Characterization of Functional Biopolymers
under Various External Stimuli
Atoosa Maleki
University of Oslo
2008
© Atoosa Maleki, 2008
Series of dissertations submitted to the
Faculty of Mathematics and Natural Sciences, University of Oslo
Nr. 785
ISSN 1501-7710
All rights reserved. No part of this publication may be
reproduced or transmitted, in any form or by any means, without permission.
Cover: Inger Sandved Anfinsen.
Printed in Norway: AiT e-dit AS, Oslo, 2008.
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PhD Thesis Atoosa Maleki
TABLE OF CONTENTS
Acknowledgments................................................................................................ 2
Abstract ................................................................................................................ 4
List of Papers ....................................................................................................... 6
List of Abbreviations and Symbols.................................................................... 7
1.
Introduction............................................................................................... 9
2.
Materials .................................................................................................. 16
3.
Experimental Techniques....................................................................... 21
3.1.
Rheology .................................................................................................................. 21
3.2.
Asymmetric Flow Field- Flow Fractionation (AFFFF)........................................ 23
3.3.
Dynamic Light Scattering (DLS) ............................................................................ 25
3.4.
Small Angle Neutron Scattering (SANS) ............................................................... 29
3.5.
Rheo-Small Angle Light Scattering (Rheo-SALS)................................................. 31
4.
Discussions ............................................................................................... 33
4.1.
Microgels ................................................................................................................. 33
4.2.
Macrogels................................................................................................................. 38
4.3.
Biopolymers in our Body......................................................................................... 40
4.3.1.
Hyaluronan ...................................................................................................................... 40
4.3.2.
Mucin ............................................................................................................................... 51
5.
Concluding Remarks and Future Aspects............................................ 56
6.
References ................................................................................................ 59
Papers (I-VII)..................................................................................................... 67
1
PhD Thesis Atoosa Maleki
Acknowledgments
My principle debt of thanks is to my supervisors; Prof. Bo Nyström and Dr. AnnaLena Kjøniksen. I would like to express my sincere gratitude to Bo for the insightful remarks,
fruitful discussions, and supportive guidance. He was always ready to listen and kindly
answer to the questions. He is indeed a wonderful professor. My expression of appreciation is
to Anna-Lena for the inspirational suggestions and be willing at any time to assist with the
experimental and technical problems during the study. In spite of lots of responsibilities she
has in the lab, she is always able to find a time to help the students.
My expression of appreciation is to Dr. Kenneth Knudsen for his support at IFE to
do the SANS measurements and his instructive comments.
I am grateful to Dr. Trond Vidar Hansen and Prof. Berit Smestad Paulsen at
Pharmacy department and Prof. Lars Skattebøl at Chemistry department for their valuable
comments and discussions.
I would like to thank Dr. Krister Thuresson and Dr. Géraldine Lafitte at Lund
University for the nice cooperation, and a special thank to Krister for his kind hospitability
during our visit in Lund.
Many thanks to the students at analytical lab for being so friendly and providing me
the instruments I had to use for some parts of my study.
I would like to thank Dr. Wilhelm Glomm and Dr. Sondre Volden at NTNU for
carrying out some measurements in their lab.
Financial support from FUNMAT (Novel functional polymer materials for drug
delivery applications) is gratefully acknowledged.
I had a very nice time in the polymer group at the University of Oslo, many thanks to
all members who still are present or have left; Wendy, Neda, Ramòn, Masoud, Kaizheng,
Nodar, Loan, Mehrdad, Zhengjun, Eirin, and Huiting for being such good friends, and for
sharing joys and knowledge.
2
PhD Thesis Atoosa Maleki
My gratitude to all faithful relatives and friends in Norway, U.S., and in Iran for
their kind support, and the joyful time we had together during all these years. Even several
pages would not be enough to write all names and the good memories we have had together,
but I want you all to know how happy I am to have you in my life.
I owe a debt of gratitude to my uncle Morteza, whom his emotional support was a
driving force to continue studying out of my homeland.
A special thank to my beloved sisters; Mahshid and Shima for being always
supportive and enthusiastic, and for their sympathetic and endless friendship.
Last but not least, a special thank to my dear parents for their everlasting support,
great affection, and their pure love. Your presences in my life were instrumental in making
this paper a reality.
"# !" . )*, *"(+
. (() .$' & $%& Atoosa Maleki
Oslo, October 2008
"/ -) 3
PhD Thesis Atoosa Maleki
Abstract
Biopolymers belong to a group of macromolecules, which exhibit both
biocompatible and biodegradable properties. In this thesis, structural and dynamical
characteristics of a number of biopolymers were studied with the aid of a rheology,
rheo-small angle light scattering (rheo-SALS), dynamic light scattering (DLS), and
small angle neutron scattering (SANS). To determine molecular weight and
polydispersity index, asymmetric flow field-flow fractionation (AFFFF) was used.
Hydroxyethylcellulose (HEC), its hydrophobically modified analogue (HMHEC), and dextran are all polysaccharides with hydroxyl functional groups along the
backbone, which can react with the difunctional cross-linker agent divinylsulfone
(DVS). Intramolecular and intermolecular cross-linking of dilute aqueous alkali
solutions of these three systems have been studied under the influence of a steady
shear rate. At the same experimental conditions, different behaviors were observed for
each system because of various chemical structures of the polymers. However, the
formation of microgels can be detected for all the systems.
An interconnected gel network is formed by cross-linking a semidilute HEC
solution in the presence of DVS. To investigate the structure of HEC networks on a
mesoscopic dimensional scale, SANS measurements were performed for the systems
in the presence of different DVS concentrations. Prior to measurement, the crosslinking reaction was quenched to a lower pH by adding HCl at various times.
Rheological and DLS experiments were carried out on semidilute sodium
hyaluronate (HA) solutions during the chemical cross-linking process. Water-soluble
carbodiimide (WSC) produce only ester bonds via carboxyl groups in HA molecules,
whereas in the presence of L-lysine methyl ester (L-lysineME) a significant fraction of
amide linkages are formed. Ester bonds are known to be weak linkages and sensitive
4
PhD Thesis Atoosa Maleki
to hydrolysis. A more stable network is evolved by using the Ugi multicomponent
condensation reaction, because of the formation of many amide bonds. AFFFF was
employed to determine the weight average molecular weight and polydispersity of HA
solution before and after the cross-linking reaction. Effects of riboflavin (RF) as a
photosensitizer and pH in a range of 1-13 on the degradation of HA chains in solution
were examined. In the domain 4 < pH < 11, virtually no scission of HA was found,
whereas outside this region disruption of the HA chains occurred. Steady shear
measurements were conducted on aqueous HA solutions in order to survey the
intermolecular interactions.
Effect of pH on the association behavior of pig gastric mucin (glycoprotein)
was investigated by means of DLS and rheo-SALS. Purified mucin was dissolved in
buffers with pH values of 1, 2, 4 and 7 at two different concentrations; 0.01 wt %
(dilute) and 1 wt % (semidilute). Strong aggregation was detected at pH 2 by DLS, as
well as higher viscosity with the aid of rheo-SALS at the same pH.
5
PhD Thesis Atoosa Maleki
List of Papers
Paper I: Atoosa Maleki, Anna-Lena Kjøniksen, Bo Nyström. Effect of Shear on
Intramolecular and Intermolecular Association during Cross-Linking of
Hydroxyethylcellulose in Dilute Aqueous Solutions. Journal of Physical Chemistry B
2005, 109, 12329-12336.
Paper II: Zhengjun Liu, Atoosa Maleki, Kaizheng Zhu, Anna-Lena Kjøniksen, Bo
Nyström. Intramolecular and Intermolecular Association during Chemical Crosslinking of Dilute Solutions of Different Polysaccharides under the Influence of Shear
Flow. Journal of Physical Chemistry B 2008, 112, 1082-1089.
Paper III: Atoosa Maleki, Anna-Lena Kjøniksen, Kenneth D. Knudsen, Bo Nyström.
Dynamical and Structural Behavior of Hydroxyethylcellulose Hydrogels obtained by
Chemical Gelation. Polymer International 2006, 55, 365-374.
Paper IV: Atoosa Maleki, Anna-Lena Kjøniksen, Bo Nyström. Effect of pH on the
Behavior of Hyaluronic Acid in Dilute and Semidilute Aqueous Solutions.
Macromolecular Symposia, in press.
Paper V: Atoosa Maleki, Anna-Lena Kjøniksen, Bo Nyström. Characterization of
the Chemical Degradation of Hyaluronic Acid during Chemical Gelation in the
Presence of Different Cross-linker Agents. Carbohydrate Research 2007, 342, 27762792.
Paper VI : Atoosa Maleki, Anna-Lena Kjøniksen, Bo Nyström. Anomalous Viscosity
Behavior in Aqueous Solutions of Hyaluronic Acid. Polymer Bulletin 2007, 59, 217226.
Paper VII: Atoosa Maleki, Géraldine Lafitte, Anna-Lena Kjøniksen, Krister
Thuresson, Bo Nyström. Effect of pH on the Association Behavior in Aqueous
Solutions of Pig Gastric Mucin. Carbohydrate Research 2008, 343, 328-340.
Additional papers not included in the thesis:
-
Maleki A., Beheshti N., Zhu K., Kjøniksen A.-L., Nyström B., Shrinking of
Chemically Cross-Linked Polymer Networks in the Postgel Region, Polymer Bulletin
2007, 58, 435-445.
-
Silioc C., Maleki A., Zhu K., Kjøniksen A.-L., Nyström B., Effect of Hydrophobic
Modification on Rheological and Swelling Features during Chemical Gelation of
Aqueous Polysaccharides, Biomacromolecules 2007, 8, 719-728
6
PhD Thesis Atoosa Maleki
List of Abbreviations and Symbols
AFFFF............................................Asymmetric Flow Field-Flow Fractionation
Af ....................................................Amplitude of the Fast Mode
As....................................................Amplitude of the Slow Mode
B.....................................................Empirical Factor
CA..................................................Citric Acid
d .....................................................Power Law Exponent (I(q) ~ q-d)
D.....................................................Diffusion Coefficient
Dc ...................................................Cooperative Diffusion Coefficient
Dm...................................................Mutual Diffusion Coefficient
DLS................................................Dynamic Light Scattering
DVS ...............................................Divinylsulfone
df.....................................................Fractal Dimension
FG ..................................................Functional Group
G´ () .............................................Storage Modulus (or G´)
G´´ ()............................................Loss Modulus (or G´´)
g1(q,t) .............................................First Order Electric Field Correlation Function (or g1(t))
g2(q,t) .............................................Measured Homodyne Intensity Autocorrelation Function (or g2(t))
HA..................................................Hyaluronan / Hyaluronic acid / Sodium Hyaluronate
HEC ...............................................Hydroxyethylcellulose
HM-HEC........................................Hydrophobically Modified Hydroxyethylcellulose
I(q) .................................................Scattered Intensity
k´ ....................................................Huggins Coefficient
kB ....................................................Boltzmann’s Constant
L .....................................................Characteristic Length
L-lysineME ....................................L-lysine Methyl Ester
Mn...................................................Number Average Molecular Weight
MW .................................................Weight Average Molecular Weight
n .....................................................Relaxation Exponent
n´ ....................................................Power Law Parameter (G´ ~ n´)
n´´...................................................Power Law Parameter (G´´ ~ n´´)
7
PhD Thesis Atoosa Maleki
nref...................................................Refractive Index
q .....................................................Wavevector
Rg ...................................................Radius of Gyration
Rh ...................................................Hydrodynamic Radius
Rheo-SALS ....................................Rheo-Small Angle Light Scattering
SANS .............................................Small Angle Neutron Scattering
T .....................................................Temperature
t ......................................................Time
tan ................................................Damping Factor or Loss Tangent
Ugi .................................................Ugi Multicomponent Condensation Reaction
WSC...............................................Water-Soluble Carbodiimide
z......................................................Power Law Exponent (I(q) ~ q-z)
.....................................................Stretched Exponent
f.....................................................Stretched Exponent for the Fast Mode
s ....................................................Stretched Exponent for the Slow Mode
.....................................................Gamma Function
J
...................................................Shear Rate
......................................................Relative Distance from the Gel Point
.....................................................Viscosity
0 ....................................................Viscosity of the Solvent
|*|..................................................Absolute Value of the Complex Viscosity (or *)
.....................................................Scattering Angle
.....................................................Wavelength
......................................................Correlation Length / Screening Length / Measure of the Mesh Size (
h)
d/d .............................................Coherent Macroscopic Scattering Cross Section
f .....................................................Fast Relaxation Time
s .....................................................Slow Relaxation Time
fe ....................................................Fast Effective Relaxation Time
se ....................................................Slow Effective Relaxation Time
.....................................................Angular Frequency (rad/s)
8
PhD Thesis Atoosa Maleki
1. Introduction
Polymers are considered to be the most important material in science and
technology for the 21st century.1 Macromolecules morphology, conformation, and
miscibility are of particular importance in order to understand their properties as
individual molecules and in intermolecular interactions with various materials. The
characterization of a polymer requires different parameters, which need to be
specified. Polymer behavior in solution is strongly dependent on the chemical and
physical structure of the polymer chain, as well as external environmental conditions.
The chain length, attractive forces and degree of branching are some factors that may
influence the features of the polymers. Increasing the chain length or molecular mass
may lead to decrease in chain mobility and increase in the viscosity. The presence of
various side groups along the chain may promote intramolecular forces in individual
molecules or intermolecular interactions between polymer chains.
Biopolymers are a class of macromolecules produced by living organisms.
Proteins, peptides, cellulose, and DNA are some examples of this family. These
macromolecules are responsible for the biological functions of molecular sensing,
homeostasis, molecular motions and catalysis.1 To improve biopolymers in the
direction of higher performance and better functionality, understanding of their
physicochemical behavior and their response to external stimuli are of great
importance. Synthesis of such polymers will also be an ultimate challenge to modern
science and technology.
The space scale plays an important role in studies of polymer systems.
Depending on the experimental technique, the system can be described directly by the
chain properties on the nanoscale, its morphology on an intermediate mesoscopic
scale, and the bulk features of the sample can be probed on the macroscopic scale.
These types of materials have been examined by a vast number of modern
9
PhD Thesis Atoosa Maleki
experimental methods. Rheology is one of the most powerful techniques to study the
characteristics of polymer solutions in bulk. Gel permeation chromatography (GPC)
and asymmetric flow field-flow fractionation (AFFFF) are used to determine
molecular weights, and molecular weight distributions of polymers. Nuclear magnetic
resonance (NMR) is utilized to characterize the composition and chemical structure of
polymers. Surface topography of samples can be explored by scanning electron
microscope (SEM). A spectrophotometer can be employed to determine the cloud
point or turbidity of a solution. Thermal properties such as the glass transition
temperature and melting point can be analyzed by differential scanning calorimetry
(DSC). Because the size of polymer chain varies from a few to several hundred
nanometers, scattering methods such as light scattering (LS), small angle X-ray
scattering (SAXS), small angle light scattering (SALS), and small angle neutron
scattering (SANS) have been widely used to characterize polymers both in dilute and
semidilute solutions.1 Rheology, AFFFF, DLS, SANS, and rheo-SALS have been
employed to study the physicochemical characteristics of different polymer systems in
this thesis.
A group of polymers that has attracted a great deal of attention is ‘responsive’
polymers. ‘Responsive’ polymers are a class of macromolecular compounds with
special characteristics. They have been used in many applications during the last
decades. They respond to changes of physical or chemical stimuli, such as
temperature, electric and magnetic filed, solvents, mechanical stress and strain,
radiation (UV, visible light, ultrasonic), and ionic strength.1-6 Specific features of these
polymers provide them to be used as ‘functional’ materials, e.g., sensors, actuators,
bioseparation, personal care products, potting compounds for civil engineering,
enhanced oil recovery, and controlled drug delivery systems.2-5,7 An example of their
application is a self-regulated drug delivery system, which has been of great interest in
the last decades, since it allows the drug to be released when it is needed.2 Figure 1
10
PhD Thesis Atoosa Maleki
shows a schematic illustration of how drugs may diffuse out from a polymer network
through the influence of various stimuli conditions.
Swelling
Solvent, T, pH,…
Degrading
Irradiation, T, pH,
Additional agent,…
Drug Trapped in the Network
Figure 1. Schematic illustration of the release of drugs (trapped in the polymer network) by
means of different stimuli conditions.
In this thesis, the characteristics of several functional biopolymers have been
investigated in the presence of external stimuli. The aim of this work is to study some
structural, dynamical, and rheological characteristics of these polymers in order to
shed light on their possible applications in one or several areas mentioned above. The
systems are divided into three categories: microgels (Paper I-II), macrogels (Paper III)
and biopolymers in our body (Paper IV-VII).
Biopolymers such as polysaccharides can be utilized to form supramolecular
structures. Hydrogels are made of physically or chemically cross-linked hydrophilic or
11
PhD Thesis Atoosa Maleki
amphiphilic polymers. The cross-links can result via intermolecular noncovalent
interactions like hydrogen bonds, hydrophobic or electrostatic interactions8-10 (physical
gel), or the transient network may be cross-linked covalently by a suitable cross-linker
agent (chemical gel)11,12. A hydrogel is able to swell and retain large volume of water
in its swollen three-dimensional structure1-4,13, as well as to shrink.14 The association
complexes can be used to produce microgels or macrogels depending on polymer
concentration and the way of preparation.15 Microgels are small particles that can be
synthesized via emulsion polymerization,16 anionic copolymerization or prepared by
the addition of a cross-linker agent to the dilute solution of polymer. Microgels can
also be produced as spherical particles with a large number of functional groups,
which is referred to as ‘reactive microgels’.17 These functional groups can be mono- or
oligosaccharides or covalently coupled drugs, which slowly can be released from the
particle via various external stimuli. Varying the cross-linking density or
environmental parameters such as pH, ionic strength, and temperature may modify the
finite pore size present in microgels.15 If a dilute polymer solution is cross-linked, the
connectivity is lost and nonlinked aggregates of various sizes are formed; these may be
referred to as microgels.15,18 There are two possible types of cross-links,19,20 namely
inter- and intramolecular cross-links (see Figure 2). Intermolecular cross-linking
occurs via the coupling of two or more polymer chains, whereas the cross-linking
takes place within the same polymer chain when intrapolymer cross-linking is
involved.19 Aggregation of polymers may occur under the influence of shear flow or
Brownian motions at quiescent conditions for dilute polymer solutions. Many
investigations have been reported on the preparation of microgels, with21 or without
18,19
cross-linker, under the influence of Brownian motions19 or shear flow.21-23 It is
possible to explore the transport mechanism in the framework of the Péclet number
(Pe) to decide whether the particle motion is governed by hydrodynamic flow or by
diffusion. The Péclet number is a dimensionless number relating the rate of advection
12
PhD Thesis Atoosa Maleki
of a flow to its rate of diffusion. It is defined through the expression Pe J R2/D =
6oR3 J /kBT, where J is the shear rate, R is the particle radius, D is the translational
diffusion, o is the solvent viscosity, kB is the Boltzmann constant, and T is the
absolute temperature.24 The formation of flocs in the presence of a shear flow is known
as ‘orthokinetic’ aggregation.25 The structure of the formed flocs is fractal, which
means that the aggregate properties such as mass, density and volume, scale as a
power of its size. Determination of fractal dimension would be a measure of how these
aggregates are filling the space.23 It has been reported26 that the measured dimension
of a dilute solution is a function of both fractal dimension and polydispersity index.
This indicates that a single measurement does not necessarily give the actual
dimension of fractal objects, and the possible effect of polydispersity has to be taken
into account. Changes in dimensions such as shrinking of cross-linked microgels can
be determined by the ratio of Rg and Rh (Rg/Rh) and its relation to Mw.19,21 The
aggregation process is important for technological problems in industry and
biochemistry.15,16,20 In the present work (Paper I-II), the effects of both shear and
cross-linker addition have been investigated for dilute polysaccharide solutions in the
course of the chemical cross-linker reaction. During the cross-linking of polymers,
competition between intrachain and interchain cross-linking is expected to occur.
Hydrophobic and hydrophilic forces present in complex fluids may generate some
specific interactions, which can affect the rheological properties of the solution.
A cross-linked semidilute polymer solution undergoes a transition from a liquid
to a solid at a critical extent of the cross-linker reaction. This phenomenon is usually
referred to as gelation. Gelation of a semidilute polymer solution can be characterized
as a process involving a continuous increase in viscosity accompanied by a gradual
enhancement of the elastic properties. Below the gelation threshold, the system
behaves like a liquid, whereas above the gel point the sample does not flow. In the
13
PhD Thesis Atoosa Maleki
vicinity of the sol-gel transition, a variety of structural and dynamical transformations
may take place.
CrossLinker
FG
(a)
Intramolecular Cross-linking
FG
X
X
X : CrossLinking Zone
FG
FG
FG: Functional Group
(b)
CrossLinker
2 x
FG
FG
FG
FG
Intermolecular Crosslinking
X : CrossLinking Zone
FG: Functional Group
FG
FG
X
X
FG
FG
Figure 2. An illustration of chemical cross-linking of polymer solutions in the presence of a
cross-linker agent. (a) Intramolecular cross-linking, where the lower part illustrates how a
single chain undergoes a cross-linking reaction via functional groups. (b) Intermolecular
cross-linking, where the lower part shows the cross-linking between two polymer chains.
Structural studies on many polymers near the gel point have demonstrated that
gelation can be described by the percolation model.27,28 This approach is a simple
lattice model for gelation. Each lattice site with X neighbors represents one
polyfunctional unit with X reaction arms. A certain molecule is randomly formed by
reacting two neighboring units. The formation of molecules and small clusters or an
infinite network depends on the fraction of reacted bonds. Macrogels are produced by
14
PhD Thesis Atoosa Maleki
physically (reversible)8,10,29,30 or chemically (irreversible)31-33 intermolecular crosslinking of a semidilute polymer solution to develop an interconnected network. In this
thesis, the effect of chemical cross-linking on the gelation behavior of a biocompatible
polysaccharide has been investigated (Paper III).
Polyelectrolytes are polymers that contain ionisable groups attached to the main
chain. In a polar solvent such as water, the charges are dissociated in a way that the
charges of one sign are localized on the chain, whereas the counterions are mobile in
the solution.34 Solution properties of polyelectrolytes differ usually from those of
uncharged polymers. The presence of charges along the chain leads to complex intraand intermolecular interactions that have considerable effect on both structural and
rheological behavior of the system. This type of interactions can partly or completely
be screened by adding salt to the solution. However, the behavior of polyelectrolytes
in the absence of salt is rather poorly understood. Over the past decades, this field of
research has attracted many scientists from both the theoretical and experimental side.
Despite all progress in this area, lack of clear molecular interpretations for some of
their characteristics cannot be ignored.35-37 Among all biopolymers with some
polyelectrolyte effects, two of which that exist abundantly in mammalian are
hyaluronan and mucin. Some structural and dynamical characteristics of these
biocompatible polymers have been investigated in this work. (Paper IV-VII)
15
PhD Thesis Atoosa Maleki
2. Materials
Hydroxyethylcellulose and its Hydrophobically Modified Analogue
Hydroxyethylcellulose (HEC) is a hydrophilic non-ionic polysaccharide,
typically prepared by the reaction of cellulose with ethylene oxide. HEC has some
surface activity in aqueous solution and is compatible with a wide range of surfactants
and salts. To change the structural and rheological behaviors of HEC, hydrophobic
groups may be incorporated into the main chain through a standard procedure.38 The
chemical structures of these polymers are depicted in Figure 3.
OR
2
CH
HO
O
OR
2
CH
OR
O
HO
OR
CH
2 OR
O
OR
HO
O
O
1-x HO
x
O
OR
O
CH
2 OCH
2 CH
n
2 OCH
2 CH(
R : "H" or "(CH2CH2O)mH"
Figure
3.
Chemical
structures
OH
)CH
Cellulose x = 0 , R = H
HEC
x=0
HM-HEC x = 0.02
of
cellulose,
hydroxyethylcellulose
2 OC
16 H
33
(HEC),
and
hydrophobically modified HEC (HM-HEC).
HEC or hydrophobically modified HEC (HM-HEC) can be cross-linked both in
dilute and semidilute solutions via hydroxyl groups in the presence of the difunctional
cross-linker divinylsulfone (DVS) at alkaline condition (pH 11.8).39 HEC and HMHEC are widely used in applications and products that require thickening, water
binding, lubricating, film forming, and protective colloid or stabilizing properties.40,41
16
PhD Thesis Atoosa Maleki
Dextran
Dextran is a bacterial polysaccharide consisting of linear -1,6-linked
glucopyranose units, with some degree of -1,2-, -1,3- and -1,4-branching42-44 (see
Figure 4). Both the molecular weight and the degree of branching affect the
physicochemical properties of these biodegradable and biocompatible polymers.43,44
The existence of large number of hydroxyl groups on the polymer chains, make them
suitable for modification and subsequent physical or chemical cross-linking.43,45
Dextran has been extensively used in drug delivery, blood volume expander, and
purification of biologics.42-44
O
O
HO
HO
OH
O
O
O
HO
O
HO
HO
OH
OH
O
O
O
HO
HO
OH
O
Figure 4. Schematic illustration of the chemical structure of branched dextran. Only (13)
side branch is shown in this figure.
Hyaluronan
Hyaluronan, also known as hyaluronate, hyaluronic acid (HA) is a natural high
molecular weight biopolysaccharide. It is an important component of extracellular
matrix and connective tissues.46,47 HA is an anionic linear polysaccharide that consists
of repeating disaccharide units of N-acetyl-D-glucosamine (GlcNAc) and D17
PhD Thesis Atoosa Maleki
glucuronic acid (GlcUA), linked by a 1-4 glycosidic bonds while the disaccharides
are linked by 1-3 bonds (see Figure 5).47 Hyaluronan is a major component of
umbilical cords, vitreous body, skin, and synovial fluid.41,47 It is degraded in vivo by
the specific enzyme called hyaluronidases.48 HA can be extracted from bovine vitreous
humor, rooster combs or umbilical cords. It can also be produced by bacteria called
Streptococcus zooepidemicus47 with a good yield and a large degree of purity. Its
excellent physicochemical properties,49 such as biocompatibility, high viscoelasticity,
nonimmunogenicity,
and
biodegradability
make
it
a
medically
important
biomaterial.47,50 HA is an attractive building block for new biocompatible and
biodegradable polymers with possible applications in drug delivery, viscosurgury,
viscosupplementation, ophthalmic surgery, wound healing, and tissue engineering.50
Hyaluronan can be cross-linked in a variety of ways due to the existence of both
hydroxyl and carboxyl functional groups in the polymer chain.50,51 The polymer
utilized in this study has a bacteria source and it is the sodium salt of hyaluronan
(sodium hyaluronate) referred to as hyaluronan with the abbreviation of HA in this
thesis.
CH3
-
+
O
O X
O
O
HO
O
HOH2C
NH
O
O
O
HO
OH
HO
O
O
NH
OH
O
HO
O
HOH2C
O- X+
O
n
CH3
GlclUA
GlcNAc
GlclUA
GlcNAc
Sodium Hyaluronate X : Na
Hyaluronan
X:H
Hydrogen bonds
Figure 5. Schematic illustration of the chemical structure of hyaluronan.
18
PhD Thesis Atoosa Maleki
Mucin
Mucus is a viscous secretion that is found on most epithelial surfaces
throughout the body, and it acts as a medium for protection, lubrication, and
transport.52,53 The most abundant macromolecule in mucus is mucin.53 Mucin is a high
molecular weight (>106) glycoprotein, composed of a polypeptide backbone and
covalently linked oligosaccharide side chains.53-55 The chemical composition of the
mucin depends on the region and species from which it is isolated.56 Interesting
viscoelastic behavior has been observed in this group of macromolecules, with 56,57 or
without55,58 additional chemical agent, because of interactions from both hydrophobic
moieties and negative charges along the chain. The mammalian stomach is a unique
biological environment in that the gastric pH drops from nearly neutrality to
approximately 1-3 during active digestion.58 However, the gastric epithelium remains
undamaged by the particular protection of the mucous gel layer where mucin has an
important role.53 Stomach mucins are of great interest to the pharmaceutical
researchers, since they form a barrier against drug adsorption.54
A non-commercial purified55 pig gastric mucin was utilized in this study. Since
the structure of mucin varies considerably depending on the site from which it has
been extracted, there is no direct scheme of its chemical structure. A schematic view of
the mucin molecule is shown in Figure 6.
H2N
COOH
Glycosylated region (linear polypeptide)
Non-glycosylated region (contains hydrophobic groups)
Oligosaccharide side chains (linear or branched)
Figure 6. Schematic view of the mucin molecule. Native mucins isolated from body
secretions or tissues are always heavily glycosilated.53
19
PhD Thesis Atoosa Maleki
Riboflavin
In recent years, the degradation of polysaccharides by light irradiation in the
presence of a photosensitizer has attracted a great deal of interest.59,60 Riboflavin or
vitamin B2 (see Figure 7) is an uncharged, water-soluble micronutrient that can induce
oxidation reactions upon exposure to UV or visible light of short wavelengths.61 In
photodynamic reactions, either energy is transferred from a triplet excited sensitizer to
1
O2 with the formation of singlet oxygen ( O2), or a hydrogen atom or an electron is
transferred from a substrate directly to a triplet excited photosensitizer, with formation
of reactive species, which under aerobic condition, may derive from molecular oxygen
( O2 x , H 2 O2 , OH x ). The reactive species generated by the photoreduction of riboflavine
can attack the glycosidic bonds of polysaccharides, causing scission of polymer
chains.59,60
O
H3C
N
H
N
H3C
N
N
O
HO
HO
OH
OH
Figure 7. Schematic illustration of the chemical structure of riboflavin.
20
PhD Thesis Atoosa Maleki
3. Experimental Techniques
3.1.
Rheology
Rheology is the science of the deformation and flow of matter. It is concerned
with the response of materials to mechanical forces. Deformation can be divided into
two types: flow and elasticity. Flow is an irreversible deformation; when the stress is
removed, the material does not revert to its original configuration. Whereas elasticity
is almost a reversible deformation; the deformed body recovers its original shape, and
the applied work is largely recoverable. Viscoelastic materials, such as semidilute
polysaccharide solutions, show both flow and elasticity features.
Steady Shear
In steady shear measurements, using a cone and plate geometry, the cone will
rotate, and each point is measured at a different shear rate, giving us the shear rate ( J
= /tan , where is the steady angular rotation speed of the cone and is the cone
angle) dependency. In this mode, the shear viscosity ( = / J ) of the sample can be
determined. The shear stress is the force that a flowing liquid exerts on a surface (per
unit area of that surface) in the direction parallel to the flow. In a Newtonian system,
the viscosity is independent of the shear rate. Some polymer solutions exhibit shearthinning behavior (the viscosity decreases with increasing the shear rate). This
behavior can often be attributed to intermolecular associations and/or entanglements
that are disrupted by shear forces. A shear-induced viscosity drop can also be observed
in dilute solutions through alignment of the polymer chains in the shear field. Many
systems exhibit shear thickening (increasing of the viscosity with rising shear rate) at
low shear rates, and shear-thinning at higher shear rates. Stretching and alignment of
the polymer chains give rise to easier access for intermolecular associations, and
21
PhD Thesis Atoosa Maleki
therefore this leads to shear-thickening. At high shear rates, the associations break up
and the system exhibits shear-thinning behavior.24 At low shear rates, the viscosity
assumes a constant value, which yields the zero-shear viscosity.
Oscillatory Shear
In order to determine the dynamic properties of the system, oscillatory shear
measurements are conducted. In oscillatory shear measurements, the sample is placed
under the influence of a periodically variation of the stress with a sinusoidal
alternation at a given angular frequency (). From this type of experiment, the storage
modulus or elastic response (G()), the loss modulus or viscous response (G()),
damping (tan ) and the complex viscosity (*) of the system can be determined.
Analyzing the Data
The ratio between the storage and loss modulus obtained from an oscillatory
shear experiment can be expressed as:
tan G
G cc
Gc
(1)
where is the phase difference between the stress and the strain in the linear
viscoelastic regime, and tan refers to the loss tangent or damping.62,63 tan is high
(>>1) for materials that are liquid-like and is low (<< 1) for materials that are solidlike.24
The gel point can be determined from oscillatory shear measurements.62
According to the model of Chambon and Winter,63,64 the gel point is characterized by
a frequency independency of tan . By plotting tan at several angular frequencies
22
PhD Thesis Atoosa Maleki
against the gel forming parameter (e.g., reaction time or temperature), the gel point can
be determined from the criterion where tan is frequency independent. The gel point
can be also determined by finding the point where the slopes (the apparent relaxation
exponents n and n) of G and G versus are the same in a log-log plot.11
G c v Z nc , G cc v Z ncc
(2)
where n ( 0 < n < 1 ) is the relaxation exponent and n = n= n at the gel point.
Another parameter that can be determined from oscillatory shear measurement
is the complex viscosity (*)24:
K*
3.2.
G c 2 G cc 2
Z
(3)
Asymmetric Flow Field- Flow Fractionation (AFFFF)
Asymmetric flow field-flow fractionation (AFFFF) is an innovative separation
method for the efficient separation and characterization of proteins, polymers and
nanoparticles in a fast and gentle way. Based on the diffusion rate of the sample, the
separation occurs in an open flow channel without using any stationary phase as is
utilized in SEC/GPC. The instrument consists of two detectors; a refractive index
detector (RI) that detects the concentration, and a multi angle light scattering detector
(MALS), which measures the molecular weight and size of the molecules. AFFFF
offers the possibility to determine a broad variety of different physicochemical
parameters of the sample,65 such as the number average molecular weight (Mn), weight
average molecular weight (Mw), and radius of gyration (Rg). The instrument used in
this study has a large separation range. Nanoparticles can be separated from 1 nm to
100 μm and proteins, peptides and polymers with molecular weights from 103 to 1012
23
PhD Thesis Atoosa Maleki
Da. The sample is injected with the tip flow, and is focused into a narrow band by a
parabolic flow profile from both sides. At the same time the sample is submitted to a
flow-field created by the cross flow, pressing the sample towards the membrane. The
diffusion of the molecules causes large molecules to be located closest to the
membrane, and smaller molecules are located further out from the membrane (Figure
8, step 1). After the focussing step, the cross flow is gradually reduced to zero causing
the small molecules to be eluted first followed by larger molecules (Figure 8, step 2).
During the measurement, the tip flow is varied in order to keep the detector flow
constant (detector flow = tip flow + focus flow – cross flow).
Laminar out to the
detectors
Laminar in (Focus flow)
Laminar in (Tip flow)
Diffusion Field
Carrier Liquid
Membrane
Porous Frit
Carrier Liquid
Flow Field
Cross Flow
Step1 : Injection and Focusing, Step 2 : Elution (without focus flow)
Figure 8. Schematic illustration of flow directions during the whole process of separation in
AFFFF.
Many studies have been reported on the characterization of biomacromolecules
such as polysaccharides with the aid of AFFFF.66-68 However, a careful attention to
correct operating conditions for each individual sample with different structural and
chemical properties must be appreciated.69
24
PhD Thesis Atoosa Maleki
Analysing the Data
The weight-average molecular weight (Mw), number-average molecular weight
(Mn), and radius of gyration (RG) can be determined by using the Postnova software
without standards (AF2000 Control, version 1.1.011),(see Paper VI).
3.3.
Dynamic Light Scattering (DLS)
Scattering in general means a random distribution of certain items, whereas it
has a more definite meaning in physic. There are many different types of objects that
may cause scattering, but a small group can be listed as particles, bubbles, droplets,
density fluctuations in fluids, defects in crystalline solids and surface roughness. A
source of energy such as light, neutrons, and X-rays pass through the sample, and the
scattering beam at different angles goes through the detector (see Figure 9).
Sample
Transmitted Beam
Incoming Beam
q
Scattered Beam
Figure 9. A simple scheme of the principle of a scattering experiment.
The angle of observation with respect to the direction of the incident light beam
is called the scattering angle and provides a measure for the length scale observed in a
scattering experiment. In other words, the scattering process allows us to explore a
system on a length scale of q-1. The wavevector q, is given by q = 4nref/ sin(/2),
25
PhD Thesis Atoosa Maleki
where nref is the refractive index of the medium, is the wavelength of the incident
light in a vacuum, and is the scattering angle. The same scattering equation can be
used for a given value of q, regardless of whether visible light, neutrons or X-rays are
employed in a scattering experiment. The information obtained from scattering
depends on the quantity qL (where L is a characteristic length), and there is an inverse
relationship between the size of the scattering object and the q values at which
scattering is observed. The value of the quantity qL indicates whether global (qL < 1)
or local (qL > 1) dimension scales are probed.70
All matters consist of atoms, which themselves are built from negative and
positive charges. Interactions of light with matter causes scattering that is the principle
of light scattering experiments. If the molecules or particles were stationary, the
scattered light intensity at each direction would be constant, whereas when they are
undergoing Brownian motions, fluctuations of the scattered intensity pattern are
generated in the solutions.1 To quantitatively analyze the particle mobility by light
scattering, it is helpful to express the scattering intensity fluctuations in terms of
correlation functions. The detailed analysis of the fluctuating intensity that is detected
as a signal by the detector is mathematically translated into an intensity autocorrelation
function.71
Analyzing the Data
A dynamic light scattering experiment probes the relaxation times of the
decay,72-74 which relax on some length scale q-1. If the scattered field obeys Gaussian
statistics, the measured homodyne intensity autocorrelation function, g(2)(t), is related
to the theoretically amenable first-order electric field correlation function; g(1)(t), by
the Siegert relation75:
26
PhD Thesis Atoosa Maleki
g ( 2 ) (t ) 1 B g (1) (t )
2
(4)
where B ( 1) is an empirical factor.
In some polydisperse systems, the decay of the correlation function is unimodal
and only one relaxation mode is observed 76:
g
1
t ª § t
exp « ¨
« ¨© W fe
¬
·
¸
¸
¹
Ef
º
»
»
¼
(5)
In most semidilute systems of associating polymers, the correlation function
exhibits both ‘fast’ and ‘slow’ relaxation modes (bimodal), which can be described as
a single exponential, followed at longer times by a stretched exponential 29:
g
1
t ª § t
A f exp « ¨
« ¨© W f
¬
·
¸
¸
¹
º
ª §
» As exp « ¨ t
¨
»
«¬ © W se
¼
·
¸¸
¹
Es
º
»
»¼
(6)
The first term (‘fast’ mode; short-time behavior) on the right-hand side is always
related to the diffusion coefficient and the second term (‘slow’ mode; long-time
behavior) is associated with disengagement relaxation of individual chains 77 or cluster
relaxation.78 In some complex systems, the correlation function is described by a
double-stretched exponential 31:
g
1
t ª § t
A f exp « ¨
« ¨© W fe
¬
·
¸
¸
¹
Ef
º
ª §
» As exp « ¨ t
¨
»
«¬ © W se
¼
·
¸¸
¹
Es
º
»
»¼
(7)
Af + As = 1 , 0 < (f, s) 1
where Af and As are amplitudes for the fast and slow relaxation modes, respectively. fe
is some effective fast relaxation time, se is some effective slow relaxation time, and is a measure of the width of the distribution of relaxation times.
27
PhD Thesis Atoosa Maleki
In some entangled solutions, the correlation function exhibits three relaxation
modes.79,80
The mean relaxation times (f, s) can be defined from the fast and slow
relaxation modes by:
f
ª § t ·E f
¸
« © W fe ¸¹
¬
º
»dt
»
¼
W fe
Ef
§ 1
*¨
¨E
© f
·
¸
¸
¹
( 8)
Es
º
»dt
»¼
W se
Es
§ 1
*¨¨
© Es
·
¸¸
¹
( 9)
W f { ³ exp« ¨¨
0
ª § t
W s { ³ exp« ¨¨
«¬ © W se
0
f
·
¸¸
¹
where is the gamma function. The fast relaxation mode is a measure of how the
whole network fluctuates and moves around in a cooperative manner, while the slow
relaxation mode defines the movement of individual chains in the network. In the case
of dilute associating systems, a bimodal population of small and large species can exist
and this leads to the appearance of a fast and a slow relaxation mode, respectively.81
The fast relaxation time is usually q2 dependent,78,80 which reveals that the
system is diffusive. The diffusion coefficient (D; Dm is mutual diffusion coefficient in
a dilute solution, and Dc is the cooperative diffusion coefficient27 in a semidilute
polymer system) is a measure of how the molecules move, or how the collective
polymer network fluctuates in the solution. The diffusion coefficient can be
determined by72,73:
W f 1
D q2
(10)
28
PhD Thesis Atoosa Maleki
When a system exhibits a diffusive mode, the hydrodynamic radius (Rh) for
dilute solutions, and hydrodynamic correlation length (h) for semidilute solutions can
be calculated from the Stokes-Einstein relationship72,73:
D#
k BT
6SK0 X
(11)
where X is identified as Rh or h.
3.4.
Small Angle Neutron Scattering (SANS)
Neutrons interact with atomic nuclei and magnetic fields from unpaired electrons,
making them a useful probe of both structure and magnetic order. Much attention has
been paid to nuclear interactions for studying biological molecules.82 Because the
neutron is an electrically neutral particle, it is deeply penetrating, and is therefore more
able to probe the bulk material. Consequently, it enables the use of a wide range of
samples that are difficult to probe with the synchrotron X-ray sources. Neutrons can be
used to analyze materials of various natures. The technique provides valuable
information over a wide variety of scientific and technological applications including
chemical aggregation, defects in materials, surfactants, colloids, ferromagnetic
correlations in magnetism, alloy segregation, proteins, biological membranes, viruses,
ribosome, and polymers. During a SANS experiment, a beam of neutrons is directed at
a sample that can be an aqueous solution, a solid, a powder, or a crystal. The neutrons
are scattered by changes of refractive index on a nanometer scale inside the sample,
which is the interaction with the nuclei of the atoms present in the sample.83 Neutron
scattering is particularly useful for investigating the shape and size of the molecules.
29
PhD Thesis Atoosa Maleki
Analyzing the Data
SANS allows investigation of the mesoscopic structure of a polymer solution,
which makes the instrument able to probe the structure of the polymer on a more local
scale. An illustration of the SANS scattering intensity over an extended q range is
shown in Figure 10. At large q values (qL >> 1), the q dependence of the scattered
intensity I(q) can be described by a power law in q (I(q) a qdf ) and the value of power
law exponent (df) gives us the information about the shape of the polymer. The
relation between molecular weight and radius of gyration of the polymers in solutions
can be defined as Rg v M, where is the excluded volume parameter and is equal to
0.33 (globular structure), 0.5 (random coil), and 0.6 (random coil) at poor, , and good
thermodynamic conditions, respectively. The value of is 1 for a rod-like shape of
the scattering object. The power law exponent df is inversely proportional to (df =
1/). This leads to values of df of 1 for rod-like, 1.7 for random coil in good
condition, 2 for random coil in condition, and 3 for globular structures.70 In some
systems, the scattered intensity profile levels out at low q values (qL << 1), and this
plateau-like domain in the dilute concentration regime is called the ‘Guinier regime’.84
At this condition, it is possible to determine the size of particles or clusters (Rg)
through the relation I(q) =I(0)(1-(1/3)q2Rg2). For a semidilute solution in the low q
region (q[ <<1), the mesh size of the network (
) can be estimated from the OrnsteinZernike law I(q) | I(0)/(1 + q2[2).70 If large association structures are considered at
intermediate q values, this leads to a strong upturn of the scattered intensity in this q
range that can be portrayed by a power law (I (q) v q-z, see Figure 10).37,85,86 The value
of the power law exponent is 4 in the Porod regime. This value characterizes the
scattering expected from macroscopic pieces of a dense polymer phase, with sharp
boundaries.87,88
30
PhD Thesis Atoosa Maleki
I (q)
Guinier regime
qL << 1
I (q ) v q
df
Z
Intermediate q regime
I (q ) v q
d f
d f |1
Rod-like
d f | 1.7
Radom Coil,( good condition )
df |2
Radom Coil,( condition )
df |3
Globular Structure
Fractal regime
qL >> 1
q
Figure 10. An illustration of the SANS scattering intensity over an extended q range.
3.5.
Rheo-Small Angle Light Scattering (Rheo-SALS)
The macroscopic behavior of complex fluids depends usually on the microscopic
structure of the systems. Rheological measurements provide information about the
macroscopic properties, but cannot give further information about the structure of the
sample. Since mechanical deformations can alter the architecture of the polymer, it is
necessary to monitor structural changes during the shear flow to further understand the
rheological features of the system.89-91 The combination of small angle light scattering
equipment and a rheometer permits a direct correlation between the rheological
behavior and the microstructure of the sample. The structural and rheological
characteristics of solutions under the given shear rate can be probed simultaneously by
rheo-small angle light scattering (rheo-SALS).
31
PhD Thesis Atoosa Maleki
Analyzing the Data
The instrument used in this study is equipped with parallel plate-plate geometry
(see Figure 11). Utilizing a prism, the laser beam is deflected and passed through the
sample placed between the transparent plates. A polarizer is placed in front of the laser
and analyzer below the sample, making both polarized (polarizer and analyzer parallel,
used in this study) and depolarized (polarizer and analyzer perpendicular) experiments
possible. The 2D scattering patterns are captured at a given time using a CCD camera,
and the data can be analyzed by the SALS software (see paper II, VII).
Polarizer
Prism
Laser
Rotating glass plate
Sample
Stationary glass plate
Beam stopper
Analyzer
Focusing optics
Aperture
Semi-transparent screen
CCD Camera
Figure 11. A schematic diagram of the rheo-SALS setup.
32
PhD Thesis Atoosa Maleki
4. Discussions
4.1.
Microgels
When a dilute solution of a cellulose derivative is chemically cross-linked with a
cross-linker agent under a steady shear, different behaviors can be observed depending
on the structure of the polymer. Figure 12 shows the time evolution of the viscosity for
a reaction mixture of 0.1 wt % HEC, HM-HEC or dextran in the presence of 30 μl/g
DVS. Intermolecular cross-linking (increase in viscosity) is observed for all systems,
whereas intramolecular compaction (decrease in viscosity) is detected for HEC before
the viscosity rises. The minimum in the viscosity-shear rate curve is favored at
moderate shear rates because the rotational motion may expose a larger number of
hydroxyl groups of a single chain to the cross-linker. This facilitates intramolecular
cross-linking. At the highest shear rate, the stretching and fast rotation of the chains
may prevent the formation of intra- and interpolymer cross-linking (see Figure 12a).
The starting time of the viscosity rise is shifted to a shorter time as the polymer and
cross-linker concentrations increase (Paper I, Figures 11-12). The compactness and
branching structure of dextran coils33,92 probably avert shrinking of the polymer chains
(see Figure 12b).
The behavior is more complicated for the HM-HEC solutions. Because of
intermolecular hydrophobic interactions, a hump is developed at intermediate times
followed by strong increase in the viscosity as for the other systems (see Figure 12b).
Enhanced association in some polymer solutions due to the hydrophobic interactions
has been previously reported.93,94 In this stage, both hydrophobic associations and a
few intermolecular cross-links may give rise to an augmentation of the viscosity before
the sharp increase is observed. The hydrophobic interactions between two or more
polymer chains in the presence of shear flow make them close to each other and
consequently the possibility of intermolecular cross-linking increases and the influence
33
PhD Thesis Atoosa Maleki
of this effect inhibits the observation of intramolecular cross-linking in the considered
range of polymer and cross-linker concentrations.
The clusters may deform, break-up, and aggregate with other clusters.95 The flocs
or clusters are expected to rupture under the influence of relatively high shear rates.
The competition between the build-up and break-up of interaggregate chains can be
observed for all systems at the peak (Paper I, II). However, the orientation and type
(flocs or network) of the clusters may also affect the rheological behavior.95 The
clusters formed via aggregation process may be highly branched and they can
-2
10
Viscosity (mPas)
Viscosity (Pas)
resemble fractals.96
3.0
0.1 wt % HEC, 30 Pl/g DVS
300
100
20
2.0
1.5
5
10
15
Time (h)
20
-3
(a)
10
Viscosity (mPas)
0.1
Viscosity (Pas)
-1
J (s )
0.0045 M NaOH
2.5
-2
10
1
Time (h)
10
0.1 wt % Polymer, 30 Pl/g DVS
0.05 M NaOH
3.0
2.5
2.0
HEC
HM-HEC
Dextran
1.5
0
5 10 15 20 25
Time (h)
(b)
-3
10
0
10
1
10
Time (min)
Figure 12. Time dependence of the shear viscosity for polymer solutions of the indicated
polymers and concentrations with or without DVS. The insets illustrate magnification of the
data around the transition zones.
34
PhD Thesis Atoosa Maleki
Intrapolymer cross-linking of HEC with DVS is not observed from DLS
measurements (Figure 13a). The reason may be the dominant role of intermolecular
cross-linking at quiescent conditions. DLS measurements at long times were not
possible due to enhanced turbidity (Paper I, Figure 3) by the formation of large
aggregates as the cross-linking reaction proceeded. Even though the concentrations of
the polymer solutions were low, the decays of the correlation functions were found to
be bimodal; a single exponential followed at longer times by a stretched exponential.
The correlation functions have been analyzed with the aid of eqs. 6 and 9. The inset
plot of Figure 13a shows that both the fast and slow relaxation functions are shifted
toward longer times, indicating the formation of large association complexes as the
cross-linker reaction continues. To study the growth of the clusters at different times,
the samples were quenched by adding several drops of concentrated HCl.
Hydrodynamic radius, (calculated by eq. 11) increases with increasing quenching time,
which shows that the clusters grow as long as cross-linking proceeds (see Figure 13b).
35
1.0
Rh (nm)
30
25
0.6
1
g (t)
0.8
Wf
-2
10
Wf , Ws (s)
0.4
20
Ws
-3
0.1 wt % HEC, 0.0045 M NaOH, 30 Pl/g DVS
o
T : 70
without
DVS
229 min
254 min
283 min
330 min
10
0.2
0.0
-9
10
15
-4
10
1 2 3 4 5
Time (h)
-7
10
-5
(a)
-3
10
10
Time (s)
-1
10
(b)
10
0
15
30
45
Quenching time (min)
60
Figure 13. (a) First-order electric field correlation function versus time for 0.1 wt % HEC
solutions without cross-linker and with 30 μl/g DVS at different stages during the crosslinking reaction. The inset plot shows the fast and slow relaxation times.
(b) The
hydrodynamic radius of 0.1 wt % HEC solution with 30 μl/g DVS at different quenching
times.
35
PhD Thesis Atoosa Maleki
Figure 14 displays 2D SALS scattered intensity patterns at different times
during the cross-linking reactions of HEC and HM-HEC at a fixed shear rate of 30 s-1
and for HEC when no shear is applied. All patterns are almost isotropic in this
wavevector range, indicating that no major structural changes occur on this
dimensional scale. For HEC, very weak signals are detected at early times, whereas the
patterns are more pronounced for HM-HEC in the same period of time (see Figure
14a). This difference is probably the effect of intermolecular hydrophobic interactions
for the HM-HEC system before the sharp rise in viscosity takes place. The overall
viscosity trends are the same for cone-plate as for the plate-plate geometry, but
because of poorer resolution for the plate-plate arrangement, the minimum of the
viscosity curve could not be observed. The scattered intensity reduces when the flocs
are disrupted by the shear at longer times (e.g., after 55 min). The rupture of the
clusters cannot be detected when no shear is applied to the system, and instead a
progressive increase in the scattered intensity is observed (Figure 14b, J = 0 s-1).
Figure 14. 2D SALS patterns at different stages during the cross-linker reaction for (a) 0.1 wt
% solutions of the indicated polymers in the presence of 30 μl/g DVS at a constant shear rate
30 s-1 and (b) 0.1 wt % HEC at the indicated shear rate in the presence of 30 μl/g DVS.
36
PhD Thesis Atoosa Maleki
The scattered intensity images for dextran are almost constant at early times
(such as for HEC) and a sharp increase is observed after 17 min (Paper II, Figure 7).
Intensity (arb. units)
100
80
60
40
q = 0.27 Pm
-1
HEC
HM-HEC
20
0.1 wt % HEC, 30 Pl /g DVS, 0.05 M NaOH
120
Intensity (arb. units)
0.1 wt % Polymer, 30 Pl /g DVS, 0.05 M NaOH
-1
J30 s
120
100
80
60
40
q = 0.27 Pm
-1
-1
0s
-1
30 s
20
(a)
(b)
0
0
0
10
20 30 40
Time (min)
50
60
0
10
20 30 40 50
Time (min)
60
Figure 15. Time evolution of the scattered intensity during the cross-linking process of (a)
0.1 wt % of the indicated polymers at a fixed shear rate of 30 s-1 and (b) 0.1 wt % HEC
solutions at zero shear rate and shear rate of 30 s-1.
To elucidate this finding, the scattered intensity at a fixed low q value (q = 0.27
μm-1) is shown in Figure 15. The increase of the scattered intensity for HM-HEC at
early times (Figure 15a) is a signal of formation of association complexes through
hydrophobic groups, since the intensity is constant over the same period of time for
HEC. After a sharp increase in the intensity, it decreases as a result of disaggregation
of the flocs with the shear flow. The different behavior of HM-HEC at long times,
probably indicates that the rates of breaking up and rebuilding the aggregates are in
balance at this shear rate. At quiescent state (Figure 15b), a sharp transition zone is
detected at an early stage, whereas at longer times the intensity levels off. This
suggests that aggregates are formed during the cross-linking process and they grow to
a limiting size through intermolecular cross-linking. At this stage, the rates of building
37
PhD Thesis Atoosa Maleki
up and breaking up of large aggregates by Brownian motion will cancel each other,
and a constant value of the intensity is observed.
4.2.
Macrogels
The gel point of a semidilute solution of hydroxyethylcellulose (HEC) in the
presence of a cross-linker (DVS) is detected with two different experimental methods
with the aid of a rheometer. In Figure 16a, the frequency independency of tan at the
gel point is illustrated for a 1 wt % solution of HEC in the presence of 10 μl/ g DVS.
The gelation time decreases as the concentration of DVS increases (Paper III, Figure
1 wt % HEC, 10 Pl/g DVS
0.0045 M NaOH
1
10
tg
0
tan G
10
Z (rad/s)
-1
10
-2
10
100
39.8
15.8
6.31
2.51
1.00
0.40
0.16
Z (rad/s)
63.1
25.1
10.0
3.98
1.58
0.63
0.25
0.10
Zero-shear viscosity (Pas)
3).
10
9
10
7
10
5
10
3
10
1
1 wt % HEC, 10 Pl/g DVS
0.0045 M NaOH
tg
-1
10
(a)
(b)
-3
10
2
3
4
Time (h)
5
0.1
1
Time (h)
10
Figure 16. (a) Viscoelastic loss tangent as a function of time for the frequencies indicated
during the cross-linking reaction of 1 wt % HEC solution in the presence of 10 μl/g DVS (b)
Time evolution of zero-shear viscosity during the gelation of the indicated system. The time
of gelation (tg) is indicated.
38
PhD Thesis Atoosa Maleki
It is possible to determine the approximate gel point by steady shear
measurements if the magnitude of the shear rate is adjusted as the viscosification of the
system is increased. Figure 16b shows the evolution of the shear viscosity at low shear
rates for the same system. To monitor an apparent zero-shear viscosity, the value of
the shear rate was continuously lowered during the gelation process. A sharp transition
of the viscosity to higher values is detected at the gel point. Over a long time, in the
post gel region, the solvent is expelled from the gel sample and this phenomenon is
usually referred to as ‘syneresis’
14,97
(Paper III, Figures 10). Scattering of the
viscosity data observed at long times may to some extend reflect this effect. A more
solid-like response is found for this system as the gel approaches the post-gel region
(Paper III, Figure 5).
q
0 Pl/g DVS
5 Pl/g DVS
10 Pl/g DVS
15 Pl/g DVS
-2.1
0
-1
d6/d: (cm )
10
q
-1.1
-1
10
-2
Quenched in post-gel regime: H | 0.4
10
-2
10
-1
q (Å)
10
Figure 17. SANS scattered intensity versus scattering vector q for 1 wt % HEC solution
without (no gel) and with (gel) different cross-linker concentrations. For the gels, the crosslinking reaction has been quenched at a certain stage 0.4, where is the relative distance
from the gel point and can be determined from = (t – tg)/tg. (t is the considered time and tg is
the time of gelation).
39
PhD Thesis Atoosa Maleki
To investigate the structure of HEC networks on a mesoscopic dimensional scale,
a series of SANS measurements have been carried out on gels with different crosslinker concentrations. The gels have been quenched in the post-gel region at certain
distance (see Figure 17) from the gel point. The progressively stronger upturn of the
scattered intensity at low q values as the cross-linker concentration increases reveals
the formation of multichain domains.37 This feature can be described by a power law
with the slope of -2.1 for the sample with the highest DVS concentration. Formation of
large-scale fluctuations as the cross-linker concentration increases, leads to a gradually
stronger upturn in the scattered intensity in this q range. Different values of the power
law exponent have been reported for polymer systems of various natures.82,93,98 In the
high q range, no effect of cross-linker concentration can be detected. A value of the
power law exponent of -1.1 shows that the polymer chains are locally stretched on this
short length scale and the cross-linking reaction does not affect the local structure of
the network.
4.3.
Biopolymers in our Body
4.3.1. Hyaluronan
Polymer degradation is a change in the properties of a polymer or polymer
based product under the influence of environmental factors such as light, heat or
external chemicals. The degradation may be undesirable or desirable.
Hyaluronan and its sodium salt derivative are known to be degraded by various
factors such as hydroxyl group,47 oxygen radicals,99,100 ultrasonication101 alkaline 102,103
and acidic103,104 conditions. Degradation of hyaluronan inside the body may cause
diseases such as mesothelioma and inflammatory arthritides.47,105 However, scientists
are considering the improvement of this property (in vitro) by making HA hydrogels
40
PhD Thesis Atoosa Maleki
with high mechanical strength by means of chemical cross-linking.106 On the other
hand, chain breakage of these biomacromolecular chains in a controlled way, makes
them good candidates for drug delivery applications.
To gain detailed insight into HA behavior at different conditions, a series of
measurements with various techniques have been conducted on dilute and semidilute
solutions of a sodium salt of hyaluronan with or without cross-linker agent.
0
0.5 wt % HA
pH = 1
pH = 5
pH = 9
pH = 12
pH = 3
pH = 7
pH = 11
pH = 13
6
10
-1
Mw
10
6
2x10
-2
10
M
w
Viscosity (Pas)
10
6
1x10
0 3 6 912
-3
(a)
pH
10
-1
10
0
pH = 1
pH = 7
pH = 13
(b)
5
10
1
2
10
10
10
-1
Shear rate (s )
3
10
1
10
Time (h)
Figure 18. (a) Viscosity as a function of shear rate for semidilute-buffered systems of
hyaluronan at the pH values indicated. The inset plot shows the effect of pH on the weightaverage molecular weight. (b) Time evaluation of the degradation of 0.5 wt % HA solutions in
terms of molecular weight from AFFFF at the indicated pH values. The dissolving of HA in
solvents at pH 1 and 7 required longer time than at pH 13. This is the reason why the data
points for these two pH values at early times are missing. (The samples were diluted during
the experiments with AFFFF; see the text for details.)
Figure 18a illustrates the effect of pH on the viscosity of 0.5 wt % buffered HA
solutions. Shear thinning is observed at high shear rates and at intermediate pH values.
In the semidilute regime, polymer molecules are strongly entangled and the viscosity
41
PhD Thesis Atoosa Maleki
depends on the rate of shear flow.27 At high shear rates, the rate of network disruption
for semidilute HA solutions exceeds the rate at which associations and entanglements
are reformed, and as a result shear thinning is registered. The drop of the zero-shear
viscosity at low and high value of pH is attributed to intramolecular rupture of polymer
chains. In addition, conformational change of HA chains at very high pH may also
contribute to the strong decrease in viscosity. The viscosity effect becomes evident by
measuring the weight average molecular weight (Mw) of the samples with the aid of
AFFFF (see the inset plot). The strong decrease in molecular weight at low and high
pH values suggests cleavage of the glycosidic bonds of the HA chains. The molecular
weight is approximately constant at intermediate pH values. The same trend of
degradation for 0.05 wt % HA solution is observed at this range of pH values (Paper
IV, Figure 2).
To monitor the kinetics of degradation of HA at different pH, the molecular
weight of HA in solutions at various times was measured with the aid of AFFFF. The
concentration of HA solutions prepared in buffers with various pH values were 0.5 wt
%. After adjusting the pH values to neutral (very acidic or basic solutions destroy the
membrane of the instrument), the concentration of HA solutions were ~ 0.3 wt %. A
strong dilution of the samples take place during all experiments with AFFFF, and the
effective concentrations of polymers probed in the measurements are approximately
100 times lower. Figure 18b demonstrates that the scission of glycosidic linkages is
much faster at pH = 13 than at pH = 1, whereas almost no degradation is observed at
pH = 7.
HA chains may be cleaved by photodegradation in the presence of a
photosensitizer such as riboflavin.60 In Figure 19, the decrease of the viscosity at a low
shear rate for 0.5 wt % HA samples dissolved in 0.1 mM RF (Supplied by SigmaAldrich) is depicted. The beam from an argon ion laser (Lexel laser, model 95),
operating at a wavelength of 457.9 nm was focused on the sample. The solutions were
42
PhD Thesis Atoosa Maleki
continuously irradiated at different times. To accomplish homogenous illumination of
the solution, both sides of the sample cells were exposed to the light in the same period
of time. Illumination of the sample in the presence of riboflavin leads to the formation
of reactive species, which may attack the glycosidic bonds of the HA molecules and
consequently lead to a reduced viscosity of the solution. The chain cleavage is
gradually arrested at long times of irradiation, which is probably due to the
consumption of all riboflavin molecules in the course of time. To elucidate this
phenomenon, a HA solution with a higher concentration of RF (1 mM) was
illuminated for 2 h. The inset plot shows that the viscosity decreases strongly for the
Viscosity (Pas)
sample with a higher amount of riboflavin.
0
10
0.5
0.4
0.3
0.2
0.1
0.0
Riboflavin
0.1 mM
1 mM
2 h illumination
Viscosity (Pas)
1
-1
10 100 1000
-1
Shear rate (s )
Time of illumination
10
No illumination
30 min
1h
2h
-1
10
0.5 wt % HA, 0.1 mM Riboflavin
0
10
1
10
-1
Shear rate (s )
2
10
3
10
Figure 19. Viscosity as a function of shear rate for non-irradiated and irradiated (at different
illumination times) of 0.5 wt % HA dissolved in 0.1 mM RF. The inset plot shows the effect
of RF concentration on the degradation of 0.5 wt % HA solution after 2 h of illumination.
43
PhD Thesis Atoosa Maleki
The time evolution of * for 0.5 wt % HA solutions with and without crosslinker is displayed in Figure 20a. Intermolecular formation of ester bonds during the
cross-linker reaction for the sample in the presence of 5 wt % and 10 wt % WSC as a
cross-linker agent will induce cross-linking and the formation of a gel network.
However, ester bonds are known to be sensitive to hydrolysis107 and this phenomenon
can be observed in the complex viscosity values versus time in Figure 20a. The
viscosity curves pass through a maximum before the network is disrupted by the
hydrolysis of the ester bonds. As was shown in Figure 18, HA is sensitive to pH and a
slight decrease in Mw was observed at pH ~ 4. Another reason for decreasing value of
* may be the degradation of HA chains in the presence of citric acid (pH ~ 4 by
adding 0.05 wt % CA). The gradual decrease of the complex viscosity is demonstrated
for 0.5 wt % HA solution with 0.05 wt % CA and without WSC. However, the
presence of a cross-linker may have a major effect on the magnitude of the
degradation, since the sample with higher cross-linker concentration (10 wt %) shows
lower * values after the peak than the one with 5 wt %. It has been reported108
that by blocking the carboxyl function in a mucopolysaccharide, hydrolysis of the acid
resistant glycosyluronic linkage may be increased. The complex viscosity is almost
constant over a long time for HA solution without CA/WSC.
Figure 20b shows the results from AFFFF measurements over a long time for
0.5 wt % HA (the samples were diluted during the experiments with AFFFF) in the
presence of 0.05 wt % CA and 5 wt % WSC. It is not possible to perform
measurements during the gel formation because of the high viscosity of the crosslinked network. The molecular weight and polydispersity of the polymer both pass
through a maximum (ca. 40 h) and decrease for several days after the cross-linking
process was started. The maximum value of Mw announces formation of large species
due to the cross-linking reaction. This competition between cross-linking and
degradation of the polymer results in an extremely polydisperse sample (Mw/Mn = 26).
44
PhD Thesis Atoosa Maleki
Low values of Mw after several days, reveal that most of the ester bonds are
hydrolyzed and scission of glycosidic bonds in the HA chains has occurred.
0.5 wt % HA, 0.05 wt % CA, 5 wt % WSC
0.5 wt % HA
30
10
Mw , Mn
Complex Viscosity (Pas)
10
0
10
M /M
w n
6
ca. 40 h
Z : 1 rad/s
1
0.0
5
-1
10
0
10
tg: 61 min
20
10
100
Time after cross-linking (days)
tg: 43 min
10
Mw
0.05 wt % CA, No WSC
0.05 wt % CA, 5 wt % WSC
0.05 wt % CA, 10 wt % WSC
-2
10
0
5
10
15
Time (h)
Mn
(a)
20
25
4
10
(b)
0.0
10
100
Time after cross-linking (days)
Figure 20. (a) Time evolution of the absolute value of the complex viscosity for 0.5 wt % HA
solutions in the presence of 0.05 wt % CA, with and without WSC as a cross-linker agent.
The gelation times are indicated (Paper V, Table 1). (b) Weight-average molecular weight
(Mw) and number average molecular weight of 0.5 wt % HA in the presence of 0.05 wt % CA
and 5 wt % WSC as a function of time after the commencement of the cross-linker reaction.
The polydispersity index (Mw/Mn) is displayed in the inset plot.
In order to stabilize and keep the cross-linked gel network of HA as long as
possible, the effect of other components and cross-linker agents were also examined.
Figure 21 depicts a comparison of the time evolution of the complex viscosity in the
course of gelation and degradation for 0.5 wt % HA in the presence of 10 wt % WSC,
with and without L-lysineME, and via the Ugi multicomponent condensation reaction.
The presence of L-lysineME leads to the formation of amide bonds, which are known
to have higher resistance against hydrolysis. It should be mentioned that both ester and
45
PhD Thesis Atoosa Maleki
amide bonds are generated during the cross-linking reaction for the sample containing
WSC and L-lysineME. As a result of this interplay, a breaking down of the network
can be observed in the presence of L-lysineME, but the network decay is postponed to
a longer time. A moderate degradation is detected for the gel network prepared
through the Ugi reaction, because most of the cross-linking bonds in this multi
component reaction are amide linkages. The possibly formation of ester bonds by the
parallel Passerini condensation reaction yields only a moderate decrease in the
complex viscosity for the sample prepared via the Ugi reaction. The possibility of the
cleavage of the glycosidic bonds of HA chains in the networks that were formed via
any of these cross-linking reactions cannot be ignored.
0.5 wt % HA
Z :1 rad/s
Complex Viscosity (Pas)
25
20
10 % WSC, 0.05 wt % CA
10 % WSC + L-LysineME,0.05 wt % CA
Ugi reaction
15
10
5
0
0
10
20
30
40
50
Time (h)
Figure 21. Time evolution of the absolute value of the complex viscosity in the course of the
gelation process of 0.5 wt % HA in the presence of 0.05 wt % CA and 10 wt % WSC, with
and without 1 wt % L-lysineME, and in the presence of 0.13 wt % 1,5-diaminopentane as a
cross-linker for the four-component Ugi condensation reaction.
46
PhD Thesis Atoosa Maleki
The results from the rheological experiments on HA samples during the crosslinking with WSC are consistent with the findings from DLS (see Figure 22). The fast
relaxation time increases as the gel network evolves and the maximum value is located
at almost in the same position as for the corresponding value of the complex viscosity
(details about the analysis of DLS data are given in Paper V). Intermolecular crosslinking gives rise to a more heterogeneous network and leads to a decrease of the value
of the diffusion coefficient. Higher heterogeneity can also be deduced from the values
of the average mesh size of the network (
), determined by the Stokes-Einstein
relationship (eq. 11)72,73 (Paper V-Figure 9a, inset). A more homogenous network may
be formed by breakage of ester linkages and scission of chains during the hydrolysis,
since the values of the fast relaxation time show a decreasing trend at longer times.
The formation of small polymer species by reorganization of the network during the
hydrolysis will cause an increase of the diffusion coefficient and faster relaxation time.
0.40
0.5 wt % HA, 0.05 wt % CA, 5 wt % WSC
Visc. maximum
Wf (ms)
0.35
0.30
tg
0.25
0.20
1
10
Time (h)
Figure 22. Time evolution of the fast relaxation time for 0.5 wt % HA in the presence of 5 wt
% WSC. The gelation time and the time at which the viscosity approaches a maximum value
is displayed.
47
PhD Thesis Atoosa Maleki
In spite of the weak glycosidic linkages of HA at the various conditions
mentioned above, the polymer chains can be quite stable against mechanical
perturbations and temperature. Figure 23a displays the effect of temperature on the
viscosity for a 1 wt % HA solution. In this case, steady shear (0.01 s-1) measurements
were conducted on the sample for 60 min at each temperature. The experiments started
at 10 oC and the temperature was raised continuously up to 45 oC, and then it was
quenched to 25 oC. The waiting time at each temperature prior to measurement was 10
min. The viscosity of the HA solution drops with increasing temperature. This effect is
probably due to higher mobility of the chains at elevated temperatures. By quenching
the temperature from 45 oC to 25 oC, the viscosity of HA goes back to virtually the
same value as before. This indicates that no hysteresis effect of HA is visible at this
concentration in this range of temperature.
The effect of a progressive raise of the shear rate and the return to the lowest
value of the shear rate on the structural behavior of a 0.2 wt % and 2 wt % HA solution
is displayed in Figure 23b. Prior to each shear rate run, the HA samples are allowed to
rest for 1 h before the next shear rate is applied. The time-induced viscosification of
0.2 wt % HA solution at the lowest shear rate may be ascribed to shear-induced
alignment and stretching of polymer chains and formation of hydrogen bonds. This
effect cannot be observed for high concentrations of HA solutions such as 2 wt %,
since the strong network that is provided by growing of entanglements at high polymer
concentrations will prevent the deformation of HA chains in this relatively weak flow
field. (Paper VI)
As the transitory shear rate perturbations become stronger, the viscosity falls
off. It passes through a minimum and increases again upon decreasing the shear rate.
The viscosity is almost restored when the shear rate approaches the lowest value, but
the viscosification of 0.2 wt % HA solution is not as pronounced as in the beginning.
48
PhD Thesis Atoosa Maleki
That may be an effect of strong shear rate that the sample has been subjected to during
the experiment. Shear-induced association of polymer solutions has been previously
reported.8
3
10
60
o
Viscosity of HA from T : 10-45 C
o
Viscosity of HA measured at 25 C
after quenching
2
10
Viscosity (Pas)
Viscosity (Pas)
50
40
30
20
10
1
10
0
0.001 s
10
-2
10
20
30
o
T( C)
(a)
40
50
-1
0.01 s
-1
0.1 s -1
1 s -1
60 min each
10 s
-1
10
-1
1 wt % HA, J : 0.01 s
-1
10
trend up
-1
100 s
wt % HA
0.2
2
30 min
-1
1000s
15 min
-3
10
0.1
The same
1
(b)
10
Time (h)
Figure 23. (a) Viscosity as a function of temperature for a 1 wt % HA solution at a fixed
shear rate of 0.01 s-1. The filled square () is the viscosity at 25 C immediately after
quenching the temperature from 45 C to 25 C. The values indicated in the graph are the
viscosities after 30 min. (b) Viscosity as a function of shear time, where the shear rate is
periodically increased up to 1000 s-1, followed by a subsequent decrease of the shear rate with
the same periodicity as in the upramp curve for 0.2 wt % and 2 wt % HA solution.
The schematic illustration of the viscosity behavior of HA solution at a
relatively low and high concentration under the influence of a low and a high shear
rate is shown in Figure 24. For a dilute HA solution (Figure 24a), the chains are
aligned under the influence of a low shear rate and the probability of forming
aggregates through hydrogen bonds is enhanced, and this leads to higher viscosity of
the solution. Formation of large aggregates in dilute hyaluronan solutions with the aid
of SANS and DLS measurements at quiescent condition has been reported.109
However, when the shear rate increases strongly, the hydrogen bonds may break down
49
PhD Thesis Atoosa Maleki
and consequently a drop of the viscosity is found. The scenario is different when the
polymer concentration is increased (Figure 24b). Low shear flow has almost no effect
on the viscosification of semidilute HA solutions, since stretching and alignment of
HA chains is prevented by strong entangled network in the weak flow field. At the
high shear rate, the network junctions as well as some hydrogen bonds may break
down and the viscosity decreases; this phenomenon is called shear thinning.
Low Shear Rate
High Shear Rate
(a)
No shear
(b)
Low Shear Rate
High Shear Rate
Hydrogen bonds
Figure 24. Schematic illustrations of shear-induced association in dilute and semidilute HA
solution at low and high shear rates. (a) At low concentration the shear-thickening effect can
be detected under the influence of low shear rate and a decrease of the viscosity is observed at
high shear rate (b) At high concentration no viscosification is found at low shear rate, whereas
at high shear rate shear-thinning is observed.
50
PhD Thesis Atoosa Maleki
4.3.2. Mucin
The effect of pH on the structural and dynamical features of the transient
network of semidilute mucin solutions, as well as the behavior of individual mucin
molecules in dilute solutions has been surveyed with the aid of DLS and rheo-SALS.
As was shown in Figure 6, there is no clear definition of the chemical structure
of mucin so far. However, the existence of anionic charged groups and hydrophobic
domains has been reported.53 This complex structure with electrostatic repulsions and
formation of hydrophobic associations will strongly affect the physicochemical and
rheological properties of this polymer. The charge density of mucin alters with the
value of pH. The electrostatic interactions will lead to the formation of complexes with
interesting characteristics, both in dilute and semidilute solutions of mucin. This is the
theme of the discussion below.
3
3
10
K0 (Pas)
10
1
10
-1
10
-3
[h (nm)
Rh (nm)
10
2
10
1
1234567
pH
2
10
0.01 wt % Mucin
(a)
10
1
2
3
4
pH
5
6
7
(b)
1 wt % Mucin
1
10
1
2
3
4
5
pH
6
7
Figure 25. (a) The hydrodynamic radius as a function of pH for 0.01 wt % mucin. (b) pH
dependence of the screening length h, calculated from the fast relaxation mode for 1 wt %
mucin. The inset shows the effect of pH on the zero-shear viscosity measured by rheo-SALS.
51
PhD Thesis Atoosa Maleki
Figure 25a shows the hydrodynamic radius (Rh), calculated from eq. 11, of a
dilute (0.01 wt %) mucin solution as a function of pH. It has been argued that at pH
around 2 the zeta-potential of mucin approaches zero.110 As a result, associations
between hydrophobic groups are dominating and they may form the large interchain
complexes. Enhanced viscosity and aggregation of pig gastric mucin at low pH have
been reported previously.55,111 At lower pH (pH < 2), mucin becomes positively
charged, leading to contraction of interchain complexes and lower values of Rh. At pH
values above 2, mucin is negatively charged and the charged density increases with
rising pH. This augmented charge density will induce higher electrostatic repulsion
and more extended chains. The intermolecular interactions will be dispersed at this
stage and Rh reduces to a much lower value (Rh 75 nm). Reduced intensity data from
DLS measurements disclose the same type of profile with a maximum value at pH
around 2 (Paper VII, Figure 2d).
The change of the characteristic mesh size of the transient network (
h,
calculated from eq 11) for 1 wt % mucin solution at different pH is depicted in Figure
25b. The enhanced hydrophobic interactions at pH 2 will form a more heterogeneous
network with the appearance of large ‘lumps’, which leads to a maximum value of h.
The increase of the charge density as the pH decreases or increases may cause a
disassociation of the ‘lumps’ and reduce the value of h because of the repulsive forces.
The longest slow relaxation time is detected for the semidilute mucin solution at pH 2 by the DLS measurements (Paper VII, Figure 4b). This signalizes strong interactions
between the chains at this pH and polymer concentration.78,112,113 A schematic
illustration of the network structures at these three stages is displayed in Figure 26.
52
PhD Thesis Atoosa Maleki
(a)
(b)
pH < 2 : Positively Charged
(c)
pH 2 : Almost Neutral
pH >> 2 : Negatively Charged
Figure 26. Schematic illustration of the possible network formation in semidilute mucin
solutions at different pH.
The contraction of the chains at pH < 2 (as shown in Figure 26a) is supported
by the viscosity results. The inset plot in Figure 25b shows the zero-shear viscosity
data from rheo-SALS experiment for a 1 wt % mucin solution.
Figure 27. SALS 2D patterns of 1 wt % solutions of mucin at a shear rate of 0.l s-1 and the
indicated pH values.
Figure 27 shows 2D SALS scattered intensity patterns for 1 wt % solutions of
mucin at 0.1 s -1 and at different pH values. The scattered intensity profile is strongest
at pH 2 and pH 4, which announces the formation of multichain structures at these pH
values. To elucidate this finding, the intensity data can be plotted as a function of q at
different pH values.
53
PhD Thesis Atoosa Maleki
SALS intensity profiles for 1 wt % mucin solutions at a shear rate of 0.1 s-1 and
different values of pH are depicted in Figure 28. The open symbols show the intensity
values along the flow direction and the solid symbols vertically across. The strong
upturn of the scattered intensity at low q values at pH 2 and pH 4 indicates the
formation of large-scale heterogeneities in the solution.85 The q dependence of the
scattered intensity (I(q) ~ q-d) of 1 wt % mucin solutions at low q values are displayed
in the inset plot with a power law exponent as a function of pH. The higher value of d
at pH 2 and pH 4 suggests the enhanced association effect of multichain domains in
the solutions. The values of the power law exponent for the mucin solution at a fixed
pH is almost independent of the shear rate (Paper VII, Figure10).
100
1 wt % mucin, 0.1 s
-1
2.0
d
1.8
80
1.6
Intensity (arb.units)
1.4
1.2
60
I (q) a q
-d
1 2 3 4 5 6 7
pH
40
pH = 1
pH = 2
pH = 4
pH = 7
20
0
-1
0
10
10
-1
q (Pm )
Figure 28. SALS scattered intensity plotted versus scattering vector q for 1 wt % mucin
solutions at a shear rate of 0.1 s-1 and the pH values indicated. The angular dependencies of
the scattering intensities along the flow direction (open symbols) and vertically across (solid
symbols) were analyzed by means of the software program. The inset plot shows q
dependency (I(q) ~ q –d ) of 1 wt % mucin solution at different pH in the flow direction.
54
PhD Thesis Atoosa Maleki
Figure 29 shows 2D SALS scattered intensity patterns for 1 wt % solutions of
mucin at pH = 2 and different shear rates. The scattered intensity profile reveals an
anisotropic pattern with an elliptical scattering pattern with a major axis parallel to the
flow direction at the highest shear rate (1000 s-1). This indicates the formation of
concentration fluctuations of large amplitude along the flow direction in this q range.
Enhanced concentration fluctuations in polymer solutions under the influence of shear
flow have been previously reported.114
Figure 29. SALS 2D patterns of 1 wt % solutions of mucin at pH = 2 and indicated shear
rates.
55
PhD Thesis Atoosa Maleki
5. Concluding Remarks and Future Aspects
In this thesis, some novel information about the influence of steady shear flows
on intramolecular and intermolecular associations in dilute aqueous alkali solutions of
HEC, dextran and HM-HEC in the presence of DVS as a cross-linker has been given.
In solutions of HEC, intrachain contraction under shear stress leads to a viscosity
decrease, followed by a sharp increase in viscosity due to intermolecular cross-linking
and formation of large aggregates. DLS measurements at quiescent conditions reveal
no contraction but only multichain aggregation under the influence of Brownian
motions. The compactness of the dextran coils prevents the effect of shrinking in this
type of branched polysaccharide, but interchain aggregation is observed as for the
HEC system. In HM-HEC solutions, no intrapolymer cross-linking occurs in the
course of the reaction. The presence of hydrophobic groups in HM-HEC generates the
hydrophobic interactions prior to intermolecular cross-linking, and this effect
overshadow the contraction of the species. Rheology and rheo-SALS demonstrates the
building up and breaking down of association structures at longer times under the
steady shear stress.
The structural behavior of HEC with DVS is different when the polymer
concentration increases. Cross-linking of semidilute HEC solutions with DVS leads to
an interconnected gel network. By increasing the cross-linker concentration, the
gelation time drops and the upturn in the SANS scattered intensity at low q values is
strengthened. This suggests that cross-linker addition promotes the growth of the
heterogeneities in the gel, but no effect of cross-linker concentration is detected in the
high q range or at short length scales.
In aqueous solution, HA solution is degraded at acidic (pH < 4) or basic (pH >
11) conditions. The degree of degradation of HA is most marked at high pH, and the
process is faster at pH = 13 than at pH = 1. AFFFF experiments show that the
56
PhD Thesis Atoosa Maleki
molecular weight decreases as the value of pH decreases or increases. In the case when
a HA solution (0.5 wt %) is exposed to light ( = 457.9 nm) in the presence of RF as a
photosensitizer, polymer degradation can also be accomplished. The reactive species
produced by riboflavin at this wavelength can attack the glycosidic bonds in the HA
chain and thereby attain cleavage of the chain. The addition of WSC as a cross-linker
to a semidilute HA solution generates a gel stabilized with many ester bonds.
Rheological results show that the absolute value of the complex viscosity passes
through a maximum and then falls off due to breaking down the network by hydrolysis
of ester bonds and chain cleavage. This degradation can be reduced by adding LlysineME to the system and consequently introducing some amide bonds, which are
known to be less sensitive to hydrolysis. When the Ugi reaction is used, most of the
cross-linking bonds are amide linkages and as a result, a more stable gel evolves.
Influence of steady shear stress has been examined on HA solutions. The system
shows shear thickening at low concentration of HA (0.2 wt %) and low fixed shear rate
(0.001 s-1) and shear thinning is observed as the shear rate increases. Almost no
hysteresis effect is found upon the gradual return to low shear rates. The results of
viscosity measurements show that 1 wt % HA solutions are chemically stable at
temperatures within the range of 10 oC to 45 oC.
The influence of pH on the structural feature of mucin in solution has been
investigated. Formation of large association complexes is observed in dilute solutions
of mucin with the aid of DLS experiments at pH 2. This indicates that the polymer
exhibits charge neutrality or low charge density at this pH. As the pH decreases or
increases, the electrostatic repulsive interactions prevent the formation of interpolymer
aggregates of the mucin molecules. The fast relaxation mode in semidilute solutions of
mucin is always diffusive. The correlation length (
h) determined by the StockEinstein relation is higher at pH 2 than other pH values, which is because of the
formation of a heterogeneous network through hydrophobic interactions at this pH.
57
PhD Thesis Atoosa Maleki
For the future work, it would be interesting to investigate the structural
properties of dilute solutions of positively or negatively charged polymers with or
without oppositely charged cosolute under the steady shear stress. For HA solutions,
the influence of pressure on its degradation process would be a topic of interest. The
effect of hyaluronidases on HA degradation in the presence and absence of pressure
can be studied. Another interesting issue that should be addressed is the effect of other
external stimuli. There are always many different aspects in the evaluation of these
types of systems. And as it is said by Pierre-Gilles de Gennes*: “There is still room for
a lot of imagination”.
* Collège de France, Nobel Prize Laureate in Physics in 1991; for discovering that methods
developed for studying order phenomena in simple systems can be generalized to more complex
forms of matter, in particular to liquid crystals and polymers.
58
PhD Thesis Atoosa Maleki
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