherical particle using the
coordinate system as shown in Scheme 7.
Laser
Top slice
d
R
0
x
Bottom slice
Scheme 7 Coordinate system of a spherical mesoporous particle (Paper II).
Assuming a negligible concentration-dependent attenuation effect and a
homogeneous concentration distribution within the particle, a further
simplified approach can be taken to estimate the light attenuation correcting
factor (Cx) as a function of distance x from the centre of the sphere. Two
optical slices at the same distance from the above and below the central slice
of a particle were taken. Since the particle is a symmetrical sphere, the
diameter of the taken slices should be similar. Fig. 9a shows the typical
intraparticle fluorescence intensity profiles of these two slices for Dex3Kcontaining spheres. The difference in fluorescence intensity at a certain point
on the top (ITx) and the same point on the bottom slice (IBx) can be used to
calculate the ratio 〈log(ITx) – log(IBx)〉. Using the coordinate system
illustrated in Scheme 4, the maximum light attenuation correction factor
〈Cmax〉 can be verified for consistency (Fig. 9b) and evaluated by
Cmax = log(I Tx ) − log(I Bx ) ⋅
2R
R2 − x2
(15)
where ITx and IBx is the fluorescence intensity of the top slice and bottom
slice, as a function of distance x from the equatorial slice, respectively. The
correcting factor at each distance x from the centre by evaluating:
C x = C max
R2 − x2
R
(16)
34
Methods and Experimental Techniques
At the centre of the slice, x = 0, Cx = Cmax, indicating a maximum value of
the light attenuation correction factor. Similarly, there is no correction at the
edge of the slice, where x = R, Cx = 0.
The intraparticle fluorescence distribution at each time point, IC,
corrected for both photobleaching and light attenuation is determined
according to the following equation:
IC = IN * (1+Cx)
(17)
where IN is the measured fluorescence intensity corrected for
photobleaching after n number of scans.
1.000
a
b
0.800
150
0.600
Cmax
Fluorescence Intensity
200
100
Top slice
0.400
0.200
Bottom slice
50
0.000
0
2
3
5
6
8
-0.200
0
0
2
3
5
6
8
-0.400
Distance from slice center (μm)
Distance from slice center (μm)
Figure 9 Intraparticle concentration profiles of the dextran loaded-sphere
and its corresponding correcting factor. a) The intraparticle fluorescence
profiles of the top and bottom optical slice of the Dex3k-loaded revealing the
higher fluorescence intensity at the slice edge compared to slice centre, and b)
the respective calculated 〈Cmax〉 calculated using eq. 18 (Paper II).
35
36
Summary of Key Results and Discussions
5. Summary of Key Results and Discussions
5.1 The effect of guest molecule-host pore wall electrostatic
interactions on molecular transport
Confocal laser scanning microscopy (CLSM) was used to image the
spatial distribution of a cationic fluorescent dye, Rhodamine 6G (Rh6G,
cationic, red) and an anionic Oregon Green 488 (OG, anionic, green) loaded
in monodisperse mesoporous silica particles. The distribution of the anionic
OG dye (Fig. 10a) within the mesoporous spheres demonstrates the
accessibility of the entire volume of the mesopores with no evidence of a
hollow core or any unavailable or blocked region. The anionic OG dye has a
higher dye concentration at the centre than near the edge of the equatorial
slice (Fig. 10b). This Gaussian-like intraparticle concentration profile of the
OG dye is typical of a diffusion controlled release.
a
Intensity (a.u.)
c
b
d
Distance from center of slice (µm)
Figure 10 Time evolution of the release of dyes from the mesoporous
spheres. CLSM optical equatorial slice at different times for the a) OG, and
the c) Rh6G-containing spheres. The respective intraparticle intensities (c
and d) decrease with time as indicated by the arrows. The intensity data are
not compensated for photobleaching (extracted from Fig. 2 and 3 of Paper I).
37
Summary of Key Results and Discussions
In contrast, the cationic Rh6G dye has a higher fluorescent intensity near
the slice’s edge (Fig. 10d) compared to the centre of the slice. It is important
to note that the inhomogeneous intraparticle profile of Rh6G is not due to
dye quenching. Dye quenching would have resulted in a different evolution
of the fluorescent intensity profiles as the release proceeds i.e. the low
fluorescent intensity in the centre of the sphere should recover to higher
values during release. Instead, the low fluorescence intensity in the middle of
the slice is retained at all time intervals.
The total amount of dye within the observed slice (at the equator of the
sphere) can be obtained by integration of the fluorescence intensity over the
entire measurement area (or more accurately, thin slice). The release of the
fluorescent dyes can thus be estimated by integrating the intensities of slices
obtained at different release times. This information is similar to the data that
can be obtained from traditional bulk release studies, where the change in
solute concentration of the liquid media is followed as a function of time.
Figure 11 present the release data for fifteen different spheres loaded with
the fluorescent dyes. The spread in the data can be due to spread in the
starting time, but also to variations in, e.g., the pore volume and the pore
structure of the individual spheres. The similarity in the decay of the
fluorescence intensity with time for different spheres suggests that they
exhibit identical release mechanism(s) despite the possible structural
variation among the spheres. We have minimized sum of the composite
squared deviation of the data and a diffusion model based on Fick’s second
law defined by90
∂C ( x, t )
∂ 2 C ( x, t )
=D
∂x 2
∂t
By modifying the spherical model used to describe adsorbate uptake in an
isothermal spherical particles,90, 91 we can calculate the effective diffusion
coefficient, Deff, of the molecular release from our spheres using the
following expression,
− n 2π 2 Deff t
It
6 ∞ 1
=
∑ exp( R 2 )
I0 π 2 1 n2
(18)
The expression can be simplified by analyzing the asymptotes at short or
long times only. The long time asymptote estimation used in this thesis for
calculating release rate is
38
Summary of Key Results and Discussions
− π 2 Deff (t + t 0 )
It
6
)
= 2 exp(
I0 π
R2
(19)
where Deff is the effective diffusion coefficient, I0 is the initial intensity, It
is the measured intensity, t is the experimental time, t0 is the time shift, and R
is the radius of the mesoporous sphere studied.125
1
1
Period 1
Fluorescence Intensity
a
ty
i
s
n
e
t
n
i
e
c
n
e
c
s
e
r
o
lu
F
0.8
0.6
0.4
0.2
0
0
1
2
3
4
5
Period 2
0.6
0.4
0.2
6
0
0
1
2
3
ty
i
s
n
e
t
n
i
e
c
n
e
c
s
e
r
o
lu
F
Period 1
Period 2
5
6
7
d
c
0.8
CENTER
EDGE
_
Edge
0.4
Average
_
_
_
_
BULK
+ + + + + + +
+
+
(sink)
+
0.6
0.2
4
t (h)
τ, h
τ, h
1
b
0.8
De
_
Near edge
_
_
_
+
+
de
+ + + + + + + +
Center
_
+
+
_
+
Dc dc +
_
_
_
Center
0
0
1
2
3
τ, h
4
5
6
7
Figure 11 Release profiles of fluorescent dyes from mesoporous silica as a
function of observation time, τ (data compensated for photobleaching).
Normalized release profiles of a) OG and b) Rh6G-containing mesoporous
spheres. Fifteen different particles were followed individually up to 15 time
steps (1 time step = 30 mins). The mean values of the normalized intensity at
each observation time (represented by filled boxes) in Period 2 were fitted
using an exponential curve (black line). c) Release curve of Rh6G from the
centre zone and the zone near the edge of the optical equatorial slices are
plotted together with global release curve. The dotted lines are fits of the
experimental data to the spherical diffusion model. The insert illustrates the
two zones. d) Schematic model describing the molecular transport of the
cationic dye within a negatively charged cylindrical mesopore. Dc and De are
the effective diffusion coefficients and dc and de are the effective pore
diameters in the core and surface zone (extracted from Fig. 5, 7 and 8 of
Paper I).
39
Summary of Key Results and Discussions
The effective diffusion coefficient of OG in the mesoporous sphere,
DeffOG = 0.62 ± 0.12 x 10-12 cm2/s, is more than 6 orders of magnitude lower
than the reported bulk diffusion coefficient in Tris-EDTA buffer126 (DOG =
1.3 × 10−6 cm2/s). The release profile for the cationic Rh6G-containing
spheres (Figure 11b) cannot be fitted to a simple exponential function. Two
different release periods are identified; Period 1 characterized by a relatively
rapid release that increases with time, and Period 2, which commenced after
approximately 2.5 h that is characterized by a relatively slow release. Fitting
the release curve during Period 2 with the simple exponential function yield
an effective diffusion coefficient of DeffRh6G = 0.24 ± 0.04 x 10-12 cm2/s. This
value is 7 orders of magnitude lower than the reported bulk diffusion
coefficient of the same molecule in water127 (DRh6G = 2.8 × 10−6 cm2/s).
Whereas previous works have shown how empirical factors can be added
to Fickian expressions to accommodate for guest molecule−host surface
interactions,32, 39 such approaches cannot describe the observed release
behaviour of the Rh6G dye from the mesoporous particles. Taking into
consideration the initial inhomogeneous distribution of the Rh6G dye within
the mesoporous silica spheres (Fig. 10c and d), the molecular transport of the
cationic dye within the mesoporous spheres was analyzed. The timedependent behaviours in the zone near the edge and at the centre of the slice
mimic the bulk release profile (Fig. 11c). The initial release burst that we
observed has also been found in previous release studies on mesoporous
materials.30-32 However, the observed increase of the release rate toward the
later part of Period 1 suggests that a second process in addition to diffusion
of initially non-adsorbed dye molecules also becomes important.
A plausible explanation is that the diffusion of the free (non-adsorbed)
dye is complemented with a delayed desorption process of some adsorbed
dye. It appears that the rate of desorption increases with time up to the end of
Period 1. At longer time, identified as Period 2, the drastic drop in the
release rate suggests that the desorption process becomes less pronounced,
which is supported by the good fit of the release curve during Period 2 to a
simple diffusion equation. However, although the molecular transport both
in the edge and the centre zone of the slice is dominated by diffusion during
Period 2, the effective diffusion coefficient in the edge zone (0.20 x 10-12
cm2/s) is almost 3 times slower than in the centre zone (0.73 x 10-12 cm2/s).
The global diffusion (DeffRh6G = 0.24 ± 0.04 x 10-12 cm2/s) is close to the
diffusion constant in the edge zone, which suggests that the permanently
40
Summary of Key Results and Discussions
adsorbed cationic dye molecules act as a semi-permeable ‘skin’,
schematically illustrated in Fig. 11d. In the edge zone, the effective pore
diameter (de) is probably reduced by the molecules permanently adsorbed
onto the pore walls that slow down the diffusion across this zone (self ratelimiting).
5.2 Intraparticle transport and release of dextran in cylindrical
mesopores
The investigation of the spatio-temporal dependencies for the molecular
transport of macromolecules inside the mesoporous silica particles is
described in this section (Paper II). Fluorescently tagged-dextran with
molecular weight (3000 g/mol, Dex3k) that gives hydrodynamic radii similar
to the diameter of the cylindrical pores (6.5 nm) of the ESE mesoporous
particles has been used. The transport of the molecules within the spheres
was observed over eighteen hours using the variation in the intensities of the
CLSM images. The time evolution of the fluorescence within the dextranloaded ESE particles (Fig. 12) shows that the dextran has penetrated to the
core of the spheres. No evidence of a hollow core or any unavailable or
blocked region has been observed.
Figures 13 present the release data for five different spheres loaded with
Dex3k. The spread in the data can be related to the inevitable spread in the
starting time but also to variations in, e.g., the pore volume and the pore
structure between the individual spheres. We have minimized sum of the
composite squared deviation of the data and fitted the curve to a diffusion
model based on Ficks second law 100 defined by eq. 19. This simplified
analysis result in apparent hindered diffusion coefficients of Dex3k (DeffDex3k
= 2.98 x 10-12 cm2/s) from the ESE spheres are at least 6 orders of magnitude
lower then their bulk diffusion coefficients in free solution (DDex3k = 23 x 107
cm2/s).
However, relating the fluorescence information only to an exponential
model for the overall release of the molecules does not give much
information on the mechanisms of the molecular transport. The information
obtained by CLSM allows us to quantify both the time-dependent
concentration variations inside the porous carrier and to follow the bulk
release.
41
Summary of Key Results and Discussions
Figure 12 CLSM images of the optical sections at the equator of the sphere
at different time. Image series showing decreasing fluorescence intensity
with release time: Note: These intensity data have not been compensated for
photobleaching. The black spots are evaporation holes.119
Fluorescence Intensity
1
P1
P2
P3
P4
P5
Mean
Exp(-0.071*t-0.162)
0.8
0.6
0.4
0.2
0
2
4
6
8
10
12
14
16
18
τ(h)
Figure 13 Normalized integrated fluorescence intensity (photobleaching
compensated) as a function of observation time, τ, for the dextran-containing
mesoporous spheres. Five different particles were followed individually at
18 time steps. The mean values of the normalized intensity at each
observation times (represented by filled boxes) were fitted using an
exponential curve (black line) (Fig. 3 of Paper II).
Quantification requires that intensity data be compensated for the effects
of light attenuation, which depends on the optical path length. We have
corrected for light attenuation by using the simplified short-cut method
proposed by Susanto et al.124 This method is based on the Lambert-Beer
42
Summary of Key Results and Discussions
equation for the intensity loss along the excitation path. After appropriate
corrections, we obtain concentration profiles inside the ESE spheres that
essentially do not vary with the distance to the external surface. The flat
concentration profile in the particles that is preserved at all times (Fig. 14)
strongly indicates that the release of dextran from the ESE spheres cannot be
well described by a Fickian-type model (characterised by Gaussian-shaped
intraparticle concentration profiles). Instead, the flat intraparticle
concentration profile suggests that the underpinning diffusion is not fully
isotropic and the release is controlled by the pores close to the external
surface of the spheres. Recent findings about on how the pores are organized
close to the external surface of mesoporous crystals.128 has shown that many
pore channels bend back into the particle (Scheme 8), leaving only a few
channels having pore mouths facing the external solution. This structural
feature that forces the dextran to traverse back into the sphere at the surface
can explain the flat concentration profile inside the particles. Since the
numbers of entrances/exits are few, the dextran molecules need to traverse
back into the sphere until it can finally reach an exit into the media.
Normalized intensity
b
1hr
4hr
7hr
10hr
13hr
17hr
Distance (μm)
Figure 14 Intraparticle intensity profile (centre to edge of sphere) as a
function of time, extracted from the sphere equatorial slices of dextrancontaining ESE sphere. Note: the intensity has been normalized, and
corrected for both photobleaching and light attenuation (Fig. 3 of Paper II).
Further analyses of the integrated fluorescent intensities revealed that the
release of the dextran molecules exhibits an initial burst, which was followed
by a truly sustained release (Fig. 15a). The release behavior is neither
exponential nor biexponential in nature. In fact the release kinetics conforms
43
Summary of Key Results and Discussions
well to a logarithmic time dependency (Fig. 15b). Logarithmic rate laws are
often observed for chemisorption and formation of, e.g., oxide films on
metals,129 and Landsberg proposed a general form of the rate law (for
adsorption) by relating the quantity of molecules adsorbed per unit time per
unit area (eq. 20)
dq
= CasN
dt
(20)
to the number of sites becoming unavailable per unit time per unit area
(eq. 21)
−
ds
= CNasbs
dt
(21)
where q is the number of molecules sticking to the surface, s is the site
density, a is the effective contact area, which is the approximated molecule
cross sectional area, C is a coefficient constant, N is number of impacts by
the molecules with the surface per unit area per unit time, b is the effective
surface area, which becomes unavailable (invalidated) due to the adsorption
by a single molecule, t0 = 1/(CNabs0), and s0 is the sites per unit area at t = 0
(experimental time zero). We reformulate this description for the case of
release (or desorption) by simply combining eq. 20 and 21, and integrating
dq/dt from q to 0, instead of 0 to q,
∫
0
q
dt
, which becomes
0 1 + CNabs t
0
dq = CNas 0 ⋅∫
t
1 ⎛
t ⎞
q ' = − ln ⎜⎜1 + ⎟⎟
b ⎝ t 00 ⎠
(22)
where q´ is the quantity of molecules that remains in the carrier, b is a
constant and t00 = 1/(CNabs0), which can be regarded as the characteristic
time of the molecule in this system (this theory is not physical in the
asymptote of very long times; however, that is not needed, at all, for our
observation times). For t >> t00 we rewrite eq. 22 to
1 ⎛ t ⎞
q* = q0 − ln⎜⎜ ⎟⎟
b ⎝ t00 ⎠
(23)
where q* may be expressed as the time-dependent concentration. Indeed,
this equation can be deduced from, and is analogous to the Temkin model.130
Please, note that both the burst and sustained release observed in Figure 15a
44
Summary of Key Results and Discussions
are natural consequences of a logarithmic time dependency. Physically, the
logarithmic dependency can be rationalized by coupling a Temkin model
with a typical release model. In fact, Johnson and Arnold have used similar a
equation based on the Temkin model to quantify reversible proteins
adsorption onto a non-uniform surface.131 They showed an increase in the
binding sites between the protein and the surface increases the apparent
binding affinities and maximum capacities i.e. increasing binding strength.
Normalized intensity
a
Time (hr)
Normalized intensity
b
log Time (hr)
Figure 15 Release profiles of Dex3k-containing sphere as a function of a)
observation time (h), and as a function of b) logarithmic time plot. (Fig. 5
Paper II).
The adsorption (energy) of macromolecules commonly displays a
variation with surface coverage.132 It is commonly found that the segment
adsorption energy decrease with increasing surface coverage. 131 Previous
work has also shown that flexible macromolecules such as dextran may
transform from a flatter confirmation to a conformation having a significant
fraction of the segments extending into the solution.117
45
Summary of Key Results and Discussions
Centre of slice
Pore channel bending
back into the sphere
Bulk
(sink)
2
6.5
nm
1
Pore channel
opening
Edge of slice
Scheme 8 Sketch showing dextran molecules within the mesoporous pores. a)
Dextran molecules traversing along liquid-filled pore channel exiting the
sphere surface, Path 1, or turning back into the sphere, Path 2 (Scheme 1 of
Paper II).
5.3 Catalytic activity and stability of TLL lipase immobilized in
functionalized mesoporous silica
The effect of hydrophobic interaction between immobilized lipase (from
the fungus Thermomyces lanuginosus, TLL), and the carrier’s pore wall have
been investigated. Two types of carrier materials were investigated; the
calcined ESE-produced mesoporous silica spheres with hydrophilic pore
walls, and the same material which has been silylated to obtain hydrophobic
pore walls (see section 4.1.2 and Paper III). The confocal images of the
hydrophilic (Fig. 16a) and hydrophobic (Fig. 16b) materials illustrate that
the Alex Fluor 488-tagged lipase has completely penetrated into the core of
the material. The relative leaching rate of the lipase was quantified from the
decrease in its fluorescence intensity with time (Fig. 16c). The specific
enzymatic activity (EU/mass lipase) of the immobilized lipase using 4nitrophenyl acetate as a reactant was evaluated according to the MichaelisMenten model
k
⎯
⎯→
1
E+S
←
⎯⎯
k
(24)
k
ES ⎯⎯→
E+P
3
2
where an enzyme, E, combines with a substrate, S, to form an ES
complex at a rate expressed by the constant k1. The reaction rate, V, is
defined as the rate of the production of the product, P, in moles per minute
per volume. V is dependent on the conversion rate constant, k3, which is
46
Summary of Key Results and Discussions
often referred to as kcat or the turnover number. The turnover number is
defined as the maximum number of molecules of the substrate that an
enzyme can convert to a product per catalytic site per unit of time. The
decrease in the enzymatic activity was measured as a function of number of
experiments (Fig. 16d).
Pore with hydrophobic wall
Alexa Fluor 488
TLL
Interfacial activation
of active site
Closed
active site
Scheme 9 Carton showing how the Alexa Fluor 488-tagged TLL enzyme
can be surface activated on a hydrophobic pore wall. The hydrophobic lid is
opened to allow access to the catalytic active site.
The CLSM results show that there is a decrease in florescent intensity as
a function of time, indicating that some of the immobilized lipase have
leaked out during the first two hours after transfer of the loaded material to a
pure electrolyte solution. However, the leakage was insignificant after this
initial release period and the total amount that remains immobilized after
four hours is relatively high. Comparing the release profiles for the two
carrier materials indicate that the hydrophilic material displays a higher
release rate and a larger total amount of lipase released compared to the
hydrophobic material. In agreement with the CLSM data, a more
pronounced decrease in activity for the hydrophilic material compared to the
hydrophobic material was observed. It is interesting to note that there is a
significant difference in the decrease in lipase concentration (between 4 and
9%), received from CLSM analysis and the decrease in enzymatic activity
(between 55 and 70%). The large discrepancy in the amount of lipase
released and the decrease in enzymatic activity indicate that denaturing of
the enzyme occurs in parallel with the physical release of lipase from the
mesoporous materials.
47
Summary of Key Results and Discussions
b)
c)
d
100
N o rm alized A ctivity (% )
Normalized Fluorescence Intensity
a)
80
1.0
0.9
0.8
60
40
Hydrophilic
Hydrophobic
20
0
0.7
0
1
2
3
4
τ (hr)
0
1
2
3
4
5
Number of experiments
Figure 16 CLSM images of the fluorescent tagged-TLL lipase loaded
spheres, and comparison of the lipase leaching with enzymatic activity. The
a) as-calcined spheres, and b) spheres with hydrophobic pore walls both
show c) leaching of the immobilized fluorescence-tagged lipase with time in
MOPS buffer (compensated for photobleaching). Their respective d)
enzymatic activity decreased after consecutive runs using 4-nitrophenyl
acetate as a reactant (Fig. 3 of Paper III).
Most of the lipases have their active site shielded by an oligopeptide
chain referred to as the ‘lid’. These lipases undergo so called interfacial
activation that relates to a dramatic increase in activity of the lipases at an
interface (Scheme 9). The higher specific activity and kcat measured for the
biocatalyst when it is immobilized onto a hydrophobic surface, corroborates
previous suggestions that a lipase of the Thermomyces lanuginosus type
have a more stable conformation onto a hydrophobic surface.12, 30, 116, 133
48
Summary of Key Results and Discussions
Note should be taken that the degree of hydrophobicity can influence the
enzymatic activity. Wannerberger et al. found that the enzymatic activity of
lipase from Humicola lanuginosus, equivalent to T. lanuginosus lipase, was
higher onto partly methylated silica surfaces compared to fully methylated
surfaces.134 Future work should thus involve an optimization of the pore size,
particle size and degree of hydrophobicity to increase the enzymatic activity
of the immobilized lipase, as well as the loading capacity of the support.
5.4 Colloidal spheres with ordered mesopores’ synthesis,
characterization, and their use as a solid supports for a bilayer
lipid membrane
Colloidal monodisperse mesoporous silica spheres have been synthesized,
via a modified Stöber method, to function as a solid support, with connected
interior cytoskeleton-like pore structure, for a bilayer lipid membrane. The
XRD spectrum and nitrogen isotherm results show that these spheres exhibit
pores with a 2D hexagonal mesostructure (p6mm) (see details in section
4.1.2 and Paper IV).
5.4.1 Mesoporous structure within the colloidal mesoporous spheres
The spatial pore arrangement was evaluated as a function of distance
from the particle’s core by analyzing thin microtome slices (40 - 60 nm thick)
taken from the powders at different distances from the core of the sphere
(Scheme 10).
iii
ii
i
SiO2 sphere
Scheme 10 Illustration showing locations where the microtome slices were
obtained from the mesostructured sphere. Position i, ii and iii, are situated at
0.13-0.16 μm (near the sphere core), 0.22-0.24 μm (between the core and the
surface) and 0.53-0.54 μm (close to the particle surface) from the sphere
core, respectively (Fig. 4 of Paper IV).
49
Summary of Key Results and Discussions
a
A
•
⊗
B
3.3 nm
b
2
3.3 nm
d
3
1
e
c
3.3 nm
f
Figure 17 TEM images of thin microtome slices of the as-synthesized
spheres cross sectioned at a) position i, b) ii and c) iii in Scheme 10, and the
corresponding FFTs. Their respective schematic illustrations of d) position i
shows (region A) domains of hexagonally arranged cylindrical channels
bending close to the particle core directed away from the page (•), and
(Region B) domains of pores crossing each other and bending towards the
particles surface into the page (⊗); of e) position 2 showing regions where
pores are; 1) in direction of, 2) at an angle with, and 3) perpendicular to the
electron beam; and f) position iii at near sphere surface where the mesopores
are perpendicular to the electron beam (extracted from Fig. 5, 6 and 7 from
Paper IV).
50
Summary of Key Results and Discussions
The slice taken at position i (near the core) of the as-synthesized powder
displays distinct domains of pore channels that extend towards the surface of
the particles (Fig. 17a). This type of radially extending pore channels have
also been found in previous studies.79, 81, 83, 135 The bright hexagonally
arranged spots at the centre of the slice are characteristic of pore channels
orientated in the direction of the electron beam.
The 6-fold symmetry of its corresponding Fourier Transform image (FFT)
at the slice centre indicates that the cylindrical pore channels have a
hexagonal order in the direction of the electron beam. Observations on
several slices obtained at different distances from the particle core suggest
that the number of the bright spots at each slice’s centre increases as we
move away from the core of the sphere when compared to the core slice.
The slice obtained at position ii has a much higher number of arrays of
bright spots at its centre than the core slice (Fig. 17b). The TEM image of
the slice taken at position iii contains mainly bright spots (Fig. 17c). This
increase in the number of bright spots, and thus pore channels oriented
towards the electron beam, as we move from the sphere core towards the
surface gives further support to the conjecture of radially aligned pores. The
diffuse scattering of the spots is likely due to the presence of domains with
pores at an angle to the electron beam, or imperfect ordering of the pores.
Some domains of the parallel pore channels appear to adopt a curved path
and some of the channels seem to be overlapping within the particles,
especially near the particle core. This indicates that the cylindrical pore
domains close to the core of the particle do not have a preferred direction.
The corresponding schematic of these slices (Fig. 17e-f) presents a simple
illustration of how the pore channels may be arranged. According to this
schematic model the core of the sphere should consist mainly of bent and
twisted bundles of hexagonally arranged cylindrical pore channels. This
corresponds well with the notion of a core structure of several randomly
orientated domains82 as we find no support for a core with an Ia3 d cubic
structure.80, 81 Simple geometrical considerations support a model where the
cylindrical domains bend and twist in the particle core to maximize the
domain density. Our observations of the pore channel structure may be
compared to the hydrophobic chain packing of surfactants within a (nonreversed) spherical micelle as described in Gruen’s structural model. 136 This
and related models showed, with support from geometric constraints of chain
packing in the core of a micelle that has a radius nearly equal to the length of
51
Summary of Key Results and Discussions
a fully extended surfactant molecule and spectroscopic measurements, that
some of the hydrophobic alkyl chains are folded and that a fraction of the
chains have to attain a less favourable conformation.
a
b
3.0 nm
3.0 nm
Figure 18 TEM images of thin microtome slices from the calcined particles
obtained at; a) near the particle core; and b) close to the particle surface. The
FFTs of these slices show a ring, which corresponds to a distance of 3.0 ±
0.1 nm between the centres of nearest neighbouring pores (Fig. 8 of Paper
IV).
The TEM images of thin microtome slices obtained from the calcined
particles (Fig. 18) show that the main feature of the radially extended
mesopores has been retained. The FFTs of the cross sections appear as a
bright ring indicating that the parallel pore channels are oriented
perpendicular to the beam in various directions. The radius of this ring is
about 3.0 ± 0.2 nm, consistent to the d10-spacing from its XRD spectrum.
The FFTs from the centre of the microtome slices do not display a distinct
hexagonal 6 fold geometry, which suggests that the thermally calcined
particles exhibit a mesostructure with a lower degree of order than the assynthesized particles, in correspondence with the analysis of the XRD and
nitrogen isotherm data.
5.4.2 Lipid bilayer membranes coating
The calcined colloidal mesoporous spheres were used as a solid support
for a bilayer lipid membrane containing a transmembrane protein (Paper V).
The formation mechanism of the supported proteolipid bilayer that surrounds
the silica particle has been investigated using optical techniques. A multisubunit, transmembrane complex "molecular machine", cytochrome c
52
Summary of Key Results and Discussions
oxidase (CytcO) was reconstituted into the small unilamellar vesicles and
these CytcO-containing vesicles were subsequently used to cover the
mesoporous silica particles.
a
c
b
Scheme 11 Schematic illustration of how small unilamellar CytcOcontaining vesicles cover the mesoporous silica sphere. Small unilamellar
vesicles (gold rings), with and without CytcO (coloured boxes), a)
approaches the silica sphere, b) disrupt and spread onto the sphere surface,
and finally c) covering the sphere with a bilayer lipid membrane containing
fully functional multi-subunit redox-driven CytcO proton pumps.
Results from the fluorescence measurement of the CytcO-containing
vesicles with fluorescein trapped within before (using a Fluorometer), and
after interaction with the silica particles (using Flow Cytometry). The drastic
drop in the fluorescence (Fig. 2 of Paper V), strongly indicates that the
vesicles break upon interaction with the particles (Scheme 11).
The turnover activity of the CytcO itself (reduction of O2 to water) was
measured to check if the membrane is continuous and defect-free (details
available in Paper V). The parameter "respiratory control ratio" (RCR) gives
a measure of the tightness of the lipid membrane. A RCR value greater than
1 indicates that the vesicles are proton tight. The CytcO-vesicles alone (Fig.
19a) and the CytcO-vesicles deposited on the particle surface (Fig. 19b) give
corresponding RCR value of 4.0 ± 0.3 and 2.7 ± 0.6 i.e. the entire particles
were covered with a membrane defining an interior that was fully isolated
from the exterior solution.
The results are consistent with other data showing that upon deposition of
membranes on silica surfaces containing nm-sized pores, the membrane is
able to span across the pore openings rather then penetrating into the pores137
(Scheme 11b). The TEM analysis showed that the pores are oriented
perpendicular to the external surface (Fig. 18); hence, it could be visualized
53
Summary of Key Results and Discussions
that the CytcO molecules may be resting onto the pore entrances. However,
this is unlikely since the pore diameter (30 Å) is smaller than the negativeside of the CytcO which has an estimated size of 40 x 80 Å. In addition, it
has also been shown that there is a water-filled space between a bilayer lipid
membrane and a (dense) silica surface that may be ~10 Å thick.47 We
speculate that the porous nature of the exterior surface of the mesoporous
spheres also could contribute to a thicker fluid film between the membrane
and the solid support because of the decrease in the attractive van der Waals
interaction between the lipids and the water-silica composite surface.
300
a
+CytcO
+ valinomycin
FCCP
200
100
[O2] μM
0
300
Vesicles
+CytcO
250
200
b
+ valinomycin
FCCP
Particles
0
100
200 300 400
Time (s)
Figure 19 The O2-consumption activity of a) CytcO vesicles b) and CytcOmembrane-covered particles before and after addition of ionophores
(valinomycin-FCCP). The ratio of the activities with and without ionophores
is the respiratory-control ratio (RCR), which is a measure of proton leaks
across the membrane.
In this context we note that CytcO turnover requires proton uptake from
the negative side of the membrane, which requires a water-filled space on
the inside of the membrane.138 In summary, the results described above show
that the mesoporous silica particles were entirely covered by defect-free
membranes containing the multi-subunit, redox-driven proton pump, CytcO.
The incorporated enzymes remained fully active, both with respect to
54
Summary of Key Results and Discussions
catalysis of the reduction of O2 to H2O and transmembrane charge separation.
The use of mesoporous silica particles may be beneficial to obtain supported
lipid bilayers with a large inner space that is sufficient to provide a proton
source and also connect the water space around the CytcO negative side
surface with the entire volume of the particle through the pores.
55
56
Conclusions
6. Conclusions
Transport kinetics within mesoporous materials have been the topic of
this thesis. The work has also been extended to utilize our synthesized and
well characterized mesoporous spheres for biotechnological applications.
The most significant contributions here are the development of a facile and
direct protocol to investigate the intraparticle molecular transport using
CLSM. The possibility to relate the bulk release to the local molecular
transport within the mesopores provides an important step toward the design
of new concepts in controlled drug delivery and chromatography using this
class of materials.
6.1 Intraparticle molecular transport studies using CLSM
We have established a protocol to utilize the confocal laser scanning
microscopy (CLSM) to evaluate the spatial distribution of two small charged
dyes (in aerosol-generated mesoporous particles), and a macromolecule (in
mesoporous particles produced by a novel ESE method) over extended
periods of time.
The time-dependent release of the anionic dye molecules from the silica
spheres is describable by a simple model, based on Fick’s second law of
diffusion, with a single diffusion constant. A significant fraction of the
cationic dye permanently adsorbed onto the negatively charged silica pore
walls, predominantly near the surface of the spheres. The semi-permeable
‘skin’ consequently formed resulted in a delayed release of the cationic dye
molecules. The self-rate-limiting mechanism imposed by the skin effect
could be further exploited for controlled release applications.
The flat intraparticle concentration profile of the macromolecule (dextran)
-containing ESE spheres was We relate such flat profiles to the complex
pore structure established for these ESE particles and similar 2D hexagonal
mesoporous crystals. The surface is featured with bundles of hexagonally
arranged pore channels that bend back into the spherical particle without
allowing a fast molecular release. This large reduction of the particles’
effective pore area in contact with the external solution explains (at least
partly) the slow release observed. A logarithmic time dependency was
observed for the release of fluorescently-tagged dextran molecules. A simple
model related both the initial burst and the sustained release to variations in
57
Conclusions
the adsorption energy at different coverage of the dextran molecules on the
internal pore walls.
6.2 Mesoporous silica spheres as supports for lipid bilayers and
TLL enzyme
A promising candidate for biocatalyst applications was developed
through hydrophobic functionalization of ESE produced mesoporous spheres.
We have utilized the CLSM protocol established to visualize the spatial
distribution of the fluorescently-tagged lipase, from the fungus Thermomyces
lanuginosus (TLL), immobilized within these spheres. Quantification of the
CLSM image series showed that lipase leakage from the mesoporous
particles was limited to an initial period of only a few hours. Both the rate
and the amount of lipase leached were reduced when the lipase was
immobilized onto the hydrophobic support. The enhanced enzymatic activity
of the lipase immobilized onto the hydrophobic support, compared to those
onto the hydrophilic support and lipase free in solution was attributed to
interfacial activation of the lipase and reduced denaturating of the lipase.
Colloidal monodisperse mesoporous silica particles have been
synthesized following a modified Stöber method. TEM analysis of thin
microtome slices of these particles revealed bundles of the hexagonally
ordered cylindrical channels extending radially to the sphere surface. These
spheres were successfully covered by a bilayer lipid membrane incorporated
with a multi-subunit, redox driven proton pump. The protein-membrane
system was proton tight, defining an interior particle compartment that is
separated from the surrounding water solution. The incorporated proteins
have been shown to remain functional. This supramolecular architecture
with an inner pore structure mimicking a cytoskeleton yields a solidsupported biofunctional cellular surface. Such surface can provide a basis for
functional studies of membrane-bound transport proteins, and also for
applications within pharmaceutical drug delivery.
58
Outlooks
7. Outlooks
The main aim of this project was to harness the spherical mesoporous
silica spheres for the transport studies of molecules within the mesoporous
materials using CLSM. Since there are only a limited number of studies on
intraparticle molecular distribution and hindered transport on the
mesoporous material, the work covered within this study can serve to trigger
ideas for further investigations.
i) It would be interesting to investigate the up-take of the cationic dye by
the negatively charged mesoporous silica at low dye concentration using
CLSM. This would provide information on the initial distribution of the
cationic dye, and if the up-take is also limited by the semi-permeable ‘skin’
form by permanently adsorbed dyes.
ii) The flat intraparticle profile of dextran in the ESE-produced
mesoporous spheres was related to the particle’s structural features. It would
be interesting to perform controlled etching of the spheres. This would allow
the verification of the effect of the particle’s structural feature on the
intraparticle concentration profile, and its influence on the dextran release
kinetics.
iii) It could also be interesting to investigate the release of small charged
OG and Rh6G dyes from the ESE-produced spheres and compare the results
with those from the neutral dextran.
iv) Applying coatings of polyelectrolyte onto colloidal mesoporous silica
particles increases the fluid space between the encapsulating lipid membrane
and the particles. This could allow incorporation of large transmembrane
proteins without impingement problems
.
59
60
Acknowledgements
Acknowledgements
This work was supported by the Swedish Science Council (VR) and by
the Foundation for Strategic Research (SSF) through the Centre for
Biomembrane Research (CBR). The I-Centre CODIRECT, the Centre for
Controlled Delivery and Release, also supported the work. The Wallenberg
Foundation is acknowledged for supporting the department to acquire
electron microscopes and a CLSM.
I extend my deepest gratitude towards my PhD supervisor Professor
Lennart Bergström for all the guidance and training during my PhD study.
Your critical but encouraging approach has made me realize my potentials,
beyond what I have perceived. Thank you for not hesitating to point out my
stubbornness when it becomes more of a hindrance than help. Most of all,
thank you for giving me the chance to pursue my doctorate study at the
Department of Physical, Inorganic and Structural Chemistry, Stockholm
University of Sweden.
Dr. Niklas Hedin (Stockholm University) is gratefully acknowledged for
good collaboration and much fruitful discussions. You have taught me much
more than you realized. Professor Hjalmar Brismar and Padideh KamaliZare from KTH, Alba Nova; Prof. Peter Brzezinski and Gustav Nordlund
from Stockholm University, and Dr. Peter Alberius and Dr. Malin Sörensen
from YKI, are thanked for good collaboration work. Acknowledgements are
to Dr. Yasuhiro Sakamoto for sharing his expertise on the TEM, Dr. Kjell
Jansson for sharing his expertise in SEM, Dr. Kazuhisa Yano (Toyota
Central Research & Development Laboratory) for sharing his synthesis
experiences on the colloidal mesoporous silica spheres, and to Dr. Peter
Oleynikov for extending his expertise in Mathcad.
Thank you to my mother, who has been a role model of inner strength
and optimism. To my father who showed me persistency. A sincere gratitude
to my brother, Ng Boon Hong, who has supported most of my education,
without him I would never have made it this far. A big thank you to my
sister, Louis Ng Boon Hiong, who has being supportive and for taking good
care of me. A warm thank you to my sister-in-law, Lily, and my brother-inlaw, Wei Weng, who have being kind to me.
I want to thank Peter Oleynikov, for being supportive and helpful during
the writing of this thesis. Your patience and calmness have helped me to
pass through this tough period when I was at the verge of nervous
breakdown. I am grateful that fate has brought us together in the most
unexpected way. I treasure all the sweet little things you have done to make
me smile, and all the moments that you have made me laugh.
Special thanks to Dr. Anders Jarfors, for introducing me to Lennart and
thus triggered my whole study journey in Sweden. I would also like to thank
him and his wife, Anna-Lena, and his two sons, Michael and Björn, for
61
Acknowledgements
receiving me in the most heart-warming manner and helping me to settle
down when I first arrived in Sweden in 2005.
I would like to give a very warm thank you to Ingrid Mary-Anne
Westerdahl, who has housed me during my first two years in Sweden. Your
kindness has lifted my heart, and your energetic and optimistic personality
has greatly inspired me. And thank you for teaching me how to cook
Swedish dishes and to bake!
My deep gratitude is towards Prof. Sven Lidin for all the great advices,
both for life and for work. Thank you for being who you are. And thank you
for helping me in so many ways, both in your words and in your actions.
A big thank you to Linnéa Andersson, both a dear friend and a colleague,
for all the fun moments shared, the encouraging and funny talks, fika etc.
Many thanks are to Padideh Kamali-Zare, also a very dear friend, for lending
a helpful hand during needy times, and for enduring my ever ongoing
complaints. Both of you have made my life bearable by lending a listening
ear. Heat warming gratitude is towards Piao Shuying, Baroz Aziz, Keiichi
Miyasaka, Zhang Daliang and Ehsan Jalilian. Your friendships have made
my daily life at the FOOS full of fun and laughter.
Also big thanks to Ann-Britt Rönnell and Eva Pettersson who have been
very helpful with the paper work.
Last but not least thank you to my friends all over the world, Joop Peng,
Nicolas Chechen, Wong May Leng, Koh Chee Keat, Soh Chai Leng, Koh
Boon Kiat, Mustafa, Kiew Chun Meng, Melody Chen and Anna Kwok, for
the moral support all these years, and for treating me lunches and dinners
since I am the only ‘poor student’ among all of you.
62
References
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
Yanagisawa, T.; Shimizu, T.; Kuroda, K.; Kato, C.; Bull. Chem. Soc.
Jpn 1990, 63, 988-992.
Beck, J. S.; Vartuli, J. C.; Roth, W. J.; Leonowicz, M. E.; Kresge, C.
T.; Schmitt, K. D.; Chu, C. T. W.; Olson, D. H.; Sheppard, E. W.;
McCullen, S. B.; Higgins, J. B.; Schlenker, J. L.; J. Am. Chem. Soc.
1992, 114, 10834-10843.
Kresge, C. T.; Leonowicz, M. E.; Roth, W. J.; Vartuli, J. C.; Beck, J.
S.; Nature 1992, 359, 710-712.
Lu, Y.; Fan, H.; Stump, A.; Ward, T. L.; Rieker, T.; Brinker, C. J.;
Nature 1999, 398, 223-226.
Rao, G. V. R.; López, G. P.; Bravo, J.; Pham, H.; Datye, A. K.; Xu, H.
F.; Ward, T. L.; Adv. Mater. 2002, 14, 1301-1304.
Yang, H.; Coombs, N.; Ozin, G. A.; Nature 1997, 386, 692-695.
Rathousky, J.; Zukalova, M.; Zukala, A.; Had, J.; Collect. Czech.
Chem. Commun. 1998, 63, 1893-1906.
Che, S.; Li, H.; Lim, S.; Sakamoto, Y.; Terasaki, O.; Tatsumi, T.;
Chem. Mater. 2005, 17, 4103-4113.
Ma, Y.; Qi, L.; Ma, J.; Wu, Y.; Liu, O.; Cheng, H.; Colloids Surf., A
2003, 229, 1-8.
Katiyar, A.; Yadav, S.; Smirniotis, P. G.; Pinto, N. G.; J. Chromatogr.
A 2006, 1122, 13-20.
Díaz, J. F.; Balkus, K. J.; J. Mol. Catal. B-Enzym. 1996, 2, 115-126.
Reis, P.; Witula, T.; Holmberg, K.; Microporous Mesoporous Mater.
2008, 110, 355-362.
Lin, V. S.-Y.; Lai, C.-Y.; Huang, J.; Song, S.-A.; Xu, S.; J. Am. Chem.
Soc. (Commun) 2001, 123, 11510 - 11511.
Vallet-Regi, M.; Ramila, A.; del Real, R. P.; Perez-Pariente, J.; Chem.
Mater. 2001, 13, 308-311.
Zhu, Y.-f.; Shi, J.-l.; Li, Y.-s.; Chen, H.-r.; Shen, W.-h.; Dong, X.-p.; J.
Mater. Res. 2005, 20, 54-61.
Goyne, K. W.; Chorover, J.; Kubicki, J. D.; Zimmerman, A. R.;
Brantley, S. L.; J. Colloid Interf. Sci. 2005, 283, 160-170.
Qu, F.; Zhu, G.; Huang, S.; Li, S.; Sun, J.; Zhang, D.; Qiu, S.;
Microporous Mesoporous Mater. 2006, 92, 1-9.
Liu, J.; Stace-Naughton, A.; Jiang, X.; Brinker, C. J.; J. Am. Chem.
Soc. 2009, 131, 1354-1355.
Lai, C. Y.; Trewyn, B. G.; Jeftinija, D. M.; Jeftinija, K.; Xu, S.;
Jeftinija, S.; Lin, V. S. Y.; J. Am. Chem. Soc. 2003, 125, 4451-4459.
Corma, A.; Moliner, M.; Díaz-Cabañas, M. J.; Serna, P.; Femenia, B.;
Primo, J.; García, H.; New J. Chem. 2008, 32, 1338-1345.
Ferry, J. D.; J. Gen. Physiol. 1936, 20, 95-104.
Deen, W. M.; AlChE J. 1987, 33, 1409-1425.
63
References
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
Pappenheimer, J. R.; Renkin, E. M.; Borrero, L. M.; Am. J. Physiol.
1951, 167, 13-46.
Renkin, E. M.; J. Gen. Physiol. 1954, 38, 225.
Liu, G.; Li, Y.; Jonas, J.; J. Chem. Phys. 1991, 95, 6892-6901.
Anderson, J. L.; Quinn, J. A.; Biophys. J. 1974, 14, 130-150.
Deen, W. M.; Bohrer, M. P.; Epstein, N. B.; AlChE J. 1981, 27, 952959.
Nicholson, C.; Tao, L.; Biophys. J. 1993, 65, 2277-2290.
Xiao, F.; Nicholson, C.; Hrabe, J.; Hrabetova, S.; Biophys. J. 2008, 95,
1382-1392.
Serra, E.; Mayoral, Á.; Sakamoto, Y.; Blanco, R. M.; Díaz, I.;
Microporous Mesoporous Mater. 2008, 114, 201-213.
Salonen, J.; Laitinen, L.; Kaukonen, A. M.; Tuura, J.; Bjorkqvist, M.;
Heikkila, T.; Vaha-Heikkila, K.; Hirvonen, J.; Lehto, V. P.; J.
Controlled Release 2005, 108, 362-374.
Manzano, M.; Aina, V.; Arean, C. O.; Balas, F.; Cauda, V.; Colilla, M.;
Delgado, M. R.; Vallet-Regi, M.; Chem. Eng. J. 2007, 137, 30-37.
Gjerdaker, L.; Aksnes, D. W.; Sorland, G. H.; Stocker, M.;
Microporous Mesoporous Mater. 2001, 42, 89-96.
Valiullin, R.; Naumov, S.; Galvosas, P.; Kärger, J.; Woo, H.-J.;
Porcheron, F.; Monson, P. A.; Nature 2006, 443, 965-968.
Benes, N. E.; Jobic, H.; Verweij, H.; Microporous Mesoporous Mater.
2001, 43, 147-152.
Roger, P.; Mattisson, C.; Axelsson, A.; Zacchi, G.; Biotechnol. Bioeng.
2000, 69, 654-663.
Kirstein, J.; Platschek, B.; Jung, C.; Brown, R.; Bein, T.; Brauchle, C.;
Nat. Mater. 2007, 6, 303-310.
Sheppard, C. J. R.; Shotton, D. M.; Confocal Laser Scanning
Microscopy; Bios Scientific Publishers: Singapore, 1997; p 106.
Fu, Q.; Rao, G. V. R.; Ista, K.; Wu, Y.; Andrzejewski, B. P.; Sklar, L.
A.; Ward, T. L.; G.P. López; Adv. Mater. 2003, 15, 1262-1266.
Buranda, T.; Huang, J.; Ramarao, G. V.; Ista, L. K.; Larson, R. S.;
Ward, T. L.; Sklar, L. A.; Lopez, G. P.; Langmuir 2003, 19, 16541663.
Cheng, K.; Landry, C. C.; J. Am. Chem. Soc. 2007, 129, 9674 -9685.
Schröder, M.; vonLieres, E.; Hubbuch, J.; J. Phys. Chem. B 2006, 110,
1429-1436.
Yiu, H. H. P.; Wright, P. A.; J. Mater. Chem. 2005, 15, 3690-3700.
Langer, R.; Nature 1998, 392, 5.
Hernandez, R.; Tseng, H.-R.; Wong, J. W.; Stoddart, J. F.; Zink, J. I.;
J.A.C.S. Communication 2004, 126, 3370-3371.
Mal, N. K.; Fujiwara, M.; Tanaka, Y.; Nature 2003, 421, 350-353.
Bayerl, T. M.; Bloom, M.; Biophys. J. 1990, 58, 357-362.
Loidl-Stahlhofen, A.; Schmitt, J.; Nöller, J.; Hartmann, T.; Brodowsky,
H.; Schmitt, W.; Keldenich, J.; Adv. Mater. 2001, 13, 1829-1834.
64
References
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
Baksh, M. M.; Jaros, M.; Groves, J. T.; Nature 2004, 427, 139-141.
Moura, S. P.; Carmona-Ribeiro, A. M.; Langmuir 2005, 21, 1016010164.
Trepout, S.; Mornet, S.; Benabdelhak, H.; Ducruix, A.; Brisson, A. R.;
Lambert, O.; Langmuir 2007, 23, 2647-2654.
Taguchi, A.; Schuth, F.; Microporous Mesoporous Mater. 2005, 77, 145.
Davis, M. E.; Nature 2002, 417, 813-821.
Thomas, J. M.; Nature 1994, 368.
Vinu, A.; Hossain, K. Z.; Ariga, K.; J. Nanosci. Nanotechnol. 2005, 5,
347-371.
Che, S.; Liu, Z.; Ohsuna, T.; Sakamoto, K.; Terasaki, O.; Tatsumi, T.;
Nature 2004, 429, 281-284.
Huo, Q.; Feng, J.; Schuth, F.; Stucky, G. D.; Chem. Mater. 1997, 9,
14-17.
Galarneau, A.; Iapichella, J.; Brunel, D.; Fajula, F.; Bayram-Hahn, Z.;
Unger, K.; Puy, G.; Demesmay, C.; Rocca, J.-L.; J. Sep. Sci. 2006, 29,
844-855.
Grun, M.; Kurganov, A. A.; Schacht, S.; Schuth, F.; Unger, K. K.; J.
Chromatogr. A 1996, 740, 1-9.
Thoelen, C.; Walle, K. V. d.; Vankelecom, I. F. J.; Jacobs, P. A.;
Chem. Commun 1999, 1841 - 1842.
Uchiyama, H.; Suzuki, K.; Oaki, Y.; Imai, H.; Mater. Sci. Eng.: B
2005, 123, 248-251.
Baca, M.; Li, W.; Du, P.; Mul, G.; Moulijn, J.; Coppens, M. O.; Catal.
Lett. 2006, 109, 207-210.
Zhang, P.; Guo, J.; Wang, Y.; Pang, W.; Mater. Lett. 2002, 53, 400405.
Lu, Y.; Ganguli, R.; Drewien, C. A.; Anderson, M. T.; Brinker, C. J.;
Gong, W.; Guo, Y.; Soyez, H.; Dunn, B.; Huang, M. H.; Zink, J. I.;
Nature 1997, 389, 364-368.
Brinker, C. J.; Lu, Y.; Sellinger, A.; Fan, H.; Adv. Mater. 1999, 11,
579-585.
Vasiliev, P. O.; Faure, B.; Ng, J. B. S.; Bergstrom, L.; J. Colloid
Interface Sci. 2008, 319, 144-151.
Bruinsma, P. J.; Kim, A. Y.; Liu, J.; Baskaran, S.; Chem. Mater. 1997,
9, 2507-2512.
Bore, M. T.; Rathod, S. B.; Ward, T. L.; Datye, A. K.; Langmuir 2003,
19, 256-264.
Andersson, N.; Alberius, P. C. A.; Skov Pedersen, J.; Bergstrom, L.;
Microporous Mesoporous Mater. 2004, 72, 175-183.
Bruinsma, P. J.; Kim, A. Y.; Liu, J.; Baskaran, S.; Chemistry of
Materials 1997, 9, 2507-2512.
Qi, L.; Ma, J.; Cheng, H.; Zhao, Z.; Chem. Mater. 1998, 10, 16231626.
65
References
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
92.
93.
Kosuge, K.; Kikukawa, N.; Takemori, M.; Chem. Mater. 2004, 16,
4181-4186.
Buchel, G.; Grun, M.; Unger, K. K.; Matsumoto, A.; Tsutsumi, K.;
Supramol. Sci. 1998, 5, 253-259.
Unger, K. K.; Kumar, D.; Grun, M.; Buchel, G.; Ludtke, S.; Adam, T.;
Schumacher, K.; Renker, S.; J. Chromatogr. A 2000, 892, 47-55.
Stöber, W.; Fink, A.; Bohn, E.; J. Colloid Interface Sci. 1968, 26, 6269.
Etienne, M.; Lebeau, B. d.; Walcarius, A.; New J. Chem 2002, 26,
384–386.
Walcarius, A.; Delacote, C.; Chem. Mater. 2003, 15, 4181-4192.
Yano, K.; Fukushima, Y.; J. Mater. Chem. 2003, 2577-2581.
Yano, K.; Fukushima, Y.; J. Mater. Chem 2004, 1579-1584.
Tendeloo, G. V.; Lebedev, O. I.; Collart, O.; Cool, P.; Vansant, E. F.;
J. Phys: Condens. Matter 2003, 15, S3037-3056.
Lebedev, O. I.; Van Tendeloo, G.; Collart, O.; Cool, P.; Vansant, E. F.;
Solid State Sci. 2004, 6, 489-498.
Tan, B.; Rankin, S. E.; J. Phys. Chem. B 2004, 108, 20122-20129.
Nakamura, T.; Mizutani, M.; Nozaki, H.; Suzuki, N.; Yano, K.; J.
Phys. Chem. C 2007, 111, 1093-1100.
Zhuravlev, L. T.; Langmuir 1987, 3, 316-318.
Widenmeyer, M.; Anwander, R.; Chem. Mater. 2002, 14, 1827-1831.
Zhao, X. S.; Lu, G. Q.; Whittaker, A. K.; Millar, G. J.; Zhu, H. Y.;
Phys. Chem. B 1997, 101, 6525-6531.
Ottaviani, M. F.; Galarneau, A.; Desplantier-Giscard, D.; Di Renzo, F.;
Fajula, F.; Microporous Mesoporous Mater. 2001, 44-45, 1-8.
Galarneau, A.; Desplantier-Giscard, D.; Di Renzo, F.; Fajula, F.;
Catalysis Today 2001, 68, 191-200.
Ruthven, D. M., Fundamentals of adsorption equilibrium and kinetics
in microporous solids. In Molecular Sieves Science and Technology,
Adsorption and Diffusion, Karge, H. G.; Weitkamp, I.-J., Eds.;
Springer: Leipzig, Heidelberg, 2008; Vol. 7, pp 1-44.
Crank, J., The Mathematics of Diffusion. In The Mathematics of
Diffusion, 2nd ed.; Oxford University Press: Oxford, 1979; p 238.
Ruthven, D. M., Diffusion in zeolite molecular sieves. In Introduction
to Zeolite Science and Practice, 3 ed.; Cejka, J.; Bekkum, H. v.;
Corma, A.; Schüth, F., Eds.; Elsevier Science: Oxford, 2007; Vol. 168,
p 748.
Ruthven, D. M.; Brandani, S.; Eic, M., Measurement of diffusion in
microporous solids by macroscopic methods. In Molecular Sieves
Science and Technology, Adsorption and Diffusion, Karge, H. G.;
Weitkamp, I.-J., Eds.; Springer: Leipzig, Heidelberg, 2008; Vol. 7, pp
45-84.
Cejka, J.; Bekkum, H. v.; Corma, A.; Schüth, F., Introduction to
Zeolite Science and Practice. Elsevier: Oxford, 2007.
66
References
94.
95.
96.
97.
98.
99.
100.
101.
102.
103.
104.
105.
106.
107.
108.
109.
110.
111.
112.
113.
114.
115.
Bansal, R. C.; Goyal, M., Activated Carbon Adsorption. In CRC Press
Taylor & Francis Group: Singapore, 2005; pp 77-135.
Solomon, A. K.; J. Gen. Physiol. 1968, 51, 335-364.
Martin, T.; Galarneau, A.; DiRenzo, F.; Brunel, D.; Fajula, F.;
Heinisch, S.; Cretier, G.; Rocca, J. L.; Chem. Mater. 2004, 16, 17251731.
Qiao, S. Z.; Bhatia, S. K.; Microporous Mesoporous Mater. 2005, 86,
112-123.
Karge, H. G.; Kärger, J., Application of IR spectroscopy, IR
microscopy, and optical interference microscopy to diffusion in
zeolites. In Adsorption and Diffusion, Karge, H. G.; J.Weitkamp, Eds.;
Springer: Verlag Berlin Heidelberg, 2008; Vol. 7, pp 135-206.
Kortunov, P.; Vasenkov, S.; Chmelik, C.; Karger, J.; Ruthven, D. M.;
Wloch, J.; Chem. Mater. 2004, 16, 3552-3558.
Ng, J. B. S.; Kamali-Zare, P.; Brismar, H.; Bergstrom, L.; Langmuir
2008, 24, 11096-11102.
Svensson-Ek, M.; Abramson, J.; Larsson, G.; Törnroth, S.; Brzezinski,
P.; Iwata, S.; J. Mol. Biol. 2002, 321, 329-339.
Davis, R. W.; Flores, A.; Barrick, T. A.; Cox, J. M.; Brozik, S. M.;
Lopez, G. P.; Brozik, J. A.; Langmuir 2007, 23, 3864-3872.
Houde, A.; Kademi, A.; Leblanc, D.; Appl. Biochem. Biotech. 2004,
118, 155-170.
Schmid, R. D.; Verger, R.; Angew. Chem. Int. Ed. 1998, 37, 16081633.
Brzozowski, A. M.; Savage, H.; Verma, C. S.; Turkenburg, J. P.;
Lawson, D. M.; Svendsen, A.; Patkar, S.; Biochem. 2000, 39, 1507115082.
Villeneuve, P.; Muderhwa, J. M.; Graille, J.; Haas, M. J.; J. Mol. Catal.
B-Enzymatic 2000, 9, 113-148.
Sheldon, R. A.; Adv. Synth. Catal. 2007, 349, 1289-1307.
Kim, J.; Grate, J. W.; Wang, P.; Chem. Eng. Sci. 2006, 61, 1017-1026.
Hudson, S.; Cooney, J.; Magner, E.; Angew. Chem. Int. Ed. 2008, 47,
8582-8594.
Takahashi, H.; Li, B.; Sasaki, T.; Miyazaki, C.; Kajino, T.; Inagaki, S.;
Chem. Mater. 2000, 12, 3301-3305.
Zhao, D.; Huo, Q.; Feng, J.; Chmelka, B. F.; Stucky, G. D.; J. Am.
Chem. Soc. 1998, 120, 6024-6036.
He, J.; Song, Z. H.; Ma, H.; YANG, L.; Guo, C. X.; J. Mater. Chem.
2006, 16, 4307-4315.
Zhao, X. S.; Bao, X. Y.; Guo, W.; Lee, F. Y.; Mater. Today 2006, 9,
32-39.
He, J.; Xua, Y.; Maa, H.; Evansa, D. G.; Wanga, Z.; Duan, X.;
Microporous Mesoporous Mater. 2006, 94, 29-33.
Zhang, Y.; Zhao, L.; Li, J.; Zhang, H.; Zheng, L.; Cao, S.; Li, C.;
Biochem. Biophys. Res. Commun. 2008, 372, 650-655.
67
References
116. Fernandez-Lafuente, R.; Armisen, P.; Sabuquillo, P.; FernandezLorente, G.; J, M. G.; Chem. Phys. Lipids 1998, 93, 185-197.
117. Kwon, K. D.; Green, H.; Bjoorn, P.; Kubicki, J. D.; Environ. Sci.
Technol. 2006, 40, 7739-7744.
118. Kloster, C.; Bica, C.; Rochas, C.; Samios, D.; Geissler, E.; Macromol.
2000, 33, 6372-6377.
119. Andersson, N.; Kronberg, B.; Corkery, R.; Alberius, P.; Langmuir
2007, 23, 1459-1464.
120. Sörensen, M. H.; Zhu, J.; Corkery, R. W.; Hayward, R. C.; Alberius, P.
C. A.; Microporous Mesoporous Mater. 2009, 120, 359-367.
121. Babel, W.; Schulz, D.; Giesen-Wiese, M.; Seybold, U.; Gareis, H. t.;
Dick, E.; Schrieber, R.; Schott, A.; Stein, W., Gelatin. In Ullmann’s
Encyclopaedia of Industrial Chemistry, Bohnet, M.; Brinker, C. J.;
Clemens, H.; Cornils, B.; Evans, T. J.; Greim, H.; Hegedus, L. L.;
Heitbaum, J.; Herrmann, W. A.; Karst, U., Eds.; Wiley-VCH:
Weinheim, 2000; Vol. A12, pp 307-317.
122. Wolf, D. E.; Edidin, M.; Dragsten, P. R.; Proc. Natl. Acad. Sci. 1980,
77, 2043-2045.
123. Susanto, A.; Herrmann, T.; von Lieres, E.; Hubbuch, J.; J. Chromatogr.
A 2007, 1149, 178-188.
124. Susanto, A.; Herrmann, T.; Hubbuch, J.; J. Chromatogr. A 2006, 1136,
29-38.
125. Roque-Malherbe, R. M. A.; Adsorption and Diffusion in Nanoporous
Materials; CRC Press: New York, 2007; p 269.
126. Berland, K. M.; J. Biotechnol. 2004, 108, 127-136.
127. Magde, D.; Elson, E. L.; Webb, W. W.; Biopolymers 1974, 13, 29-61.
128. Che, S.; Lund, K.; Tatsumi, T.; Iijima, S.; Joo, S. H.; Ryoo, R.;
Terasaki, O.; Angew. Chem. Int. Ed. 2003, 42, 2182-2185.
129. Landsberg, P. T.; J. Chem. Phys. 1955, 23, 1079-1087.
130. Temkin, M.; Pyzhev, V.; Jour. Phys. Chem. (U.S.S.R.) 1939, 13, 851867.
131. Johnson, R. D.; Arnold, F. H.; Biochimica et Biophysica Acta (BBA) Protein Structure and Molecular Enzymology 1995, 1247, 293-297.
132. Ball, V.; Schaaf, P.; Voegel, J.-C., Mechanism of interfacial exchange
phenomena for proteins adsorbed at solid-liquid interfaces. In
Biopolymers at Interfaces, 2nd ed.; Malmsten, M., Ed. Marcel Dekker,
Inc.: New York, 2003; Vol. 110, p 295.
133. Blanco, R. M.; Terreros, P.; Fernández-Pérez, M.; Otero, C.; DíazGonzález, G.; J. Mol. Catal. B: Enzym. 2004, 30, 83-93.
134. Wannerberger, K.; Welin-Klintstrom, S.; Arnebrant, T.; Langmuir
1997, 13, 784-790.
135. Pauwels, B.; Tendeloo, G. V.; Thoelen, C.; Rhijn, W. V.; Jacobs, P. A.;
Adv. Mater. 2001, 13, 1317-1320.
136. Gruen, D. W. R.; J. Colloid Interface Sci. 1981, 84, 281-283.
68
References
137. Simon, A.; Girard-Egrot, A.; Sauter, F.; Pudda, C.; Picollet D'Hahan,
N.; Blum, L.; Chatelain, F.; Fuchs, A.; J. Colloid Interface Sci. 2007,
308, 337-343.
138. Brzezinski, P.; Reimann, J.; Adelroth, P.; Biochem. Soc. Trans. 2008,
36, 1169-1174.
69